3  Test

from google.colab import drive
drive.mount('/content/drive')
Drive already mounted at /content/drive; to attempt to forcibly remount, call drive.mount("/content/drive", force_remount=True).
!pwd
/content
import os
# checking if the directory demo_folder
# exist or not.
design_build_dir = "/content/drive/MyDrive/design_build"
if not os.path.exists(design_build_dir):

    # if the demo_folder directory is not present
    # then create it.
    os.mkdir(design_build_dir)
os.chdir(design_build_dir)
!pwd
/content/drive/MyDrive/design_build

3.1 Udocker for Colab

  • docker 설치 (colab 사용시 udocker 활용)
  • https://github.com/drengskapur/docker-in-colab
  • 코랩에서 다음 코드 복사, 붙여넣기
  • 서열 분석 툴을 모아둔 이미지 GATK (https://gatk.broadinstitute.org/hc/en-us) container 사용
def udocker_init():
    import os
    if not os.path.exists("/home/user"):
        !pip install udocker > /dev/null
        !udocker --allow-root install > /dev/null
        !useradd -m user > /dev/null
    print(f'Docker-in-Colab 1.1.0\n')
    print(f'Usage:     udocker("--help")')
    print(f'Examples:  https://github.com/indigo-dc/udocker?tab=readme-ov-file#examples')

    def execute(command: str):
        user_prompt = "\033[1;32muser@pc\033[0m"
        print(f"{user_prompt}$ udocker {command}")
        !su - user -c "udocker $command"

    return execute

udocker = udocker_init()
Docker-in-Colab 1.1.0

Usage:     udocker("--help")
Examples:  https://github.com/indigo-dc/udocker?tab=readme-ov-file#examples

3.1.1 Image pulling from the hub

!udocker --allow-root pull broadinstitute/gatk:4.5.0.0
Info: downloading layer sha256:a0350cd371d6d1507fc4cf617e175eb5bd4c3b204c333c94d185bf1ea0795e28
Info: downloading layer sha256:0ed77206bc58a5c4253dc48f63c49ae2f6a3dfef68374e35b8906e397b8b3a34
Info: downloading layer sha256:4da44515c0282c753dea8e5d620f840683b9be744fc3558aa200a3b6801d6d02
Info: downloading layer sha256:a3d1f390828eebbdf0dd57dde549378ccefa065824953f433e83a7c90782f18d
Info: downloading layer sha256:e28bcb90193018e21503133c0c383ae7124a6407a607045140b41cf81f2eb850
Info: downloading layer sha256:0258c7d63f9241bccdb6655a641406158b0a0d6cedc40219f8f8eb98d84590ba
Info: downloading layer sha256:b3625edcebc02c6a07a068a9bb09e929f281041bf1929c3fc914e22a01c7db76
Info: downloading layer sha256:8744436b9dbf91ddefe776c5aa6d061fa93655ff265edc3c6cfcd2ca5398e5e0
Info: downloading layer sha256:f34824c486eb930b1bf0bdd34c45fbb7d237e6c22fdaf716a65c9e9a2fc6f194
Info: downloading layer sha256:4f084384c353a3bf1e2d8a7c6855261feced9060e1ccfcb572243777d87b5741
Info: downloading layer sha256:891945c414afb02f8aa7f1ca2688134147fdd3f8108372851fbb996c3c96d8b2
Info: downloading layer sha256:cffcd2ae2fcdfcfb2ee1886960f2a4fffdc20d4f7a53ea5cb8b107fce7f2acdd
Info: downloading layer sha256:cf7105ec9c451c3c31b518f90a63fcd21e409dcf99a8c23c2e6b45fb0e3404ef
Info: downloading layer sha256:134b348e3b5e7720f8e80c492939d15d4b15338fb03cd3417a651c0d79b7f6aa
Info: downloading layer sha256:5bb37dfbacf38a543a3b8322ede27dc48c7abd9346a53633913c95356d1ca32e
Info: downloading layer sha256:90226c0b8ca9b1c5bdfb48f0625f151e2acbffcf793aad9319f81fa795749236
Info: downloading layer sha256:91e6f2d6d5602796597c6b4f77786421b207d0746b8265ead7357f543facbc02
Info: downloading layer sha256:762ff3861584e4482f79ebaeef7c0a5b5fd2f6ce91d9190b54353b614a5e2b26
Info: downloading layer sha256:043eca7b7dd5fb53ae84f5e3e94d3bdee177fbfe702bfef8502ccf5062794939
Info: downloading layer sha256:1a799190d815f77920c9963278e0e8b1a3c2f66bf5850bc46ce6fb9da3273258
Info: downloading layer sha256:5e8117c0bd28aecad06f7e76d4d3b64734d59c1a0a44541d18060cd8fba30c50
!udocker --allow-root pull staphb/minimap2:2.28
Info: downloading layer sha256:858eac332b979bf2408f3c3c67dd281593cb8219212009d75944e0fbfc8a93a1
Info: downloading layer sha256:4f4fb700ef54461cfa02571ae0db9a0dc1e0cdb5577484a6d75e68dc38e8acc1
Info: downloading layer sha256:c4c71a0bb4e0776f27a061207311a5b51348e03aa9fa0b71783d1c647431536a
Info: downloading layer sha256:b3d5e5561d0719e43f9195e30076f07b92c6552dc888d4f9a62b9d47100c38f1
Info: downloading layer sha256:bccd10f490ab0f3fba61b193d1b80af91b17ca9bdca9768a16ed05ce16552fcb
!udocker --allow-root images -l
REPOSITORY
broadinstitute/gatk:4.1.3.0    .
 /root/.udocker/repos/broadinstitute/gatk/4.1.3.0
    /sha256:b48fdadebab0cc3a4ed00c1f522658ebb68047690d1426a98bbdfec9dff1755b (384 MB)
    /sha256:5ad56d5fc14905886c560200ab69f905b5c5287eaf12f8f761a7ab54f7a61c1b (0 MB)
    /sha256:16fb14f5f7c998f3cdd435d57a870e25866d6856f3e94ed8f37ebc452c37e5b8 (988 MB)
    /sha256:395460e233f5bdcd910d618a3b615e0d881e09ad27d58f3065eef53ecae6a808 (0 MB)
    /sha256:170e558760e8b2e484a022b7d7272cf284fc4e1936ba7a0a671fc586440ad272 (0 MB)
    /sha256:ae79f251470513c2a0ec750117a81f2d58a50727901ca416efecf297b8a03913 (45 MB)
    /sha256:0ef3d00baa098a5253ed18b54451748d16e9d0e26f32d7f4eac882640afe89ac (0 MB)
    /sha256:6f01dc62e444044e3ce494269837ef0aedb80fef69c679416137f17812d2eb9c (0 MB)
broadinstitute/gatk:4.5.0.0    .
 /root/.udocker/repos/broadinstitute/gatk/4.5.0.0
    /sha256:cf7105ec9c451c3c31b518f90a63fcd21e409dcf99a8c23c2e6b45fb0e3404ef (701 MB)
    /sha256:1a799190d815f77920c9963278e0e8b1a3c2f66bf5850bc46ce6fb9da3273258 (363 MB)
    /sha256:762ff3861584e4482f79ebaeef7c0a5b5fd2f6ce91d9190b54353b614a5e2b26 (49 MB)
    /sha256:4da44515c0282c753dea8e5d620f840683b9be744fc3558aa200a3b6801d6d02 (0 MB)
    /sha256:8744436b9dbf91ddefe776c5aa6d061fa93655ff265edc3c6cfcd2ca5398e5e0 (0 MB)
    /sha256:891945c414afb02f8aa7f1ca2688134147fdd3f8108372851fbb996c3c96d8b2 (0 MB)
    /sha256:5e8117c0bd28aecad06f7e76d4d3b64734d59c1a0a44541d18060cd8fba30c50 (28 MB)
    /sha256:a3d1f390828eebbdf0dd57dde549378ccefa065824953f433e83a7c90782f18d (0 MB)
    /sha256:134b348e3b5e7720f8e80c492939d15d4b15338fb03cd3417a651c0d79b7f6aa (0 MB)
    /sha256:4f084384c353a3bf1e2d8a7c6855261feced9060e1ccfcb572243777d87b5741 (0 MB)
    /sha256:5bb37dfbacf38a543a3b8322ede27dc48c7abd9346a53633913c95356d1ca32e (0 MB)
    /sha256:043eca7b7dd5fb53ae84f5e3e94d3bdee177fbfe702bfef8502ccf5062794939 (0 MB)
    /sha256:f34824c486eb930b1bf0bdd34c45fbb7d237e6c22fdaf716a65c9e9a2fc6f194 (0 MB)
    /sha256:b3625edcebc02c6a07a068a9bb09e929f281041bf1929c3fc914e22a01c7db76 (0 MB)
    /sha256:0258c7d63f9241bccdb6655a641406158b0a0d6cedc40219f8f8eb98d84590ba (0 MB)
    /sha256:cffcd2ae2fcdfcfb2ee1886960f2a4fffdc20d4f7a53ea5cb8b107fce7f2acdd (0 MB)
    /sha256:a0350cd371d6d1507fc4cf617e175eb5bd4c3b204c333c94d185bf1ea0795e28 (0 MB)
    /sha256:0ed77206bc58a5c4253dc48f63c49ae2f6a3dfef68374e35b8906e397b8b3a34 (1095 MB)
    /sha256:e28bcb90193018e21503133c0c383ae7124a6407a607045140b41cf81f2eb850 (0 MB)
    /sha256:90226c0b8ca9b1c5bdfb48f0625f151e2acbffcf793aad9319f81fa795749236 (0 MB)
    /sha256:91e6f2d6d5602796597c6b4f77786421b207d0746b8265ead7357f543facbc02 (123 MB)

3.1.2 Create a container (it takes > 2min)

!udocker --allow-root create --name=gatk broadinstitute/gatk:4.5.0.0
43258f40-d39f-316f-8a89-594ce69950cc
!udocker --allow-root create --name=minimap2 staphb/minimap2:2.28
b3f70d3a-fd01-3b04-a386-87cfbbf7c997
!udocker --allow-root ps
CONTAINER ID                         P M NAMES              IMAGE               
43258f40-d39f-316f-8a89-594ce69950cc . W ['gatk']           broadinstitute/gatk:4.5.0.0
b3f70d3a-fd01-3b04-a386-87cfbbf7c997 . W ['minimap2']       staphb/minimap2:2.28
0e74f2dd-6e30-3ea9-b8c0-bf38ddc81fe4 . W                    broadinstitute/gatk:4.1.3.0
!udocker --allow-root run -v=/content/drive/MyDrive/design_build:/design_build gatk ls -al /design_build
 
 ****************************************************************************** 
 *                                                                            * 
 *               STARTING 43258f40-d39f-316f-8a89-594ce69950cc                * 
 *                                                                            * 
 ****************************************************************************** 
 executing: ls
total 82809
-rw------- 1 root root 18903978 Jun  4 03:50 FAT01036_pass_barcode01_5eb57f2c_2b75e8f5_0.fastq.gz
-rw------- 1 root root 19758840 Jun  4 03:50 FAT01036_pass_barcode01_5eb57f2c_2b75e8f5_1.fastq.gz
drwx------ 2 root root     4096 Jun  4 03:53 bam
drwx------ 2 root root     4096 Jun  4 03:53 info
-rw------- 1 root root      466 Jun  4 10:33 mapping_stats.log
-rw------- 1 root root    16343 Jun  4 16:34 my.vcf
-rw------- 1 root root      277 Jun  4 16:34 my.vcf.idx
-rw------- 1 root root    15019 Jun  4 03:50 pACBB-PL-from_EcoRI_hs.gb
-rw------- 1 root root      108 Jun  4 16:00 pACBB_PL.dict
-rw------- 1 root root     7363 Jun  4 07:20 pACBB_PL.fasta
-rw------- 1 root root       23 Jun  4 13:58 pACBB_PL.fasta.fai
-rw------- 1 root root     9280 Jun  3 04:26 pUC19-J23100.gb
-rw------- 1 root root     8592 Jun  3 04:26 pUC19-L2U3H03.gb
-rw------- 1 root root     9040 Jun  3 04:27 pUC19-RB0030.gb
-rw------- 1 root root    11934 Jun  3 04:27 pUC19-egfp.gb
-rw------- 1 root root 46033492 Jun  4 16:18 reads_with_RG.bam
-rw------- 1 root root       96 Jun  4 16:19 reads_with_RG.bam.bai
-rw------- 1 root root       16 Jun  4 13:58 regions.bed
drwx------ 2 root root     4096 Jun  4 03:53 sam
drwx------ 2 root root     4096 Jun  4 10:23 sort

3.2 Data Preparation

3.2.1 Download

!wget https://raw.githubusercontent.com/greendaygh/kaist_design_build_2024/main/data/parts/pACBB-PL-from_EcoRI_hs.gb
!wget https://github.com/greendaygh/kaist_design_build_2024/raw/main/data/fastq/FAT01036_pass_barcode01_5eb57f2c_2b75e8f5_0.fastq.gz
!wget https://github.com/greendaygh/kaist_design_build_2024/raw/main/data/fastq/FAT01036_pass_barcode01_5eb57f2c_2b75e8f5_1.fastq.gz
--2024-06-05 01:53:18--  https://raw.githubusercontent.com/greendaygh/kaist_design_build_2024/main/data/parts/pACBB-PL-from_EcoRI_hs.gb
Resolving raw.githubusercontent.com (raw.githubusercontent.com)... 185.199.109.133, 185.199.111.133, 185.199.108.133, ...
Connecting to raw.githubusercontent.com (raw.githubusercontent.com)|185.199.109.133|:443... connected.
HTTP request sent, awaiting response... 200 OK
Length: 15019 (15K) [text/plain]
Saving to: ‘pACBB-PL-from_EcoRI_hs.gb’

          pACBB-PL-   0%[                    ]       0  --.-KB/s               pACBB-PL-from_EcoRI 100%[===================>]  14.67K  --.-KB/s    in 0.002s  

2024-06-05 01:53:18 (7.83 MB/s) - ‘pACBB-PL-from_EcoRI_hs.gb’ saved [15019/15019]

--2024-06-05 01:53:19--  https://github.com/greendaygh/kaist_design_build_2024/raw/main/data/fastq/FAT01036_pass_barcode01_5eb57f2c_2b75e8f5_0.fastq.gz
Resolving github.com (github.com)... 140.82.114.4
Connecting to github.com (github.com)|140.82.114.4|:443... connected.
HTTP request sent, awaiting response... 302 Found
Location: https://raw.githubusercontent.com/greendaygh/kaist_design_build_2024/main/data/fastq/FAT01036_pass_barcode01_5eb57f2c_2b75e8f5_0.fastq.gz [following]
--2024-06-05 01:53:19--  https://raw.githubusercontent.com/greendaygh/kaist_design_build_2024/main/data/fastq/FAT01036_pass_barcode01_5eb57f2c_2b75e8f5_0.fastq.gz
Resolving raw.githubusercontent.com (raw.githubusercontent.com)... 185.199.108.133, 185.199.109.133, 185.199.110.133, ...
Connecting to raw.githubusercontent.com (raw.githubusercontent.com)|185.199.108.133|:443... connected.
HTTP request sent, awaiting response... 200 OK
Length: 18903978 (18M) [application/octet-stream]
Saving to: ‘FAT01036_pass_barcode01_5eb57f2c_2b75e8f5_0.fastq.gz’

FAT01036_pass_barco 100%[===================>]  18.03M  51.8MB/s    in 0.3s    

2024-06-05 01:53:19 (51.8 MB/s) - ‘FAT01036_pass_barcode01_5eb57f2c_2b75e8f5_0.fastq.gz’ saved [18903978/18903978]

--2024-06-05 01:53:19--  https://github.com/greendaygh/kaist_design_build_2024/raw/main/data/fastq/FAT01036_pass_barcode01_5eb57f2c_2b75e8f5_1.fastq.gz
Resolving github.com (github.com)... 140.82.113.3
Connecting to github.com (github.com)|140.82.113.3|:443... connected.
HTTP request sent, awaiting response... 302 Found
Location: https://raw.githubusercontent.com/greendaygh/kaist_design_build_2024/main/data/fastq/FAT01036_pass_barcode01_5eb57f2c_2b75e8f5_1.fastq.gz [following]
--2024-06-05 01:53:20--  https://raw.githubusercontent.com/greendaygh/kaist_design_build_2024/main/data/fastq/FAT01036_pass_barcode01_5eb57f2c_2b75e8f5_1.fastq.gz
Resolving raw.githubusercontent.com (raw.githubusercontent.com)... 185.199.111.133, 185.199.110.133, 185.199.109.133, ...
Connecting to raw.githubusercontent.com (raw.githubusercontent.com)|185.199.111.133|:443... connected.
HTTP request sent, awaiting response... 200 OK
Length: 19758840 (19M) [application/octet-stream]
Saving to: ‘FAT01036_pass_barcode01_5eb57f2c_2b75e8f5_1.fastq.gz’

FAT01036_pass_barco 100%[===================>]  18.84M  36.2MB/s    in 0.5s    

2024-06-05 01:53:20 (36.2 MB/s) - ‘FAT01036_pass_barcode01_5eb57f2c_2b75e8f5_1.fastq.gz’ saved [19758840/19758840]
!ls -alh
total 37M
-rw------- 1 root root 19M Jun  5 01:53 FAT01036_pass_barcode01_5eb57f2c_2b75e8f5_0.fastq.gz
-rw------- 1 root root 19M Jun  5 01:53 FAT01036_pass_barcode01_5eb57f2c_2b75e8f5_1.fastq.gz
-rw------- 1 root root 15K Jun  5 01:53 pACBB-PL-from_EcoRI_hs.gb

3.2.2 Install python packages

!pip install pydna
!pip install biopython
Collecting pydna
  Downloading pydna-5.2.0-py3-none-any.whl (115 kB)
     ━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━ 115.9/115.9 kB 3.2 MB/s eta 0:00:00
Collecting appdirs>=1.4.4 (from pydna)
  Downloading appdirs-1.4.4-py2.py3-none-any.whl (9.6 kB)
Collecting biopython>=1.80 (from pydna)
  Downloading biopython-1.83-cp310-cp310-manylinux_2_17_x86_64.manylinux2014_x86_64.whl (3.1 MB)
     ━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━ 3.1/3.1 MB 13.1 MB/s eta 0:00:00
Requirement already satisfied: networkx>=2.8.8 in /usr/local/lib/python3.10/dist-packages (from pydna) (3.3)
Requirement already satisfied: prettytable>=3.5.0 in /usr/local/lib/python3.10/dist-packages (from pydna) (3.10.0)
Collecting pyfiglet>=0.8.post1 (from pydna)
  Downloading pyfiglet-1.0.2-py3-none-any.whl (1.1 MB)
     ━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━ 1.1/1.1 MB 18.8 MB/s eta 0:00:00
Requirement already satisfied: pyperclip>=1.8.2 in /usr/local/lib/python3.10/dist-packages (from pydna) (1.8.2)
Requirement already satisfied: numpy in /usr/local/lib/python3.10/dist-packages (from biopython>=1.80->pydna) (1.25.2)
Requirement already satisfied: wcwidth in /usr/local/lib/python3.10/dist-packages (from prettytable>=3.5.0->pydna) (0.2.13)
Installing collected packages: appdirs, pyfiglet, biopython, pydna
Successfully installed appdirs-1.4.4 biopython-1.83 pydna-5.2.0 pyfiglet-1.0.2
Requirement already satisfied: biopython in /usr/local/lib/python3.10/dist-packages (1.83)
Requirement already satisfied: numpy in /usr/local/lib/python3.10/dist-packages (from biopython) (1.25.2)

3.2.3 Reference fasta file preparation

from pydna.readers import read

records = read("pACBB-PL-from_EcoRI_hs.gb")
display(records.list_features())
/usr/local/lib/python3.10/dist-packages/Bio/GenBank/Scanner.py:1528: BiopythonParserWarning: Attempting to parse malformed locus line:
'LOCUS       pACBB-PL-from_Ec        7214 bp DNA     circular SYN 02-JUN-2024\n'
Found locus 'pACBB-PL-from_Ec' size '7214' residue_type 'DNA'
Some fields may be wrong.
  warnings.warn(
/usr/local/lib/python3.10/dist-packages/Bio/SeqFeature.py:230: BiopythonDeprecationWarning: Please use .location.strand rather than .strand
  warnings.warn(
Ft# Label or Note Dir Sta End Len type orf?
0 nd --> 0 7214 7214 source no
1 L:L1U4H07 --> 133 162 29 misc_feature no
2 L:opd <-- 169 1144 975 CDS yes
3 L:BBa_0034 <-- 1148 1169 21 misc_feature no
4 L:BBa J23100 <-- 1173 1208 35 misc_feature no
5 L:L3S1P56 --> 1326 1378 52 misc_feature no
6 L:dmpR <-- 1385 3074 1689 CDS no
7 L:BBa_B0032 <-- 3078 3100 22 misc_feature no
8 L:BBa_J23103 <-- 3104 3138 34 promoter no
9 L:Po --> 3250 3421 171 misc_feature no
10 L:BBA_B0030 --> 3425 3449 24 misc_feature no
11 L:sfGFP --> 3454 4168 714 CDS no
12 L:L3S1P56 --> 4175 4226 51 misc_feature no
13 L:CmR <-- 5250 5910 660 CDS yes
14 L:cat promoter <-- 5910 6013 103 promoter no
15 L:p15A ori <-- 6538 7083 545 rep_origin no
from Bio.SeqRecord import SeqRecord
from Bio import SeqIO

# SeqRecord(records)
print(records)

record = SeqRecord(
    records.seq,
    id="pACBB_PL",
    description="for part assembly"
)

SeqIO.write(record, "pACBB_PL.fasta", "fasta")
Dseqrecord
circular: True
size: 7214
ID: .
Name: pACBB-PL-from_Ec
Description: synthetic circular DNA
Number of features: 16
/molecule_type=DNA
/topology=circular
/data_file_division=SYN
/date=02-JUN-2024
/accessions=['.']
/keywords=['']
/source=synthetic DNA construct
/organism=synthetic DNA construct
/taxonomy=[]
/references=[Reference(title='Direct Submission', ...)]
Dseq(o7214)
AATT..GATG
TTAA..CTAC
1
!ls -alh
total 37M
-rw------- 1 root root  19M Jun  5 01:53 FAT01036_pass_barcode01_5eb57f2c_2b75e8f5_0.fastq.gz
-rw------- 1 root root  19M Jun  5 01:53 FAT01036_pass_barcode01_5eb57f2c_2b75e8f5_1.fastq.gz
-rw------- 1 root root 7.2K Jun  5 01:56 pACBB_PL.fasta
-rw------- 1 root root  15K Jun  5 01:53 pACBB-PL-from_EcoRI_hs.gb

3.2.4 Create directories

import os

sam_dir = "/content/drive/MyDrive/design_build/sam"
bam_dir = "/content/drive/MyDrive/design_build/bam"
info_dir = "/content/drive/MyDrive/design_build/info"
sort_dir = "/content/drive/MyDrive/design_build/sort"

if not os.path.exists(sam_dir):
    os.mkdir(sam_dir)
if not os.path.exists(bam_dir):
    os.mkdir(bam_dir)
if not os.path.exists(info_dir):
    os.mkdir(info_dir)
if not os.path.exists(sort_dir):
    os.mkdir(sort_dir)

3.3 Mapping

  • One fastq file example
!udocker --allow-root run -v=/content/drive/MyDrive/design_build:/design_build minimap2 bash -c "minimap2 \
        -ay /design_build/pACBB_PL.fasta \
        /design_build/FAT01036_pass_barcode01_5eb57f2c_2b75e8f5_0.fastq.gz > \
        /design_build/sam/0.sam"
 
 ****************************************************************************** 
 *                                                                            * 
 *               STARTING b3f70d3a-fd01-3b04-a386-87cfbbf7c997                * 
 *                                                                            * 
 ****************************************************************************** 
 executing: bash
[M::mm_idx_gen::0.014*0.37] collected minimizers
[M::mm_idx_gen::0.015*0.46] sorted minimizers
[M::main::0.015*0.46] loaded/built the index for 1 target sequence(s)
[M::mm_mapopt_update::0.015*0.47] mid_occ = 10
[M::mm_idx_stat] kmer size: 15; skip: 10; is_hpc: 0; #seq: 1
[M::mm_idx_stat::0.015*0.47] distinct minimizers: 1270 (98.27% are singletons); average occurrences: 1.034; average spacing: 5.494; total length: 7214
[M::worker_pipeline::13.192*1.42] mapped 4000 sequences
[M::main] Version: 2.28-r1209
[M::main] CMD: minimap2 -ay /design_build/pACBB_PL.fasta /design_build/FAT01036_pass_barcode01_5eb57f2c_2b75e8f5_0.fastq.gz
[M::main] Real time: 13.194 sec; CPU: 18.793 sec; Peak RSS: 0.263 GB
  • Multiple files
from glob import glob
fastq_filenames = ["/design_build/"+fn for fn in glob(f"*.fastq.gz")]
print(fastq_filenames)
['/design_build/FAT01036_pass_barcode01_5eb57f2c_2b75e8f5_0.fastq.gz', '/design_build/FAT01036_pass_barcode01_5eb57f2c_2b75e8f5_1.fastq.gz']
from glob import glob

## -------
fastq_filenames = ["/design_build/"+fn for fn in glob(f"*.fastq.gz")]
sam_filenames = [fn.replace(".fastq.gz", ".sam").replace("/design_build/", "/design_build/sam/") for fn in fastq_filenames]
## -------

zipdata = zip(fastq_filenames, sam_filenames)
reference_filename = "/design_build/pACBB_PL.fasta"
#print(list(zipdata))

for fnfile, samfile in zipdata:
    exestr = f"minimap2 -ay {reference_filename} {fnfile} > {samfile}"
    print(exestr)
    !udocker --allow-root run -v=/content/drive/MyDrive/design_build:/design_build minimap2 bash -c "{exestr}"
minimap2 -ay /design_build/pACBB_PL.fasta /design_build/FAT01036_pass_barcode01_5eb57f2c_2b75e8f5_0.fastq.gz > /design_build/sam/FAT01036_pass_barcode01_5eb57f2c_2b75e8f5_0.sam
 
 ****************************************************************************** 
 *                                                                            * 
 *               STARTING b3f70d3a-fd01-3b04-a386-87cfbbf7c997                * 
 *                                                                            * 
 ****************************************************************************** 
 executing: bash
[M::mm_idx_gen::0.012*0.45] collected minimizers
[M::mm_idx_gen::0.013*0.55] sorted minimizers
[M::main::0.013*0.55] loaded/built the index for 1 target sequence(s)
[M::mm_mapopt_update::0.013*0.56] mid_occ = 10
[M::mm_idx_stat] kmer size: 15; skip: 10; is_hpc: 0; #seq: 1
[M::mm_idx_stat::0.013*0.56] distinct minimizers: 1270 (98.27% are singletons); average occurrences: 1.034; average spacing: 5.494; total length: 7214
[M::worker_pipeline::12.149*1.55] mapped 4000 sequences
[M::main] Version: 2.28-r1209
[M::main] CMD: minimap2 -ay /design_build/pACBB_PL.fasta /design_build/FAT01036_pass_barcode01_5eb57f2c_2b75e8f5_0.fastq.gz
[M::main] Real time: 12.150 sec; CPU: 18.809 sec; Peak RSS: 0.263 GB
minimap2 -ay /design_build/pACBB_PL.fasta /design_build/FAT01036_pass_barcode01_5eb57f2c_2b75e8f5_1.fastq.gz > /design_build/sam/FAT01036_pass_barcode01_5eb57f2c_2b75e8f5_1.sam
 
 ****************************************************************************** 
 *                                                                            * 
 *               STARTING b3f70d3a-fd01-3b04-a386-87cfbbf7c997                * 
 *                                                                            * 
 ****************************************************************************** 
 executing: bash
[M::mm_idx_gen::0.011*0.44] collected minimizers
[M::mm_idx_gen::0.013*0.51] sorted minimizers
[M::main::0.013*0.51] loaded/built the index for 1 target sequence(s)
[M::mm_mapopt_update::0.013*0.51] mid_occ = 10
[M::mm_idx_stat] kmer size: 15; skip: 10; is_hpc: 0; #seq: 1
[M::mm_idx_stat::0.013*0.52] distinct minimizers: 1270 (98.27% are singletons); average occurrences: 1.034; average spacing: 5.494; total length: 7214
[M::worker_pipeline::11.232*1.77] mapped 4000 sequences
[M::main] Version: 2.28-r1209
[M::main] CMD: minimap2 -ay /design_build/pACBB_PL.fasta /design_build/FAT01036_pass_barcode01_5eb57f2c_2b75e8f5_1.fastq.gz
[M::main] Real time: 11.233 sec; CPU: 19.912 sec; Peak RSS: 0.227 GB
!ls -al sam/
total 116192
-rw------- 1 root root 39055100 Jun  5 02:04 0.sam
-rw------- 1 root root 39055100 Jun  5 02:15 FAT01036_pass_barcode01_5eb57f2c_2b75e8f5_0.sam
-rw------- 1 root root 40868986 Jun  5 02:15 FAT01036_pass_barcode01_5eb57f2c_2b75e8f5_1.sam
sam_filenames
['/design_build/sam/FAT01036_pass_barcode01_5eb57f2c_2b75e8f5_0.sam',
 '/design_build/sam/FAT01036_pass_barcode01_5eb57f2c_2b75e8f5_1.sam']

3.4 Merging sam files

merged_samfile = sam_filenames[0].replace("_0", "_merged")
sam_files_str = " ".join(sam_filenames)

execstr = f"samtools merge -f {sam_files_str} -o {merged_samfile}"
#print(execstr)
!udocker --allow-root run -v=/content/drive/MyDrive/design_build:/design_build gatk bash -c "{execstr}"
 
 ****************************************************************************** 
 *                                                                            * 
 *               STARTING 43258f40-d39f-316f-8a89-594ce69950cc                * 
 *                                                                            * 
 ****************************************************************************** 
 executing: bash
! ls -alh sam/
total 186M
-rw------- 1 root root 38M Jun  5 02:04 0.sam
-rw------- 1 root root 38M Jun  5 02:15 FAT01036_pass_barcode01_5eb57f2c_2b75e8f5_0.sam
-rw------- 1 root root 39M Jun  5 02:15 FAT01036_pass_barcode01_5eb57f2c_2b75e8f5_1.sam
-rw------- 1 root root 73M Jun  5 02:24 FAT01036_pass_barcode01_5eb57f2c_2b75e8f5_merged.sam
!head -n 20 sam/FAT01036_pass_barcode01_5eb57f2c_2b75e8f5_merged.sam
@HD VN:1.6  SO:unsorted GO:query
@SQ SN:pACBB_PL LN:7214
@PG ID:minimap2 PN:minimap2 VN:2.28-r1209   CL:minimap2 -ay /design_build/pACBB_PL.fasta /design_build/FAT01036_pass_barcode01_5eb57f2c_2b75e8f5_0.fastq.gz
@PG ID:minimap2-4B3706C2    PN:minimap2 VN:2.28-r1209   CL:minimap2 -ay /design_build/pACBB_PL.fasta /design_build/FAT01036_pass_barcode01_5eb57f2c_2b75e8f5_1.fastq.gz
@PG ID:samtools PN:samtools PP:minimap2-4B3706C2    VN:1.13 CL:samtools merge -f -o /design_build/sam/FAT01036_pass_barcode01_5eb57f2c_2b75e8f5_merged.sam /design_build/sam/FAT01036_pass_barcode01_5eb57f2c_2b75e8f5_0.sam /design_build/sam/FAT01036_pass_barcode01_5eb57f2c_2b75e8f5_1.sam
c8444849-ddea-4bed-b1df-80b1981aac85    0   pACBB_PL    4292    60  112S108M1D54M1I1M1I50M2I43M1D73M2D21M1D32M3I36M1D86M2I5M1I4M2D10M1D13M2I4M2D25M1D5M1D110M1D11M3I19M1D4M1D49M3D31M2I3M2I2M1D5M2D4M1I90M1I74M2I7M2I70M2I19M1D2M1D4M1D12M1D48M1I54M2I1M1D10M2D2M2D4M3D2M1I2M1I8M1I6M2D20M1I12M2D5M1D19M1D5M1D23M2D2M1I6M1D12M2I34M1D7M1I30M3D15M1D11M1D8M3D11M1D7M1D101M1D48M1D50M3D24M2D58M5I74M1D1M1D24M2I18M3D68M1I5M2D3M1D9M2D51M1D49M2D4M1D51M2D59M1D1M1D17M1I17M1D27M4D50M2I8M1D3M2D18M2I3M1D70M1D5M1I35M1D88M1D1M1D17M3D47M1D43M3D38M1I7M1D6M1D7M4D72M3I22M3D2M1D1M3I40M1D20M5S *   0   0   AATTCATCCTGAACTTATCTAGACCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATCGTCTCCAGTGAGCCTTGAGACCTTTACACTTTACAAGTAAAGGTCTCTTCGCATAGTGAGACGATTCTCCTTACACATCTGTGCGGTATTTCACACCGCATGTACTAGTTAACTTCAGGACCTGCAGCTCGGGCAGCGTTGGGTCCTGGCCACGGGTACGCTGATCGTGCTCCTGCCGTTGAGGACCCGGCTAGGCTGGCGGGGTTGCCTTACTGCGATTAGCAGAATGAATCACCGATACGCGAGCGAACGTGAAGCGACTGCTGCTACACAAAACGTCTGCGACCTGAGCAACAACATGAATGGTCTTTGGTTCCGTGTTTCGTAAAGTCTGGAAACGCGGAAGTCCCCTACGTGCTGCTGAAGTTGCCCGCAACAGAAAGTCGCCAACCGGTGATACCACGATATAAGGCTGAGAGTCAACGCCATGAGCGGCCTCCTGTTTTCTTATTCTGAGTTACAACAGTCCGCACCGCTGCCGGTAGCTCCTTCCGGTGGGCGCGGGGCATGACTATCGTCGCCGCACTTATGACTGTCTTCTTTATCATGCAACTCGTAGGACCGCCGTTCCGGGCAACCCGACGGTCCCTGGCCACGGGACACCTTACCATACCCACGCCGAAACAAGCGCCTGCTCATTATGTTCCGGATCTGCATCGCAGGATGCTGCTGGCTACCCTGTGGAACACCTACATCTGTATTAACGAAGCGCTAACCGTTTTTATCAGGCTCTGGGAGGCAGAATGACGATCATATCGACGTCAATTATTACCTCCACGGGAAGCCTGAGCAAACTGGCCTCAGGCATTTGAGAAGCACACGGTCACACTGCCGGTAGTCAGTAAACCGGTAAACCAGCAATGAAGACGCAAAGCGTATTCCAGCGACCCTGCCCTGAACCGACGACCGGGTCGAATTTGCTTTCGAATTTCTGCCATTCATCCGCTTATTATCACTTATTCAGGCGTAGCAACCAGGCGTTTAAGGGCACCAATAACTGCTGTAAAAAAATTACGCCCCGCCCTGCCACTCATGCCGGTACTGTTGCGTAATTCGTACCAAGCATTCTGCCGACATGGAAGCCATCACAAACGGCATGATGAACCTGAATCGCCAGCGGCATCGGCATACCTTGTCGCACTGCGTATATGTTGCCATGGTGAAAATGGAGCGAAGAAGTTCTCCATATTGGCCACGTTTAAATCAAAACTGGTGGCAGCTCTTCCGGGGATTGGCTGGGCTGAAAAACATATTCTCAATAAACCCTTTGCAGGAAATAGGCGGTTCATATGCTGCGCCACACTCTTGCAGATATGTGTAGAAACTGCCGGGAAATCGTCATTGTTCCTCCAGAGCGATGAAAACGTTCAGTTGCTCATGGAAAACGTTCTGTCGGTGCGAACCTATCCCATATCCTGCCAGCTCACCGTCTTTCATTGCCATACGGAACTCGGATGAGGCATTCATCAGCCAGGCAAGAATGTGAATAGACAGATAAAACTTGGCAACGTTTTCTTACAGTCACAAAGCCCGTATATCCAATGAACGGTCTGGTTATAGGTACATTGAGCAACTGACTGAAATGCCTCAAAATGTTCTTTACGATGCCATTGGGATATATCAACGGTGGTATATCCAGTGATTTTTTCTCCATTTTAGCTTCCTTAGCTCCTGAAAATCTCGATAACTCAAAAATACGCCCGGTAGTAATCTTATTTCATTATGGTGAAAGTTGGAACCTACGTGCCGATCAGCATCTCATTTGCCAAAAGTTGGCCCAGGGCTTCCCGGTATCAACAGGGACACCAGGATTTATTTATTCTGCCTTGCGAAGTGATCTTCCGTCACAGGTATTTATTCGGCGCAAAGTGCGTCGGGTGATGCTGCCAACTTACTGATTTGGTATGATGGTGTTTTTGAGGTGCTCCCCGGTGGCTTCTGTTTCTGGCTGTCCCTCCTGTTCAGCTACTGACGGGGTGGTGCGTAACGGCAAAAGCACCGCCGGACATCAACGCCTAACCGTTATCCTGACACTATGTTGGCACTGATGAGGGTGTCAGTGAAGTGCTTCATGTGGCAGGAGAAAAAGGCTGCACCGGTGCGTCAGCAGAATATGTGATACAGGATATGTTACTTCACGCTCACTGACTCGCTACGCTCGGTCGTTCGACTGCGGCGAGCGGAAATAATACGAACGGGGCGGAGATTTCCTGGAAGATGCCAGGAAGATACTTAACAGGGAAGTGGGGGCCGCGGCAAAGCCGTTTTTTCCATAGGCTCCGCCCCCTGACAAACATCACGAAATCTGACAAATCAGTGGTGGCGAAACCCGACGGGACTATAAAGATACCAGGCGTTTTCCCCCTGGCAATATAATGCGCTCTCCTGTTCACCTTATTTCGGTTTACCGGTGTCATTCCGCTGTTATGGCCGCGTTTGTCTCATTCCACGCCTGACACTCAGTTCGGGTAAGGCAGTTCGCTCCAAGCTGGACTGTATGCACGAACCCCCGTTCAGTCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGAAAGACATGCAAAAGCACCACTGGCGGCGTACTGTTAATTGATTTAGAGTTAGTCTTGAAGTCATGCGCCGGTTAAGGCTAAACTGAAAGGACAGTTTTGGTGACTGCGCTCCTCCAAGCCAGTTACCTCGGTTCAAGTTGGTAGCTCAGAGAACCTTCGAAAAACCGCCCTGCAAAGGCGGTTTTTCATTTCAGTGAGATTACGCGCAGACCAAAACGATCTCAAGAAGATCATCTTATTAATCAGATAAAATATTTCAAGATTTCCGAAGTGCAATTTATCTCTTCAAATGCCTTTCTGAAGTCGGCCCCATACGATATAAGTTGTAATTCTCATGTTGACAGCTTATCATCGATGAATTA    &,;ADDDDB@@BCEBBCCBA******55656...59=9=;<6677:7644411111?@A@A@A@7779::8899:;;;;>:::;?@0///,+)(((),0/(213;<>>=('&&%%%%%&&(*+./3:>111**(((,->@@@CCEBBBBEGCC>==>?B>>B?88/////88000))))*DBCE8666745566?@@ABC<:;:943:;<4410//((&%$$$%&&'+,,0.-)%%%%0.-7501?@FDA-,,,-38=@.-...>88432,(''$$$$$$$%&'(*:@ADAD<;8422-****-36:<>>>?=988;>@?=>><<=<//+**,1:@?@@==???A<??@;:;972)'''(.0589B@==+***+0189;CCBAABBCA0??AC???BCABA@99<>;<1-01237=>?B=;:>A=?>?>@@?>==<1--./..0'&&&&+188;<????@@@?;=8.-&$$$%&(4478?>?>=<<;<8644.../09)'')--.489:<ABBCEABCCCDDBAB??>>?@BBABACAA>>@A>@?@,/==A>=<<<;<=>@A@<<:9880001>BBCBCF??>>>AACDDDDB@@?@A444<>?AA88888>?.--668<311+*()(%%&&'&####$&'&&()))%%%%&''&&&%'&%%%%&&&'19?@@CCCED<;;;A==995551-,'&&%&0000010--12799999;666<<54ABCDB>;55411136COMAHG{JG{IJKHHFDDDEFHGKJH{{HGARBCMKFFIIGHG{HG32222IDCC=:?86)((()'''''&('*++,*(&&&))))))*+01211.-,--*('&&'))))),-44//.2456-0:<<=?@BFFEG?>>?<>?GIM\LEGEDG,,,,-333*''''()--.8BDCAA?..,,))*)())'(())&))$$%%%%%%%&'(+'')*'&&-366611254349877744449@0?@@A15578AEDGM{H@????EA*((%%%%&'''&**+/<9B332337755356:9;?@BECCCC+*10233335BD<7,,(''(()>;2.8:?@???B:=AA><=>?22222313100145?931*('&&&()+%%%&&()+-013;<FEDDDFADC;78@ACBCD?@?@@E////0LIGGEDGHDGIEDFNF@-,,++/119+*)'&&&%%'&&%%&%%%&*....+))))'&+))+**+,-/<<52--(((//.*))*&&&&&,*++,,333356;B>ILFE=<<EDC?@<0))(''',-/,,('((()+))+**)))&'''(/0/12235>BF:97311)1*(*)&&''(()21.---)(&'&&))()*....//..*'&&')))''&&&&(+('&%%&).455+),'&+/4822-'&&&&(*''')**)))+471122/.+++(&'%%&&2312,+**+,.+))'&$$$$$%&'&&$$&&'+565233451.&&&()=@@DCBCECFIHF>>>>=>BB=>===BCAC222<732.-,,.,,11*'&&%%&''/--0..,,('*.&&&&%%%&(,,,''&$$$%%$$&%%&&&$%$%&)&'&''&'&&&'''&&'%%$%&&&+++,,,HGGEFG????>?BEHAB9999:ADBA@@BB@BBC:999>@>EFEIQFPLSKDGGHFLIJIFIKFIHKHJJGC0/=??@@FJKKGH::9:;CCEGD<@ADEACAF@???>CEDCHBEIGFFEC=B=AA{DDBDCB>;55556A8<265553,,,,+-,,-/1256?B2<A>CAAFEB??==<B=6211((&',27:;55441(('''((1--,&())-/8<34@B>;41412259=0///00-./,,+++05@AA?1110/1*(%%%%&'(10/)'&%$$%&)-38;CDGED::C=<50//58FNJ@GDEB@@ABED?ADCDCC0/.-./.+*)''())6@BCFEDCHE=)(((()-2B5422222785341100/*(%&&(&()))))/4BD6<>ADA>D?:722212=BC;::>=>>??@200002222(&&&'(+'(-02399;;;<=<<<?B>::.''&&%%%&&$###$$(%%%&'''(*/,18434-,,,-12220/.01<655556FEHGDFDDBC??>BAFBDA@??@FF:9CDGEFHHH{FEFDEA?@@AABDIGGH{OFFGC@?===?*))''&&*''&&%(*3368:<=------:;?-,,,->?A@>><9832254;;;:9877,*)))((++**,,-7D?AA?D<@@C?F;7@>BA?;-,01557C?AAA@EE+******+-&&&&&+<A0//121(''&'(/072355633469:C;<FF==603644***+-=?<76.-/(%%&%&&)-***6?A;7750//65467:0///02((&%%%&)+,/**/52///+/79420.//,))))(&&%&&++-0:=,,*)(&'&&&%%%%&/45BCBDA>70/1311134((922222<99---.781/11**)24((333445582.--.,--02)((03*((*78>AAAB@>6667;<>==>A??ACAC??==>>>?=366>C<99:?@BBC>?EGECHDEEDHIFGFFGHFIELHJFFEDB=><@)(&&'*+6;;/.4644445;;@;211123++++&$$$$%'&&'*++-.--/5'&&&&')+>??@@::989<65566>>GGIFCECBDDDFHEF@9;433320*))34:>=>>@=;;<=>D<43.../=<1111118887:;<50/+),*)**+23:74446:AADD@9989::+**+31111..)'*+)*++-1/0133)('%$$%%%-/03ACAAA@AAC@CDEECEFEBD>=44322333?BDEEGEEGEFB??CDDMIEFFDDGFCACCE=<+((*))+47<>@CCEB7//23*(((+,'&&&'&&,3589)(''(0565655520/----(((((2/06..*))%%'()*7:@AFDCBCBBBA@<;+    NM:i:270    ms:i:4320   AS:i:4260   nn:i:0  tp:A:P  cm:i:257    s1:i:1768   s2:i:0  de:f:0.0706 SA:Z:pACBB_PL,1,+,73M1I2914S,26,1;  rl:i:0  ru:i:0
c8444849-ddea-4bed-b1df-80b1981aac85    2048    pACBB_PL    1   26  63M1I10M2914H   *   0   0   AATTCATCCTGAACTTATCTAGACCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATCGTCTCCAGTGAGCCT  &,;ADDDDB@@BCEBBCCBA******55656...59=9=;<6677:7644411111?@A@A@A@7779::8899  NM:i:1  ms:i:140    AS:i:140    nn:i:0  tp:A:P  cm:i:7  s1:i:56 s2:i:0  de:f:0.0135 SA:Z:pACBB_PL,4292,+,112S2871M52D5S,60,270; rl:i:0  ru:i:0
5337d681-20a1-405d-9224-74596d8164d3    0   pACBB_PL    4287    60  107S25M2I125M1D111M1D42M1D2M1D119M1D29M4I1M1D2M1I74M1D20M1I3M2D43M1D65M1I64M1D62M1I81M1D45M1I428M1D118M1D33M1D104M4D446M1D102M3D182M1I18M1D128M2D95M2I11M1I75M1D4M2I63M1D122M1I46M2D10M5S   *   0   0   AATTCATCCTGAACTTATCTAGACCAGGCTGGAAAGCGGGCAGTGAGCGCAACGCAATCGTCTCAGTGAGCCTTGAGACCTTTACACTTTACAAGTAAAAGGTCTCTTCACTTAGTGAGACGATTCTCCTTACCCGCATCTGTGCGGTATTTCACACCGCATGTACTAGTTAACTTCAGGACCTGCAGCTCGGGCAGCGTTGGGTCCTGGCCACGGGTGCGCATGATCGTGCTCCTGTCGTTGAGGACCCGGCTAGGCTGTGGGGTTGCCTTACTGGTTAGCAGAATGAATCACCGATACGCGAGCGAACGTGAAGCGACTGCTGCTGCAAAACGTCTGCGACCTGAGCAACAACATGAATGGTCTTCGGTTCCGTGTTTCGTAAAGTCTGGAAACGCGGAAGTCCCCGATGGCACTGAAGTTGCCCGCAACAGAGAGTGGAACCAACCGGTGATACCACGATACTATGACTGAGAGTCAACGCCATGAGCGGCCTCATTTCTTATTCTGAGTTACAACAGTCCGCACCGCTGCCGGTAGCTCCTTCCGGTGAGCGCGGGGCGCGGAGAGCTATCGTCGCCGCACTTATGACTGTCTTCTTTATCATGCAACTCGTAGGACAGGTGCCGGCAGCGCCCAACAGTCCCCGGCCACGGGGCCTACCTGCCACCCACGCCGAAACAAGCGCCCTGCACCATTATGTTCCGGATCGCCTCGCAGGATGCTGCTGGCTACCCTGTGGAACACCTACATCTGTATTAACGAAGCGCTAACCGTTTTTTATCAGGCTCTGGGAGGCAGAATAAATGATCATATCGTCAATTATTACCTCCACGGGGAGGCCTGAGCAAACTGGCCTCAGGCATTTGAGAAGCACACGGTCACACTGCTTCCGGTAGTCAACCAAACCGGTAAACCAGCAATAGACATAAGCGGCTATTTAACGACCCTGCCCTGAACCGACGACCGGGTCGAATTTGCTTTCAATTTCTGCCATTCATCCGCTTATTATCACTTATTCAGGCGTAGCAACCAGGCGTTTAAGGGCACCAATAACTGCCTTAAAAAAATTACGCCCCGCCCTGCCACTCATCGCAGTACTGTTGTAATTCATTAAGCATTCTGCCGACATGGAAGCCATCACAAACGGCATGATGAACCTGAATCGCCAGCGGCATCAGCACCTTGTCGCCTTGCGTATAATATTTGCCCATGGTGAAAATGGGGGCGAAGAAGTTGTCCATATTGGCCACGTTTAAATCAAAACTGGTGAAACTCACCCAGGGATTGGCTGAGACGAAAAACATATTCTCAATAAACCCTTTAGGGAAATAGGCCAGGTTTTCACCGTAACACGCCACATCTTGCGAATATATGTGTAGAAACTGCCGGAAATCGTCGTGGTATTCACTCCAGAGCGATGAAAACGTTTCAGTTTGCTCATGGAAAACGGTGTAACGGGGTGAACACTATCCCATATCACCAGCTCACCGTCTTTCATTGCCATACGGAACTCCGGATGAGCATTCATCAGGCGGGCAAGAATGTGAATAAAGGCCGGATAAAACAGCCGCCCATTTTCTTTACGGTCTTTAAAAAGGCCGTAATATCAGCTGAACGGTCTGGTTATAGGTACATTGAGCAACTGACTGAAATGCCTCAAAATGTTCTTTACGATGCCATTGGGATATATCAACGGTGGTATATCCAGTGATTTCTCCATTTTAGCTTCCTTAGCTCCTGAAAATCTCGATAACTCAAAAAATACGCCCGGTAGTGATCTTATTTCATTATGGTGAAAGTTGGAACCTCTTACGTGCCGATCAACGTCTCATTTTCGCCAAAAGTTGGCCCAGGGCTTCCCGGTATCAACAGGGACACCAGGATTTATTTATTCTGCGAAGTGATCTTCCGTCACAGGTATTTATTCGGCGCAAAGTGCGTCGGGTGATGCTGCCAACTTACTGATTTAGTGTATGATGGTGTTTTTGAGGTGCTCCAGTGGCTTCTGTTTCTATCAGCTGTCCCTCCTGTTCAGCTACTGACGGGGTGGTGCGTAACGGCAAAAGCACCGCCGGACATCAGCGCTAGCGAGGTATATACTGGCTTTATATGTTGGCACTGATGAGGGTGTCAGTGAAGTGCTTCATGTGGCAGGAGAAAAAGGCTGCACCGGTGCGTCAGCAGAATATGTGATACAGGATATATTCCGCTTCCTCGCTCACTGACTCGCTACGCTCGGTCGTTCGACTGCGGCGAGCGATGGCTTACGAACAGGGCGGAGATTTCCTGGAAGATGCCAGGAAGATACTTAACAGGGAAGTGAGAGGGCCGCGGCAAAGCCGTTTTTCCATAGGCTCCGCCCCCCTGACAAGCATCACGAAATCTGACGCTCAAATCAGTGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGCGGCTCCCTCGGTATACTCCTGTTCCTGCCTTTCGGTTTACCGGTGTCATTCCGCTGTTATGGCCGCGTTTGTCTCATTCCACGCCTGACACTCGGATTCGGGTAGGCAGTTCGCTCCAAGCTGGACTGTATGCACGAACCCCGTTCAGTCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGAAAGACATGCAAAAGCACCACTGGCAGCAGCCACTGAAGCCATTGATTTAAGAGGAGTTAGTCTTGAAGTCATGCGCCGGTTAAGGCTAAACTGAAAGGACAAGTTTTGGTGACTGCGCTCCTCCGGCCGTAGTTACCTCGGTTCAAAGAGTTGGTAGCTCAGAGAACCTTCGAAAAACCGCCCTGCAAGGCGGTTTTTCGTTTTCAGAGCAAGAGATTACGCGCAGACCAAAACGATCTCAAGAAGATCATCTTATTAATCAGATAAAATATTTCAAGATTTCAGTGCAATTTATCTCTTCAAATGTAGCACCTGAAAGTCAGCCCCATACGATATAAGTTGTAATTCTCATGTTTGACGACATCATCGATGAATTA    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11820868-051c-4c6b-ae74-aea5790dc92b    0   pACBB_PL    1   60  132M8I11M1D9M6I2M2I2M6I1M1I14M1I33M1D69M1D6M2I839M3D29M1D8M2I6M1D157M9D10M1D9M6D2M2D2M6D26M2D27M1D327M1D152M1I138M2D200M1I2M1D34M1D33M6D8M1I1M3I65M1I110M1D113M1D87M2D209M1D5M2I29M2D123M1I8M2D27M1I89M1D25M1D16M1I78M1D94M74I7M3D13M1D578M1I148M5D7M2D2M6D4M1I12M1D3M9D365M2I22M2D99M1D24M1I79M1I48M1D7M3I98M1I19M1I162M1I58M1I32M3D71M1I142M1D39M1I70M4D178M1I195M2D36M2I64M1D56M1I51M3I104M2I1M1D14M5D66M1D145M1I20M5D18M1D8M1I2M1I3M1I12M4D36M1I3M2I177M2D249M3D5M1D103M1D3M1I5M1D56M11S    *   0   0   AATTCATCCTGAACTTATCTAGACCCGACTGGAAAGCGGGCGATGAGCGCAACGCAATCGTCTCAGTGAGCCTCTAGTACATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAATCGTCTCAGCCTGTGATTTTGTTATCAATAAAAGGCCCCCCGATTTGGGAGGCCTTTTTTCGAGAAATTAGTTATCATTTTCTGGTTAACAACAGAGTAGTTAACCGGAGCAAACAGTAACCTTTACCTTCCGCCTGGATGTGACCGATACCCGGGAACGGTAAATGGGAAGCCGCGATCGGAAGCCACACCTTTCGCCGCGTCCGCGAACGCTTTTTTACGTTCAACCGCCGCAGATTTACCGTCGATGTCCAGCTGGTTGGTAACAGACGGGTCGTCGAACTGAACCGCCGCAACCAGGATCAGGTCACCCAGCAGCGCCAGTTTCTGACCCTGAGATTCAACAACGTAGGTGGTGTGACCCGGGGTGTGGCCATGGGAAGCCAGCGCTTTGATACCCGGAACCAGGTCGGTGTTACCAGAGAACGGTTTGAATTTACCCGCTTTAACGTACGGATTTAAGGAAGCCATCGCACCTTTGAAGAAACCTTTAGATTCGTCGTCCGGCGCTTTGTCCAGGTTGGTCTGAGACAGCCAGAAGTCCGCTTCTTTCTGGTCCGCACGAACAACCGCGTTCGGGAACGCCAGCTGTTCACCAACCATCAGACCACCAACGTGGTCCGGGTGCATGTGGGTGATGTAGATTTCGTCAACCTGTTCCGGCTGGTAACCCGCCGCTTTCAGGTTCGCCGCCAGACGACCCAGGGTCGGACCGAACAGACCCGCCGCACCGGTGTCAACCAGAACCAGTTTAGAACCGGTGTTAACCAGGTAACCGGTAACAGAGGTTTCCAGCGGCGCTTTCTGGAAAGATTTCGCCAGCGCAGACTGGGTTTTCGGCACCGGCTGGTTCAGACGTTTGTCAACCGGCAGCGCAACGGTACCGTCAGACAGCGCGGTGATTTCGAAGTCACCCAGCAGCATACGGTAGTAACCCGGCGCAGAGGTACGAACCTGCGGCGCCGCCGCAGACGCGTGGGTAACGAAAACCATCTGCGCCGCGGTGCACAGACCCGCCAGCGGCAGAGTTTTGGTCAGGTTACGCATCTGCTACTTTCCTGTGTGACTCTAGAAAGGCTAGCACTATACCTAGGACTGAGCTAGCCGTAAAAGGCCTTTTGAGACGATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGTCTACTTTCTAGTACATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAATCGTCTCACTTTGTGACAAGAAAAGATCCAGCCGAAGCTGGATCTTAGTTAGCCTTCTATGCCGATTTTTTAACCGATAAGCTAGTGCCGGTGGCTTAGGCCGAGCAATCGTGCAGCACCGGAGACATTTTGGTTGGCCTGTTGCATGGCTTTGCGCATCAGAGATTCCTCGATTTCATCGAGTGACAGGCCGCTGCTCAACAATTGGCTGATCCAACTGCCTTGGCCATTGCCTGGCTGAATAAGAACGCCGTCCGAAGACACCCGCTCAGAGGCGGTCTGGGGCTCTTCCGGCGCGCGTGGAAACAGCGCCTGTACGCTGATAGATTCGTTCGGATCGGTTAAGATTATACCACGCTCGATTACATTTTCTAACTCACGGATATTGCCCGGCCAGCTGTAATGCAGGCAGGCCTCCAGGCTTTATCTGAAAGGCCTAATGTTCTCTTCCCGTATTCCTGGTGGAAGCGCTGAAGAAAATGCTCAACCAGCAGAGGAATGTCTTCACGGCGTTCGCGAAGGGCTGGGATCGCCACCGGAAAAACATTCAGCCGATAATACAGATCGGCACAGAAAACGACCGGCTTTAACCGCTTCGGCCAGATCTTCGTGTGTGGCTGCTATAACCCTTACGTCTATTTTGCGCGTCCTGTTGTCACCAACTCGCTCCAGCTCTCCTTCTTGGAGCACGCGCAGCAAGCTGGCTTGAGCTCGGGCTTAACTCGATCACCTCGTCAAGAAATATAGTCCCTTTATCGGCCCGTTCAAAGCGTCCCATGCGTGATTGGGTGGCGCCGGTAAAGGCTCCTTTCTCCACGCCAAACAATTCGGACTCGATCAGGTCCGGCGGTATCGCCACACAGTTCACAGCAACAAAAGGCTCTGCGGCGCGTTTGAAGCGCCGATGCACTACGCGCGCGATGACCTCTTTGCGCACTGCAGTTTCTCAAGTAACAGCACACTAACTTTGCCCTGTGCTGCCACAATACTCCATGCGCACCGTTTGATAGGCTGGGGTCTGACCAATGCCGTAGTACTGTCATTCCTGTTTGTCTAGGTTTTGTACGCAGCGACACCAGTTGTGATTGTAACTCGTATAACTCCTCGATGATGGGGTCATTCTTAAAATACTGTTTGAAAGAGGCAACATCGTCCCATTCTTCGGCCGGTTTCCAATGACCCGACACTTATCACCTCCACAGCCGCGGCAACTTACTTCCTTAAAAATGATTTCTCTGCCCATAAAAGCAGAAGAATAGGCGCAGGCGTAGCCGAGCAGAGTCAACATACCGGATCTTGCATCTGCCCCAGATCGGTCTGACAGATTTTCACTTCAAAGGAGTCGATCCATTCCATCTCGGCATAGAAACATACATTCCTTATCGATATCAACTTCCGTAGGGCGTACTTTGACCAAACCCTTGAGCGAATGCAGCTGCGGCCCTGCGAGGAACATATCATACTCGCTGGCGTGGAGTCTAAGCTTCCTGGCCAGTTCGGCATCTTTCAGGCCTGACTGATAACCCTGTCTTAAGAAAAAGCCTTTCGCTCGTTCGATTCCCAGTGTATTGACGCATTCCGGCTTGAAAACTCGCCATTGCTGAAACCTGCAGTAAGCATTCTTTGTTCGCCAAGCCAGATTTTGCCTTCCGTGCTTTGGAAGTGAATCAGGTTAGTCAGGTCCTTAAAATCGGAATGCTGGATTTCAGGCTTATATTTTATTGGCATCTGCTATTTCTCCTCTTTCTCTAGAAAAGGCTAGCACAGTCCCTAGGACTGAGCTAGCTGTAAAAGGCGCAGTGAGACGATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGTCTAGCAGTAGACCCGACTGGAAGCGGGCAGTGAGCGCAGCGCAATGTCTCACTGCGCCTTGAAGCGTTTGCTCAAGCGGCCTTGGGCAATTGATCAAATGCTTAAAAAGCCTGCGCAAGCGCGGCTTAATTTCGCTCGCTCGATCATTCTAAAAATTAGAAACACATTGAAAAACAATACCTTGAAGTCGGTTTTCAGACCTTGGCACAGCTGTTGCACTTTGTCCTGCGCTTTAGCTGTCACCGGATGTGCTTTCCGGTCGATGAGTCCGTGAGGACGAAGCAGCCTCTACAAATAATTTTGTTTAATCTAGAGAAAGAGGAGAAATAGCAGATGCGTAAAGCTGAGAAACTGTTCACTGGTGTCGTCCCTATTCTGGTGGAACTGGATGGTGATGTCAACGGTCATAAGTTTTCCGTGCGTGGCGAGGGTGAAGGTGACGCAACTAATGGTAAACTGACGCTGAAGTTCATCTGTACTACTGGTAAACTGCCGGTACCTTGGCCGACTCTGGTAACGACGCTGACTTATGGTGTTCAGTGCTTTGCTCGTTATCCGGACCATATGAAGCAGCATGACTTCTTCAAGTCCGCCATGCCGGAAGGCTATGTGCAGGAACGCACGATTTCACTGGAGGATGACGGCACGTACAAAACGCGTGCGGAAGTGAAATTTGAAGGCGATACCCTGGTAAACCGCATTGAGCTGAAAGGCATTGACTTTAAAGAAGACGGCAATATCCTGGGCCATAAGCTGGAATACAATTTTAACAGCCACAATGTTTACATCACCGCCGATAAACAAAAAAATGGCATTAAAGCGAATTTTAAAATTCGCCACAACGTGGAGGATGGCAGCGTGCAGCTGGCTGATCACTACCAGCAAAACACTCCAATCGGTGATGGTCCCTGTTCTGCTGCCAGACAATCACTATCTGAGCACGCAAAGCGTTCTGTCTAAAGATCCGAACGAGAAACGCGATCATATGGTTCTGCTGGAGTTCGTAACCGCAGCGGGCATCACGCATGGTATGGATGAACTGTACAAATAACTAAGATCCAGCTTCGGCTGGATCTTTTCTCTGTCACTCACTGAGACGATTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATGTACTAGTCACTTAGTGAGACGATTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATGTACTAGTTAACTTCAGGACCTGCAGCTCGGGCAGCGTTGGGTCCTGGCCACGGGTGCGCATGATCGTGCTCCTGTCGTTGAGGACCCGGCTAGGCTGGCGGGGTTGCCTTACTGGTTAGCAGAATGAATCACCGATACGCGAGCGAACGTGAAGCGACTGCTGCTGCAAAACGTCTGCGACCTGAGCAACAACATGAATGGTCTTCGGTTTCCGTGTTTCGTAAAGTCTGGAAACGCGGAAGTCCCCTACGATTGCTGCTGAAGTTGCCCGCAACAGAGTGGAACCAACCGGTGATACCACGATACTATGACTGAGAGTCAACGCCATGAGCGGCCTCATTTCTTATTCTGAGTTACAACAGTCCGTACCGCTGCCGGTAGCTCCTTCCGGTGGGCGCGGGGGCATGACTATCGTCGCCGCACTTATGACTGTCTTCTTTATCATGCAACTCGTAGGACAGGTGCCGGCAGCGCCCAACCAGTCCCCCGGCCACGGGGCCTGCCACCATACCCACGCCGAAACAAGCCTTTTCACCGCCATTATGTTCCGGATCTGCATCGCAGGATGCTGCTGGCTACCCTGTGGAACACCTACATCTGTATTAACGAAGCGCTAACCGTTTTTATCAGGCTCTGGGGAGGCAGAATAAATGATCCTTATCGTCAATTATTACCTCCACGGGGAGAGCCTGAGCAAACTGGCCTCAGGCATTTGAGAAGCACACGGTCACACTGCTTCCGGTAGTCAATAAACCGGTAAACCAGCAATAGACATAAGCGGCTATTTAACGACCCTGCCCTGAACCGACGACCGGGTCGGAATTTGCTTTCGAATTTCTGCCATTCATCCGCTTATTATCACTTATTCAGGCGTAGCAACCAGGCGTTTAAGGGCACCAATAACTGCCTTAAAATTACGCCCCGCCCTGCCACTCATCGCAGTACTGTTGTAATTCATTAAGCATTCTGCCGACATGGAAGCCCATCACAAACGGCATGATGAACCTGAATCGCCAGCGGCATCGGCACCTTGTCGCCTTGCGTATAATATTTGCCCATGGTGAAAATGGGGGCGAAGAAGTTGTCCATATTGGCCACGTTTAAATCAAAACTGGTGAAACTCACCAGGGATTGGCTGAGACGAAAAACATATTCTCAATAAAGCCCTTTAGGGAAATAGGCCAGGTTTTCACCGTAACACGCCACATCTTGCGAATATATGTGTAGAAACTGCAATCGTCGTGGTATTCACTCCAGAGCGATGAAAACGTTTCAGTTTGCTCATGGAAAACGGTGTAACAAGGGTGAACACTATCCCATATCACCAGCTCACCGTCTTTCATTGCCATACGGAACTCCGGATGAGCATTCATCAGGCGGGCAAGAATGTGAATAAAGGCCGGATAAAGCTTATTGCTTATTTTTCTTTACGGTCTTTAAAAAGGCCGTAATATCCAGCTGAACGGTCTGGTTATAGGTACATTGAGCAACTGACTGAAATGCCTCAAAATGTTCTTTACGATGCCATTGGGATATATCAACGGTGGTATATCCAGTGATTTTTTTCTCCATTTTAGCTTCCTTAGCTCCTGAAAATCTCGATAACTCAAAATACGCCCGGTAGTGATCTTATTTCATTATGGTGAGAAAGTTGGAACCTCTTACGTGCCGATCAACGTCTCATTTTCGCCAAAAGTTGGCCCAGGGCTTCCGGTATCAACAGGGACACCAGGATTTATTTATTCTGCGAAGTGATCTTCCGTCACGAATTGTTTATTCGGCGCAAAGTGCGTCGGGTGATGCTGCCAACTTACTTTTTGGTTAGTGTATGATGGTGTTTTTGAGGTGCTCCAGTGGCTTCTGTTTCTATCAGCTGTCCCTCCTGTTCAGCTACTGACGGGGTGGTGCGTAACGGCAAAAGCACCATGAGGACGTCAGCGCTAGTGTATACTGGCTTACTATGTTGGCACTGATGAGGGTGTCAGTGAAGTGCTTCATGTGGCAGGAGAAAAAGGCTGCACCGGTGCGTCAGCAGAATATGTGATACAGGATATATTCCGCTTCCTCGCTCACTGACTCGCTACGCTCGGTCGTTCGACTGCGGCGAGCGGAAATGGCTTACGAACGGGGCGGAGATTTCCTGGAAGATGCCAGGGAAGATACTTAACAGGGTAAGGGCCGCGGCAAAGCCGTTTTCCATAAGAGCTCCCGCCCCCCTGACATCACGAAATCTGACGCTCAAATCAGTGGTGGCGAGAGCTTCCGACAGGACTATAAAGATACCAGGCGTTTCCCCTGGCGGCTCCCTCGTGCGCTCTCCTGTTCCTGCCTTTCGGTTTACCGGTGTCATTCCGCTGTTATGGCCGCGTTTGTCTCATTCCACGCCTGACGCTCAGTTCCGGGTAGGCAGTTCGCTCCAAGCTGGACTGTATGCACGAACCCCGTTCAGTCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGAAAGACATGCAAAAGCACCACTGGCAGCAGCCACTGGTAATTGATTTAGAGGAGTTAGTCTTGAAGTCATGCGCCGGTTAAGGCTAAACTGAAAGGACAAGTTTTGGTGACTGCGCTCCTCCAAGCCAGTTACCTCGGTTCAAAGAGTTGGTAGCTCAGAGAACCTTCGAAAAACCGCCCTGCAAGGCGGTTTCGTTTCAGAGCAAGAGATTACGCGCAGACCAAAACGATCTCAAGAAGATCATCTTATTAATCAGATAAAATATTTCAAGATTTCAGTGCAATTTATCTCTTCAAAGTACGCTGCGAAGTCAGCCCCATACGATATAAGTTGTAATTCTCATGTTTGACAGCTTATCATCGGCGATGAATTA 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8b7db6e7-70c6-4a09-b37e-0de040b76a19    0   pACBB_PL    4287    60  105S10M1D85M1I120M2I212M1D107M1I343M1D46M2I10M1D365M2I108M1D21M3D2M2I3M1D2M1D167M2D81M2I64M1I1M1I318M1I55M2I2M1I109M1D105M1I106M1D11M2I6M1D22M1D180M1D77M1I119M6D48M5S  *   0   0   AATTCATCTGAACTTATCTAGACCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATCGTCTCTAGTGAGCCTTGAGACCTTTACACTTTACAAGTAAGGTCTCTTCACTTAGTGGACGATTCTCCTTGCGCATCTGTGCGGTATTTCACACCGCATGTACTAGTTAACTTCAGGACCTGCAGCTCAGGCAGCGTTGAGTCCCCGGCCACGGGTGCGCATGATCGTGCTCCTGTCGTTGAGGACCCGGCTAGGCTGGCGGGGTTGCCTTACTGGTTAGCAGAATGAATCACCGATACGCGAGCGAACGTGAAGCGACTGCTGCGCTGCAAAACGTCTGCGACCTGAGCAACAACATGAATGGTCTTCGGTTTCCGTGTTTCGTAAAGTCTGGAAACGCGGAAGTCCCCTACGTGCTGCTGAAGTTGCCCGCAACAGAGAGTGGAACCAGCCGGTGATACCACGATACTATGACTGAGAGTCAACGCCATGAGCGGCCTCATTTCTTATTCTGAGTTACAACAGTCCGCACCGCTGCCGGTAGCTCCTTCCGGTGGGCGCGGGGCATGACTATCGTCGCCGCACTTATGACTGTCTTCTTTATCATGCAACTCGTAGGACAGGTGCCGGCAGCGCCCAACAGTCCCCCCGGCCACGGGGCCTGCCACCATACCCACGCCGAAACAAGCGCCCTGCACCATTATGTTCCGGATCTGCATCGCAGGATGCTGCTGGCTACCCTGTGGAACACCTGCATCTGTATTAACGAAGCGCTAACCGTTTTTATCAGGCTCTGGGAGGCAGAATAAATGATCATATCGTCAATTATTACCTCCACGGGGAGAGCCTGAGCAAACTGGCCTCAGGCATTTGAGAAGCACACGGTCACACTGCTTCCGGTAGTCAATAAACCGGTAAACCAGCAATAGACGCAAGCGGCTATTTAACGACCCTGCCCTGAACCGACGACCGGGTCGAATTTGCTTTCAATTTCTGCCATTCATCCGCTTATTATCACTTATTCAGGCGTAGCAACCCAGGCGTTTGGGGCACCAATAACTGCCTTAAAAAAATTACGCCCCGCCCTGCCACTCATCGCAGTACTGTTGTAATTCATTAAGCATTCTGCCGACATGGAAGCCATCACAAACGGCATGATGAACCTGAATCGCCAGCGGCATCAGCACCTTGTCGCCTTGCGTATAATATTTGCCCATGGTGAAAATGGGGGCGAAGAAGTTGTCCATATTGGCCACGTTTAAATCAAAACTGGTGAAACTCACCCAGGGATTGGCTGAGACGAAAAACATATTCTCAATAAACCCTTTAGGGAAATAGGCCAGGTTTTCACCGTAACACGCCACATCTTGCGAATATATGTGTAGAAACTGCCGGAAATCGTCGTGGTATTCGCACTCCAGAGCGATGAAAACGTTTCAGTTTGCTCATGGAAAACGGTGTAACAAGGGTGAACACTATCCCATATCACCAGCTCACCGTCTTTCATTGCCATACGGAGCTCGGATGAGCATTCATCAGGCGAACGGAAGTAATAAAGGCCGGATAAAACTTGTGCTTATTTTTCTTTACGGTCTTTAAAAAGGCCGTAATATCCAGCTGAACGGTCTGGTTATAGGTACATTGAGCAACTGACTGAAATGCCTCAAAATGTTCTTTACGATGCCATTGGGATATATCAACGGTGGTATATCCAGTGATTTTTCTCCATTTTAGCTTCCTTAGCTCCTGAAAATCTCGATAACTCAAAAAATACGCCCGGTAGTGATCTTATTTCATTATGTGGTGAAAGTTGGAACCTCTTACGTGCCGATCAACGTCTCATTTTCGCCAAAAGTTGGCCCAGGAGATTTCCCGGTATCAGCAGGGACACCAGGATTTATTTATTCTGCGAAGTGATCTTCCGTCACAGGTATTTATTCGGCGCAAAGTGCGTCGGGTGATGCTGCCAACTTACTGATTTAGTGTATGATGGTGTTTTTGAGGTGCTCCAGTGGCTTCTGTTTCTATCAGCTGTCCCTCCTGTTCAGCTACTGACGGGGTGGTGCGTAACGGCGAAAGCACCGCCAGACATCAGCGCTAGCGGAGTGTATACTGGCTTACTATGTTGGCACTGATGAGGGTGTCAGTGAAGTGCTTCATGTGGCAGGAGAAAAAAGGCTGCACCGAGTGCGTCAGCAGAATATGTGATACAGGATATATTCCGCTTCCTCGCTCACTGACTCACGCCTGCGCTCGGTCGTTCGACTGCGGCGAGCGGAAATGGCTTACGAACGGGGCGGAGATTTCCTGGAAGATGCCAGGAAGATACTTAACAGGGAAGTGAGAGGGCCGCGGCAAGCCGTTTTTCCATAGGCTCCGCCCCCCTGACAAGCATCACGAAATCTGACGCTCAAATCAGTGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGCGGCTCCCTCGTGCGCTCTCCTGTTCCTGCCTTTCGGTTTACCGGTGTCATTCCGCTGTTATGGCCGCGTTTGTCTCATTCCACGCCTGACACTCAGTTCGGGTAGGCAGTCTTCGCTCAAGCTGGACTGTATGCACGAACCCCCGTTCAGTCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGAAAGACATGCAAAAGCACCACTGGCAGCAGCCACTGGTAATTGATTTAGAGGAGTTAGTCTTGAAGTCATGCGCCGGTTAAGGCTAAACTGAAAGGACAAGTTTTGGTGACTGCGCTCCTCAAGCCAGTTACCTCGGTTCAAAGAGTTGGTAGCTCAGAGAACCTTCGAAAAACCGCCCTGCAAGGCGGTTTTTTCGTTTTTCAGAGCAAGAGATTACGCGCAGACCAAAACGATCTCAAGAAGATCATCTTATTAATCAGATAAAATATTTCAAGATTTCAGTGCAATTTATCTCTTCAAATGTAGCACCTGAAATTATTTTATATAAGTTGTAATTCTCATGTTTGACAGCTTATCATCGATGAATTA   ((((''(*'''((*,399:A???>==><<<4/.....36366888<9::;>A;:::;?A@B52+)((),,3339<=<>?@@CFCC?>>>>BC@>==>?<<==@AAB?5212122421/++./22<=;;,,))),79<<??BGJAAAB?BC===;:>=43///./8979555452129????65555)((((-./246,,,,,.--.16<=71)))*0::::;:@BACABCBBBBAAC@>:;;;97>@A?A6433389;99999?>@?>>??@AAAA=>A@ABBCCDB@A@??@?@BAAB>>>>>@B???==>>?>=<:;;70/(''-..23?@>>>@@@??@@AEB?@?@AA99889??A44444AA9878;@@?>>?@@?ABADA@ABA@A@=<;6/../6886:99:;=;==???>>;++++++-.967867988;79=;=<:;:9(((()@@@>;:;;;>==<<<=>>@@?8>C<<>D<<;=<>>>>A@?>;;;;<>?AD*08:;??211@@@@@==::::;<<>??<=>>>><<<<<>>>>>??@B?????@@@A@AA==;;;=:::::=?@>>=<<<<>@?ABB@?>>===>@C@@AA>?/....<<;<>=;=<<<>><<;::<::;;<=83/.,,-433560;:;????:98CADGFE?>=:966677CEDGA@B?BBABCDDC85=A+*)++-88:@=)(-4;?F?MJGFEG{CCCBBLEJFEC{F@999<=79AFGGFCBA@3*)))*//19<????:9..-/35;;>@BA=GFADGH??=A@HCENJGFA@-----::EENOGHCCBBCOHEBAB?AACBC@BBD?>:2101327468DGFA??=;84203399999::<221/..--0112....68<>>ADIPIDB@B@:KJDBCHEDCFGFC=>=;<;55211//-,((%%%&+:?JBDBB??:87666:;?DBCHFHKMLMIJFM@)((((2111249:<:9/++++FFLJGEFEFDFOJEGFG{DEBAFD@@@E?EDB110022?DCECBC213???AB700001>?BBCHFEDEBEC==DEDH{B@<=BA@A@C+4?>=@{G{HJOPNNNGFDCGDFGFHEBDFJFDMEHVCEGCEEG{KF{HH{IEFGEBBCDE@QKMGGGGJI{{PIGE@====??G{{FKIJDB@;-,,+-3///01<AADIKQNJJL{\FFHFIFH????@JDDEFFBBA?CEIFEG@@@@>A?=3337;;@DCCEBDBIEAEAA679@06@?C?@?<==>===@@=DGGH{=F9==:(,;>>DC?D><74456;:=8EFEGIJH{JJCFHFKPOKE933367<5=HDE1111178;>?<@..=?@??>@B>==<=BB@>;-++,,+.9>?@@CCDF>?@>>@;;:77466:666223368...-254=>??==>>>CBDIB@@@?A>>>??@@@@?>?>;<?>A??>==>CAAAACGA@DEGHEICEDIH>65556@ABCAEBDIFLEHIIFGHC:>?E54.)&%%%%%&)/0;>ABBBDFF<<<44554-,,)''&&&&&)4=A<HDGGF{IEHLEFEGEGEEGF{FCIH{{MF{GDAFGBBBDBDIHIHJAC9999787:ACADEFEIJF{HG{{GLNP{FEEGDD00000011111139889;;ADGIUGDFHGEE@CDIJEEDCABCFE{GFFHJGKIGQHOQEDEBCFDE{GJJJG)))))CCEG:9=CA>>>===6==BCNFGMCCED?>=AGFDCF:::F{HNIBLAD@F]?ADDDDC><<==>EECBE@EIQFHHDEDDB<*))+3=ABA=F@GKSIQNHLHPSJGFMHIGHMDQIHJKENFKLFK{IFCFDG?B@=E??>>..--**'''')(),37=EC(((((24?<;9=CB><CDBEGAEGLBHE<)(((*1678?DIN@?@BA?@?@?FDGEEDBD98777<>>DCB@A@66566@@AAC<<=A??@@@CCFDDFGEJIE@FECFGDHCBDCBB?A:9811<=@8545667222123<=<>BA>=<<;;>@@@AAA+*@DEGDCB:@@?>=----.?DCHEF@??E844)))))./0,,,*'''&&')),,-.>B@A?@?>===@BFBDBBDGFHEA?>=>9:AAB==>>>6666<2?ABCBBBABCCDDFFGDEDBEFF88766645466735;<;4312+((()-<?ABCDDAACABCFEEEBB:766;?@77783/...078;=BFEBBBBA?B921''%%%'&&(,.?@CADBA@CA@ACGEFCCDDB?>>DC=EDDCDB@?==,+++,DBACBAHAFPAF<B>))=@@>.-3217;>---122119889<<<>>A@AA@+>;<>??=;((822:;;;=:76689;BCFEHFJHFDD??@@D44434@AA@@==E?>;>=@BJIOGHVGGGJHHN{{NGK{E{II{LEDGDFGGIGEE;?0011..//07AC49;;;5/666;;;<86269===<??@BCDDDGHB@>>?@BFGGEGDFD>CBEPFFDECEFHGJPLDIGCDEDFBBC?@@A@EHFDFEGGEABBBECEF>2003575::('&('(**)((''((*..0,++5:=DCBC?>??@D><<<;;;;:8;?C=000@>>?>>BEBBB?@>99?B>????@@GDDGJHKHFGF{DACA@==>;:=<<<<A@@BBBCCGACGKCFEFHKFOIKVFFFDKGIJFG><===EEF@=))(*)+,-0;<?ID@FC@@?>>DFFGE>>HD{EDBDEDD=;<<<EF@@?@BB@ACEEJBAACDEGDEDDD>88(((5@65555A>99;;=><4/*&()*,./ACCDCD;::?@?@BFME>?>@EMFFCDEQOGEE?98339:;<:866898>A0.,-****),-04378:F{EC@AB@@?B@A@FGAIBCBB;;6555=:BBBBCDBCEDA433>AABN{FEFHJCA@AAACCE@???>ABCECGBC@@@CFFEB:/.--.1<***)()-../*().23>.**2;@?CADAA@AAACDCBB>>==?ABBFDEB@@@?;;;:;   NM:i:67 ms:i:5483   AS:i:5468   nn:i:0  tp:A:P  cm:i:432    s1:i:2603   s2:i:0  de:f:0.0177 SA:Z:pACBB_PL,1,+,73M2964S,22,2;    rl:i:0  ru:i:0
8b7db6e7-70c6-4a09-b37e-0de040b76a19    2048    pACBB_PL    1   22  7M1D56M1I9M2964H    *   0   0   AATTCATCTGAACTTATCTAGACCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATCGTCTCTAGTGAGCCT   ((((''(*'''((*,399:A???>==><<<4/.....36366888<9::;>A;:::;?A@B52+)((),,333   NM:i:2  ms:i:132    AS:i:132    nn:i:0  tp:A:P  cm:i:6  s1:i:54 s2:i:0  de:f:0.027  SA:Z:pACBB_PL,4287,+,105S2927M1D5S,60,67;   rl:i:0  ru:i:0
b3846fc3-30c8-488c-a0b7-b99500971507    16  pACBB_PL    4287    60  104S92M2D334M1D7M2D39M1I121M1I140M2I61M2I126M1I480M3I51M2D7M1D2M1D3M1I18M1D8M2I135M1I275M1D87M1D265M1D31M1I16M1D37M1I64M1I1M2I47M4D31M1D145M1D8M1I64M1I8M2I61M1I144M4S  *   0   0   TAATTCATCCTGAACTTATCTAGACCCGACTGGAAAGGGCAGTGAGCGCAACGCAATCGTCTCAGTGAGCCTTGAGACCTTTACACTTTACAAGTAAGGTCTCTTCACTTAGTGAGACGATTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATGTACTAGTTAACTTCAGGACCTGCAGCTTCGGCAGCGTTGTCCTGGCCACGGGTGCGCATGATCGTGCTCCTGTCGTTGAGGACCCGGCTAGGCTGGCGGGGTTGCCTTACTGGTTAGCAGAATGAATCACCGATACGCGAGCGAACGTGAAGCGACTGCTGCTGCAAAACGTCTGCGACCTGAGCAACAACATGAATGGTCTTCGGTTTCCGTGTTTCGTAAAGTCTGGAAACGCGGAAGTCCCCTACGTGCTGCTGAAGTTGCCCGCAACAGAGAGTGGAACCAACCGGTGATACCACGATACTATGACTGAGAGTCAACGCCATGAGCGGCCTCATTTCTTATTCTGAGTTACAACAGTCCGCACCGCTGCCGGTAGCCTCCCGGTGGGCGCGGGGCATGACTATCGTCGCCGCACTTTATGACTGTCTTCTTTATCATGCAACTCGTAGGACAGGTGCCGGCAGCGCCCAACAGTCCCCCGGCCACGGGGCCTGCCACCATACCCACGCCGAAACAAGCGCCCTGCACCATTATGTTCCCGGATCTGCATCGCAGGATGCTGCTGGCTACCCTGTGGAACACCTACATCTGTATTAACGAAGCGCTAACCGTTTTTATCAGGCTCTGGGAGGCAGAATAAATGATCATATCGTCAATTATTACCTCCACGGGGAGAGCTTCCGAGCAAACTGGCCTCAGGCATTTGAGAAGCACACGGTCACACTGCTTCCGGTAGTCAATACAAACCGGTAAACCAGCAATAGACATAAGCGGCTATTTAACGACCCTGCCCTGAACCGACGACCGGGTCGAATTTGCTTTCGAATTTCTGCCATTCATCCGCTTATTATCACTTATTCAGGCGTAGCAACCAGGCGTTTAAGGGCACCAATAACTGCCTTAAAAAAATTACGCCCCGCCCTGCCACTCATCGCAGTACTGTTGTAATTCATTAAGCATTCTGCCGACATGGAAGCCATCACAAACGGCATGATGAACCTGAATCGCCAGCGGCATCAGCACCTTGTCGCCTTGCGTATAATATTTGCCCATGGTGAAAATGGGGGCGAAGAAGTTGTCCATATTGGCCACGTTTAAATCAAAACTGGTGAAACTCACCCAGGGATTGGCTGAGACGAAAAACATATTCTCAATAAACCCTTTAGGGAAATAGGCCAGGTTTTCACCGTAACACGCCACATCTTGCGAATATATGTGTAGAAACTGCCGGAAATCGTCGTGGTATTCACTCCAGAGCGATGAAAACGTTTCAGTTTGCTCATGGAAAACGGTGTAACAAGGGTGAACACGGTCCCATATCACCAGCTCACCGTCTTTCATTGCCATACGGAAGAACTCCGGATGAGCATTCATCAGGCGGGCAAGAATGTGAATAAAGGCCGGACAACTTAGTTATTTTTTCTTTACGGTCTTTAAAAGGCCGTGTAATATCCAGCTGAACGGTCTGGTTATAGGTACATTGAGCAACTGACTGAAATGCCTCAAAATGTTCTTTACGATGCCATTGGGATATATCAACGGTGGTATATCCAGTGATTTTTTTCTCCATTTTAGCTTCCTTTAGCTCCTGAAAATCTCGATAACTCAAAAAATACGCCCGGTAGTGATCTTATTTCATTATGGTGAAAGTTGGAACCTCTTACGTGCCGATCAACGTCTCATTTTCGCCAAAAGTTGGCCCAGGGCTTCCCGGTATCAACAGGGACACCAGGATTTATTTATTCTGCGAAGTGATCTTCCGTCACAGGTATTTATTCGGCGCAAAGTGCGTCGGGTGATGCTGCCAACTTACTGATTTAGTGTATGATGGTGTTTTTGAGGTGCTCCAGTGGCTTTGCTTCGGTCAGCTGTCCCTCCTGTTCAGCTACTGACGGGGTGGTGCGTAACGGCAAAAGCACCGCCGGACATCAGCGCTAGCGGAGGGGTACTGGCTTACTATGTTGGCACTGATGAGGGTGTCAGTGAAGTGCTTCATGTGGCAGGAGAAAAAAGGCTGCACCGGTGCGTCAGCAGAATATGTGATACAGGATATATTCCGCTTCCTCGCTCACTGACTCGCTACGCTCGGTCGTTCGACTGCGGCGAGCGGAAATGGCTTACGAACGGGGCGGAGATTTCCTGGAAGATGCCAGGAAGATACTTAACAGGGAAGTGAGAGGGCCGCGGCAAAGCCGTTTTTCCATAGGCCTGCCCCCCTGACAAGCATCACGAAATCTGATTGCCCAAATCAGTGGTATGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGCGGCTCCCTCGTGCGCTCTCCTGTTCCTGCCTTTCGGTTTACCGGTGTCATTCCGTTCGAAATATGGCCGCGTTTGTCTCATTCCACGCCTGACACTCAGTTCCGGGTAGTTCGCTCCAAGCTGGACTGTATGCACGAACCCCCGTTCAGTCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGAAAGACATGCAAAAGCACCACTGGCAGCAGCCACTGGTAATTGATTTAGAGGAGTTAGTCTTGAAGTCATGCGCCGGTTAAGGCTAACCGAAAGGGATAAGTTTTGGTGACTGCGCTCCTCCAAGCCAGTTACCTCGGTTCAAAGAGTTGGTAGCTCAGGAGAACCTTCTCGAAAAACCGCCCTGCAAGGCGGTTTTTTCGTTTTCAGAGCAAGAGATTACGCGCAGACCAAAAACGATCTCAAGAAGATCATCTTATTAATCAGATAAAATATTTCAAGATTTCAGTGCAATTTATCTCTTCAAATGTAGCACCTGAAGTCAGCCCCATACGATATAAGTTGTAATTCTCATGTTTGACAGCTTATCATCGATGAATT    CCDC@????@?@?>???;6***<;<<;?2//1))))**67;==>66566@?;5.***+:7766677=76777?B@@@;44?????BCBABA@@?>>=<<=:7722.***68::7336:?<8333033::;=;=>>@A?=<=;:::;B00001A@;9621387;:45/---./04222222-'%%'%'/<;.-+''(:;:::;HCA?:87622243488)((((235688444//0<<@;;:98988-+++,B@D<:A>>;:9877::=@ABBA@@A@<ABABCCECAABDFCBBDEDGF@>===>BBBAA>>>77778BBEHEBBBABDDACBECEE>>>>?A@AA@AA@?<4399889:?@A=>88==??>;;;?:99431/,++,=A@>>>>ACBA:C777>;9::==AA@@=;<<=>>77778ED?>======<<@@?C6====?@@@??>@@@@@A@A@@11111@<;<<<?A=>==>?>:8889?@?CABBBBACB10//0D@ABA@@@;;98788>>?22222@??=8444522244=>>@<===96=?>><;;;;===<<=>?@::;+()===>??===;8;<8ABABAAA97,+)(((**--8855643655:::<:999:<?>==77777>@A>:87.+0.---.:;BAA@?>>:::::?@?@@@?@AA@A>>>>=@?>?@@=6-,,,,,-56>>A?><;76677:;@??>>@?<=<:;<==?@C@@B@?<:;::;==<<==<BA?>>>>=?>=>??>>;;=>??@=6334899974104556;<<5553?@@=>>>>?=>==?ADHJFGA0-**,-8/.,+0.+,,())(())8::ACC>>==>?CCB@@<72@@@A>=>>>AA>>>>?AAAB@@@@@BBBA??>??A?=;90**<=;<<=7@@??@977787?>??>??>>@@@ACHFBA><<<;;<==45555>>941002111338?AABB?<:795???>@><=BCBDEBCEEAACB@ABBBDCBCAAABCCCACA@A>=7610,,//0)''((<=D>?DGCCCBCEEECBDB@C>>A>@EXNLHEGFCA++++,BDIFJOEIFBBB@BCDDBBBCCFEEGKEGAL^ID{DDGJDCDCAGBBBFJLHIMGMFT{EI{QIHGJHFIDH<<<<=;;;CECIBDEHGEKGIKHFDFEFEIGFEEGCE?NILLB=:((()788<6=7:::9;9989;A=C<?=:DCDIGFDCDCBCBDCD{{PFKJHIHHIO{J{KFOHEH?>BAAAAEFFDELFD@3023///A@F?>>::;FD=?A<:A?+***+:;=H=;FKDGGFIF:D?A=@@A553255=8=<>=>FB8>{GFMLIIN{E{JGJIGEIEGLECB;233202=?;?<;:::;ACCEHHIKFB@FECDE988FB:@BCA<43345BBFXJRIGFGABDEDGGJNHLHFGC9{FDBG@@@?EGGJGFD922/0.,,,,,/6;BGHHFDBCB:511.,,,,0834A;?=/----6511-))+21100000E?>FCCCEQFDEA767546>?..-))))=?DDWGC@;92/)(((*/+,&%$%%&)+--,*)((,-02766)6786400)('&&&))--.>AB@@BADDEGEDFFFGFGKFFGHBABA@AFDDB>99889>===EDB@88368B@??:99<;667655CBEDC@DGEABCABD>8//A<;<;<ACBB?;:55678D@GGb@<7535@AB;;;;;=<875+.B>>>??FEBGDBB?A@@@GE@>=;>GKGIFAA@?>@C<=BCD@B<D@@=<=4<>ACCFABADD?EA@A88896,,8868DBA>>?CYABDEG<;;;888=9?@@=<32/,10/1111128978>:@4435656566BAA656BACBDF<@ADIDHEDJJMIIG{GJIIHHIFB@A@@DBEGJH@ABEEFDF=7...9>>?HCSN{{JIIHKIFJNKJGCCAHDGDCDFEEDEGHHBDBACDADDEFHA>=<==>;:844666669<A@983(''&%%$%%&(,+++-.6??EHCDDEFTHFEHN>===;<7;>@AAAAEDEFGFEE?<8=8?CCCDDCEFEEHGBF=65/.-*)))((+-*&'),./.-//0211113377?<;7667..28<@?@@BBB???BADEE@@?@7556:FIKMFCBECKI@DBEHDLEBABACBAAA?>>?@EFEILBBBDCIF@;55;5556;9:332234,----=<?@<988668=>==>>AGFHEEECBFDFSINKIFIF@HCBC?4KE;<*))):::D<<:7>?==BCDC=C3*****A@A@920:99:CCF=FA?EBE>F42497889744<59:...44;:9<30000A:;9798$##%**+++3+13()))@@@ABDFC>====?<::0.*(')***3?AB@?<9&&&&&&>C/////BBA?==<97312=>@>?>=1,*))+*,645****+,,:::11111:=>A8BEC22223<@?>?<>?:>KHDFEIBGIHERGFF=<:81-*('&&&%&'+/--,,,2@@==333334A>=<;:;7333=44221228962+)(&$%'''()'&''((99>BA:87779:H{HEHFCA<<<<DBCAB@<<99:::DCG000(&&&'(((4;<EFDCFCDDEIHGFFIFIGIFIAAEIFEE@>76631237555535555ABCCF{GH;;;;;DHMCBB?BEHEFDKJJH?DB?>842223??FGILISC667C;8742.-)(&&((+,,(()+34>@HF?=<=>?HFEB,++**@=<<=;=CB?:??54444<;;;>7BF88888AAABDCD2..0B64/+))&'(*,,,<><<7/'&&&',,EJHGEBATDD999:HCBFC@>>;>>@B>;9888:9:H{FICA;/.:DECBIR?CDBGFHHHEFA?743/--..--17DC>EEDDE76111BC>GCAE?EHHIHIGJJHF<77>?EICDAB>==:/----245<<=;:::<=@B<<=DECJECFDECDEDCH@@@@BCDGFAHGB<889CBABBCCILTGGEC>    NM:i:68 ms:i:5472   AS:i:5460   nn:i:0  tp:A:P  cm:i:445    s1:i:2663   s2:i:44 de:f:0.0187 SA:Z:pACBB_PL,1,-,1S71M2D2968S,26,2;    rl:i:0  ru:i:0
b3846fc3-30c8-488c-a0b7-b99500971507    2064    pACBB_PL    1   26  1H35M2D36M2968H *   0   0   AATTCATCCTGAACTTATCTAGACCCGACTGGAAAGGGCAGTGAGCGCAACGCAATCGTCTCAGTGAGCCT CDC@????@?@?>???;6***<;<<;?2//1))))**67;==>66566@?;5.***+:7766677=76777 NM:i:2  ms:i:135    AS:i:134    nn:i:0  tp:A:P  cm:i:7  s1:i:56 s2:i:0  de:f:0.0139 SA:Z:pACBB_PL,4287,-,104S2928M4I4S,60,68;   rl:i:0  ru:i:0
bb6e8f07-a2c0-4485-b535-6c41c3fed79b    0   pACBB_PL    1   60  89M3I43M8I12M1I9M6I2M2I2M6I169M1I460M1I338M2I37M3I7M1D36M2D5M4I29M1D24M1I2M1I56M1D1M3D4M1D23M2D3M9D2M5D21M2D21M1D6M1D3M3I1M1D235M1I86M1D201M1I93M2I87M1I11M2D292M1D676M1I89M1I13M3D1M3D24M2D1M4D172M3D13M1D245M1I26M1D9M2I16M1I19M2D11M1I4M1I133M2I266M1I23M1I2M2D48M1I21M4D112M2I321M1D99M1I22M4I121M1I254M1I17M1D297M2I8M2I553M2I53M4I45M1D7M1D82M3D4M3D16M1I1M1I7M1I28M1D12M3D23M2I2M1I22M1D7M1D16M1D11M1I9M1I10M3I3M2D9M1D3M1D3M2I6M2D5M1D5M1D5M1D10M1D5M2I2M2D25M1D10M4D1M2D13M1I28M1I173M1D229M1D20M3D5M1I15M2D1M2D20M3D72M2D5M2I51M5S    *   0   0   AATTCATCCTGAACTTATCTAGACCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATCGTCTCAGTGAGCCTCTAGTACATGCGGTGTGAGGAAATACCGCACAGATGCGTAAGGAGAATCGTCTCAGCCTGTGATTTTGTTATCAATAAAAAAGGCCCCCCGATTTGGGAGGCCTTTTTTCGAAAATTAGTTATCATTTCGGGTTAACAACAGAGTAGTTAACCGGAACGAAGCAGTAACCTTTACCTTCCGCACGGATGTGACCGATACCCGGGAACGGTAAATGGGAAGCCGCGATCAGGTAACCACCTTTCGCCGCGTCCGCGAACGCTTTTTTACGTTCAACCGCCACCAGATTTACCGTCGATGTCCAGCTGGTTGGTAACAGACGGGTCGTCGAACTGAACCGCCGCAACCAGGATCAGGTCACCCAGCAGCGCCAGTTTCTGACCCTGAGATTCAACAACGTAGGTGGTGTGACCCGGGGTGTGGCCATGGGAAACCAGCGCTTTGATACCCGGAACCAGGTCGGTGTTACCAGAGAACGGTTTGAATTTACCCGCTTTAACGTACGGATTTAAGGAAGCCATCGCACCTTTGAAGAAACCTTTAGATTCGTCGTCCGGCGCTTTGTCCAGGTTGGTCTGAGACAGCCAGAAGTCCGCTTCTTTCTGGTCCGCACGAACAACCGCGTTCAGGAACGCCAGCTGTTCACCAACCATCAGACCACCAACGTGGTCCGGGTGCATGTGGGTGATGTAGATTTCGTCAACCTGTTCCGGCTGGTAACCCGCCGCTTTCAGGTTCGCCGCCCAGACGACCCAGGGTCGGACCGAACAGACCCGCCGCACCGGTGTCAACCAGAACCAGTTTAGAACCGGTGTTAACCAGGTAACCGGTAACAGAGGTTTCCAGCGGCGCTTTCTGGAAAGATTTCGCCAGCGCAGACTGGGTTTTCGGCGCCGGCTGGTTCAGACGTTTGTCAACCGGCAGCGCAACGGTACCGTCAGACAGCGCGGTGATTTCGAAGTCACCCAGCAGCATACGGTAGTAACCCGGCGCAGAGGTACGAACCTGCGGCGCCGCCACGAACGCGTGGGTAACGAAAACCATCTGCGCCGCGGTGCACAGACCCGCCAGCGGCAGAGAGGAGTTTTGGTCAGGTTACGCATCTGCTATTTCTCCTCTTTAATCTCTAGAAAGGCTAGCACTATACCTAGGACTGAGCTAGCCGTAAGGCGTGACCTTTGAGACGATTGCGTTGCGCTCACTGCCGCTTTCCAGTCGGGTCTGTTTTCCTAAGTACATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAATCGTCTCACTTTGTATGTTTGAAGAAAAGATCCAGCCCGGCTGGATCTTAGTTAGCCTTCTATGCCGATTTTTTAACCGATAAGCTAGTACGGTCGCTCCCTGGCCGAGCAATCGTGCAGCACCGGAGACATTTTGGTTGGCCTGTTGCATGGCTTTGCGCATCAGAGATTCCTCGATTTCATCGAGTGACAGGCCGCTGCTCAACAATTGGCTGATCCAACTGCCTTGGCCATTGCCTGGCTGAATAAGAACGCCGTCCGAAGACACCCGCTCAGAGGCGGTCTGGGGCTCTTCCGGCGCGCGTGGAAACAGCGCCTGTACGCTGATAGATTCGTTCCGGATCGGTTAAGATTATACCACGCTCGATTACATTTTCTAACTCACGGATATTGCCCGGCCAGCTGTAATGCAGGCGGGCCTCCAGGCTTTATCTGAAAGGCCTAATGTTCTCTTCCCGTATTCCTGGTGGAAGCGCTGAAGAAAATGCTCAACCAGCAGAGGAATGTCTTCACGGCGTTCGCGAAGGGCTGGGATCGCCACCGGAAAAACATTCAGCCGATAATACAGATCGGCACGAAAACGACCGGCTTTAACCGCTTCGGCCAGATCTTCATGTCCGGCACCATATAACCCTTACGTCTATTTTGCGCGTCCTGTTGTCCGCAACTCGCTCCAGCTCTCCTTCTTGGAGCACGCGCAGCAAACTGGCTTGAGCTCTCCCAGGCTTAACTCGATCACCTCGTCAAGAAATATAGTCCCTTTATCGGCCCGTTCAAAGCGTCCCATGCGTGATTGGGTGGCGCCGATAAAAGGCTCCTTTCCACGCCAAACAATTCGGACTCGATCAGGTCCGGCGGTATCGCCGCACAGTTCACAGCAACAAAAGGCTCTGCGGCGCGTTTAGAGCGCAAATGCACACTACGCGCGATGACCTCTTTGCCTACCCCGGTTTCTCCAAGTAACAGCACACTAACTTTGCCCTGTGCTGCTTTGTCCATCATATTGCGCACCGTTTGATAGGCTGGGGTCTGACCAATGCCGTAGTACTGTCCTTCCTGTTTGTCTAGGTTTGTACGCAGCGACACCAGTTGTGATTGTAACTCGTATAACTCTCGATGATGGGGTCATTCTTAAAATACTGTTTGAAAGAGGCAACATCGTCCCATTCTTCGGCCAGTTTTCCAATGACCCGACACTTATCACCTCCACAGCCGCGGCAACTTACTTCCTTAAAAATGATTTCTCTGCCCATAAAAGCCGAAGAATAGGCGCAGGCGTAGCCGAGCAGAGTCCAGCATACCGGATCTTGCATCTGCCCCAGATCGGTCTGACAGATTTTCACTTCAAGAGAGTCGATCCATTCCATCTCGGCATAGAAACGCCCACATTCCTTATCGATATCAACTTCCGTAGGGCGTACTTTGACCAAACCCTTGAGCGAATGCAGCTGCGGCCCTGCGAGGAACATATCATACTCGCTGGCATTAGGTCTAAGCTTCCTGGCCAGTTCGGCATCTTTCAGGCCTGACTGATAACCCTGTCTTAAGAAAAAGCCTTTCGCTCGTTCGATTCCCAGTGTATTGACGCATTCCCGGCGAAAACTCGCCATTGCTGAAACCTGCAGTAAAAGCATTCTTTGTTCGCCAAGCCAGATTTTGCCTTCCGTGCTTTGGAAGTGAATCGGGTTAGTCAGGTCCTTAAAATCGGAATGCTGGATTTCAGGCTTATATTTTATTGGCATCTGCTACTTTCCTGTGTGACTCTAGAAAAGGCTAGCACTGTACCTAGGACTGAGCTAGCCGTCAAAGGCGCAGTGAGACGATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGTCTAGCAGTAGACCCGACTGGAAAAGCGGGCAGTGGCGCAATCGTCTCACTGCGCCTTTAACTCAAGCGGCCTTGGGCAATTGATCAAATGCTTAAAAAGCCTGCGCAAGCGCGGCTTAATTTCGCTCGCTCCATTCATTCTAAAAATTAGAAACACATTGAAAAACAATACCTTGAAGTCGGTTTTCAGACCTTGGCACAGCTGTTGCACTTTGTCCTGCGCTTTTCTAGAGAAAGAGGAGAAATAGCAGATGCGTAAAGGCGAAGAGCTGTTCACTGGTGTCGTCCCTATTCTGGTGGAACTGGATGGTGATGTCAACGGTCATAAGTTTTCCGTGCGTGGCGAGGGTGAAGGTGACGCAACTAATGGTAAACTGACGCTGAAGTTCATCTGTACTACTGGTAAACTGCCGGTACCTTGGCCGACTCTGGTAACGACGCTGACTTATGGTGTTCAGTGCTTTGCTCGTTATCCGGACCATATGAAGCAGCCGCGACACCTTTAATTCCGCCATGCCGAAGGCTATCCGTACAGGAACGCACGATTTTACTTTAAGGATGACGGCGTACAAAACGCCGTGCCGGAAGTGAAATTTGAAGGCGATACCCTGGTAAACCGCATTGAGCTGAAAGGCATTGACTTTAAAGAAGACGGCAATATCCTGGGCCATAAGCTGGAATACAATTTTAACAGCCACAATGTTTACATCACCGCCACGATAAACAAAAAAATGGCATTAAAGCGAATTTTAAAATTCGCCACAACGTGGAGGATGGCAGCGTGCAGCTGGCTGATCACTACCAGCAAAACACTCCAATCGGTGATGGTCCTGTTCTGCTGCCAGACAATCACTATCTGAGCACGCAAAGCGTTCTGTCTAAAGATCCGAACGAGAAACGCGATCATATGGTTCTGCTGGAGTTCGTAACCGCAGCGGGCATCACGCATGGTATGGATGAACTGTACAAATAACTAATTTTCGAAAAAAGGCCTCCCAAATCGGGGGCGCTTTTTATTGATAACAAAATCACTCACTGAGACGATTCTCCTTACGCATCCTGTGCGGTATTTCACACCGCACTAGTCACTTAGTGAGACGATTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATGTACTAGTTAACTTCAGGACCTGCAGCTCGGGCAGCGTTGGGTCCTGGCCACGGAAGTGCGCATGATCGTGCTCCTGTCGTTGAGGACCCGGCTAGGCTGGCGGGGTTGCCTTACTGGTTAGCAGAATGAATCACCGATACGCGAGCGAACGTGAAGCGACTGCTGCTGCAAAACGTCTGCGACCTGAGCAACAACATGAATGGTCTTCGGTTTCCGTGTTTCGTAAAGTCTGGAAACGCGGAAGTCCCCTACGTGCTGCTGAAGTTGCCCGCAACAGAGAGTGGAACCAACCGGTGATACCACGATACTATGACTGAGAGTCAACGCCATGAGCGGCCTCATTTCTTATTCTGAGTTACAACAGTCCGCACCGCTGCCGGTAGCTCCTTCCGGTGGGCGCGGGGCATGACTATCGTCGCCGCACTTATGACTGTCTTCTTTATCATGCAACTCGTAGGACAGGTGCCGGCGGCGCGCCAACAGTCCCCCGGCCACGGGGCCTGCCTGCCACCATACCCACGCCGAAACAAGCGCCCTGCACCATTATGTTCCGGATCTGCATCGCAGGATGCTGCTGGCTACCCTGTGGAACACCTACATCTGTATTAACGAAGCGCTAACCGTTTTTTATCAGGCTCTGGGAGGCAGAATAAATGATCATATCGTCAATTATTACCTCCACGGGGAGAGCCTGAGCAAACTGGCCTCAGGCATTTGAAGAGCACACGGTCACACTGCTTCCGGTAGTCAATAAACCGGTAAACCAGCAATAGACATAAGCGGCTATTTAACGACCCTGCCCTGAACCGACGACCGGGTCGAATTTGCTTTCGAATTTCTGCCATTCATCCGCTTATTATCACTTATTCAGGCGTAGCAACCAGGCGTTTAAGGGCGCAATAACTGCCTTAAAAAAATTACGCCCCGCCCTGCCACTCATCGCAGTACTGTTGTAATTCATTAAGCATTCTGCCGACATGGAAGCCATCACAAACGGCATGATGAACCTGAATCGCCAGCGGCATCAGCACCTTGTCGCCTTGCGTATAATATTTGCCCATGGTGAAAATGGGGGCGAAGAAGTTGTCCATATTGGCCACGTTTAAATCAAAACTGGTGAAACTCACCCAGGGATTGGCTGAGACGAAAAACATATTCTCAATAAACCCTTTAGGGAAATAGGCCAGGTTTTCCTGCCGTAGCTTACGCCACATCTTGCGAATATATGTGTAGAAACTGCCGGAAATCGTCGTGGTATTCACTCCAGAGCGATGAAAACGTTTCAGTTTGCTCATGGAAAACGGTGTAACAAGGGTGAACACTATCCCATATCACCAGCTCACCGTCTTTCATTGCCATACGGAACTCCGGATGAGCATTCATCAGGCGGGCAAGAATGTGAATAAAGGCCGGATAAAACTTGTGCTTATTTTTCTTTACGGTCTTTAAAAAGGCCGTAATATCCAGCTGAACGGTCTGGTTATAGGTACATTGAGCAACTGACTGAAATGCCTCAAAATGTTCTTTACGATGCCATTGGGATATATCAACGGTGGTATATCCAGTGATTTTTTTCTCCATTTTAGCTTCCTTAGCTCCTGAAAATCTCGATAACTCAAAAAATACGCCCGGTAGTGATCTTATTTCATTATGGTGAAAGTTGGAACCTCTTACGTGCCGATCAACGTCTCATTTTCGCCAAAAGTTGGCCCAGGGCTTCCCGGTATCAACAGGGACACCAGGATTTATTTATTCTGCGAGAAGTGATCTTCCGTCACAGGTATTTATTCGGCGCAAAGTGCGTCGGGTGATGCGCACTGCCAACTTACTGATTTAGTGTATGATGGTGTTTTTGAGGTGCTCAGTGGCTATGTTTCTATCAGCTGTCCCTCCTGTTCAGCTACTGACGGGGTGGTGCGTAACGGCAAAAGCACCGCCGGACATCAACGCTGAAGACACTGGCTTACTATGCTCTGGCACTGGCTGGGGGTGTCGGTGCGATGCTTCATGCGCCGGAGAAAAGATGCACCGGTGCGTCTGACGAAACCAGCGTGATACAGGATATATTCCGCTCCTCGCCACTGACTCGCTACGCCCGTCGTTCGAGCTGCGGCAACGCGGAAATGGCCCCTTGAACGAGGCGAGTTTCACCCGGAATGCCGGAAGGCCTTGCTCGGAAGTAGAGGCTGCCGGCAAAGCCTGATCACCGCAGGCTCGCCCCGGTGGTTACGAAATCTGACCGCTCAAATCAGTAGTGGCGAAACCCGACCAGGACCGCAAAGATACCAGGCGTTTCCCCTGGCGGCTCCCTCGTGCGCTCTCCTGTTCCTGCCTTTCAGTTTACCGGTGTCATTCCGCTGTTATGGCCGCGTTTGTCTCATTCCACGCCTGACACTCAGTTCCGGGTAGGCAGTTCGCTCCAAGCTGGACTGTATGCACGAACCCCCGTTCAGTCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGAAAGACATGCAAAAGCACCACTGGCAGCAGCCACTGGTAATTGATTTAGAGGAGTTAGTCTTGAAGTCATGCGCCGGTTAAGGCTAAACTGAAAGGACAAGTTTTGGTGACTGCGCTCCTCCAAGCCAGTTACCTCGGTTCAAAGAGTTGGTAGCTCAGAGAACCTTCGAAAACCGCCCTGCAAGGCGGTTTCGTTTTTTAAAACAAAAGTGCGCAGACCAAAACGATCTCAAGATCATCTTATTAATCAGATAAAATATTTCAAGATTTCAGTGCAATTTATCTCTTCAAATGTAGCACCTGGTCAGCCCCCCATACGATATAAGTTGTAATTCTCATGTTTGACAGCTTATCATCGATGAATTA 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  NM:i:330    ms:i:12736  AS:i:12630  nn:i:0  tp:A:P  cm:i:1005   s1:i:5984   s2:i:99 de:f:0.0319 rl:i:0  ru:i:0
a91040bf-a1de-40b4-8e89-d3bc6c86dc6f    4   *   0   0   *   *   0   0   TTGTACTTCGTTCAGTTACGTATTGCTAAGGTTAACGCAAAGACACCAAAACCAGCACTATTTACTTTATTACGAGTGCAGCTATATATTAAGCTAACAAAAATGCCAATAAAGTAAATAAAAAAAGCATTACCTGACGAATATATAGCGCCCCGAATTTAAAGGTATGAGCGGAGCAGGTGCTATAGAAGTAGATAAAAATCTGACCTTCAGGATTCGCGGATTAAATAACATTCATGTCCTTGACTGCTTTATTCGTGTTGATGGAACCAGCTGATGGCGTTGTCGACTTTGGTAAAATAAATTCCCGAACAATTAAAAATACCAGCGCGGAATTGAGACGTTTAGCGTAGTCATGACCAAAGATCCGGGTGCGGCTAAACAGTTTAATATTTTAGGAGTTTTTTATTTACTTCTGCTTGTTTTAGCTAAAGCAATAGCTGCACTCGTAC    '''(45520///61////-+++++66..-.049;88('''(**+)(&%%(&'('%%&'(**+*&&&&')/00100000066656;87/.*)('().555788JC?99999?=:+++++2><;//.-*((&%%',,///((((()76422112331.---./41(4479:;<;89322/.../0022/----020//5=>B?<@76666:;>86689<=@?@>@BECJKDGCA:>@;<=>>>1000126+''&&%%%%$$##$$$('&&)8?@88777<77776889:<<<:;:8544489???>?AB@@:?>>===?AAAABB?=<98976-+**,,,,*+++++*+3456>?>>77778>>>60100/035:;</++'&%%%$$&,.+99?BCFE?4100123<;12:<=::65,*'%%%%&)*,445444334664455633+****&&&    rl:i:0  ru:i:0
139107cf-6440-4422-a923-6133588b2d94    0   pACBB_PL    4290    60  98S195M1D7M2I1M1I12M1D26M2I56M1D2M1I8M2D1M1D17M1D16M1D71M1I5M1D20M2D105M2D19M3D1M1D28M2D90M1I12M1D14M1D4M1D37M3D2M2D79M1I2M1D36M1I28M1I32M3D93M1D24M1I1M1I80M2I4M3I3M1I160M1I12M1I8M1I7M1D50M1D41M1D25M1D17M1I37M1D73M3I10M1D8M1D122M2I19M2D18M2D6M2I5M3D12M1D25M2D8M1I104M1I42M1I11M1D73M1I9M1I25M1I31M1I43M3D34M2D26M1D16M1D32M1D53M3D6M1I129M1D1M1D10M3D4M5I99M1I58M2D76M2I37M1I140M11S  *   0   0   GAACTTATCTAGACCCGACTGGGAAAGCGGGCGGTAGGCATAACGCAATCGTCTCGAGTGAGCCTTGAGACCTTTTACTTTACAAGTAAGGTCTCTTCCTTAGTGAGACGATTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATGTACTAGTTAACTTCAGGACCTACGGCTCAGGCAGCGTTGGGTCCTGGCCACCAGTGCGCATGATCATGCTCCTGTCGTTGAGGACCCGGCTAGGCTGGCGGGGTTGCCTTACTGGTTAGCAGAATGAATCACCGATACGCGGGCAACTTTATTAGGCGACTGCTGCTAAAAACGTCTGCGACCTGAGCAACAACGCTTGAATGGTCTTCGGTTTCCGTGTTTCGTAAAGTCTGGAAACGCGGAAGTCCCCTCGATAACACTGGTGCCCGCAACAGGGGGTGAACCAACCGGTGATATACGATACTATGACTGAGAGTCAACGCCATGAGCGGCCTCATTTCTTATTCTGAGTTACAACAGTCCGCACACTTTGCCGGTGACTCCTTCGGATGGGCAGGGCATGACTATCGTCGCCGCACTTATGACTGTCTTCTTTATCATGCAACTCGTAGGACAGGTGCCGGCAGCGCCCAACAGTCCCCCGGCCACGGGGCCTGCCTGTACCCACGCCGAAACAGCCTGCACCATTATGTTCCGGATCTGCATCATGGTGCTGCTGGCTACCCTGTGGAACACCTACATCTGTATTAACGAAGCGCTAACCGTTTTTATCAGGCTCTGGGAGGCAGAATAAATAAATCATATAATCGTTATTACCTCCACGGGAAGCCTGAGCAAACTGGCCTCAGGCATTTGAGAAGCACGTTACTGCTTCCGGTAGTCAATAAACCGGTAAACCAGCAATAGACATAAGCGGCTATTTAACGACCCTGCCCTGAACAAACTGACGGGTCAGATTTGCTTTCGAATTTCTGCCATTCATCACCCAGCCCATCACTTATTCAGGCGTAGCAACCAGGCGTTTAAGGGCACCAATAACTGCCTTAAAATTACGCCCCGCCCTGCCACTCATCGCAGTACTGTTGTAATTCATTAAGCATTCTGCCGACATGGAAGCCATCACAAACGGCATGATGAACTGAATCGCCAGCGGCATCGGCACACGCTGTCGCCTTGCGTATAATATTTGCCCATGGTGAAAATGGGGGCGAAGAAGTTGTCCATATTGGCCACGTTTAAATCAAACCCCTGAAAGTGCGACCTCACCCAGGGATTGGCTGAGACGAAAAACATATTCTCAATAAACCCTTTAGGGAAATAGGCCAGGTTTTCACCGTAACACGCCACATCTTGCGAATATATGTGTAGAAACTGCCGGAAATCGTCGTGGTATTCACTCCAGAGCGATGAAAACGTTTCCAGTTTGCTCATATGAAAGCGAACATAACAGGGTGAACACTATCCCATATCACCAGCTCACCGTCTTTCATTGCCATACGAACTCCGGATGAGCATTCATCAGGCGGGCAAGAATGTGAACAAGGCCGGATAAAACTTGTGCTTATTTTCTTTACGGTCTTTAAAAAAGGCCGTAATATCCAGCTGAACGGTCTGGTTATGGTACATTGAGCAACTGACTGAAATGCCTCAAAATGTTCTTTACGATGCCATTGGGATATATCAACGGTCGAATGCTCTCCAGTGATTTTTCCTCATTTTAGCTTCCTTAGCTCCTGAAAATCTCGATAACTCAAAAAATACGCCCGGTAGTGATCTTATTTCATTATGGTGAAAGTTGGAACCTCTTACGTGCCGATCAACGTCTCATTTTCGCCATAAAAGTTGGCCCGGGGCTTCAGTATCAGCAGGGACACGGATTTCGGCCCATGCGAAGTGATCTCCGTCACAGGTATTTATTCGGCGCAGGCCGTCAGGGTGATGCTACCAACTTACTGATTTAGTGTATGATGAATTGTTTGAGGTGCTCCAGTGGCTTCTGTTTCTATCAGCTGTCCCTCCTGTTCAGCTACTGACGGGAGTGGTGCGTAACGGCAAAAGCACCGCCGGACATCAGCGCTAGCCGGAGTGTATATGGCTTACTATGTTGGCACTGATGAGGGTGTCAGTGAAGTGCTTCATGTGGCAGGAGAAAAAAGGCTGCACCGAGTGCGTCAGCCAGAATATGTGATACAGGATATATTCCCGCTTCCTCGCTCACTGACTCGCCACGCTCAGTTAGTTCGACTGCGGCGAGCGGAAATGGCTTACGAACAGGGCGATTTCCTGGAAGATGCCAGGAAGATACTTAACAGAAGTGAGAGGGCCGCGGCAAAATCGTTTTCCATAGGCTCCGCCCCCTGACCAACATCACGAAATCTGACGCTCAATCAGTGGTGGCGAAACCCGACAGGACTATAAAGATACCGGGCGTTTCCCCTGGCTCCACTCGTGCACTCTCCTGTTCCTGCCTTTCGGTTTACCGGTGTCATTCCGCTGTTATGGCCGCGTTTGTCTCATTCCACGCCTGACACTCAGTTCCGGGTAGGCAGTTCGCTCCAAGCTGGACTGTATGCAGACACCCCGTTTTCGAACAAACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGAAAGACATGCAAAAGCACCACTGGCAGCAGCCACTGGTAATTGATTTAGAGGGGATTAGTCTTGAGGTAACGCAGCAGTTAAGGCTAAACTGAAAGGACAAGTTTTGGTGACTGCTCCTCCAAGCCAGTTACCTCGGTTCAAAGAGTTGGTAGCTCAGAGAACCTTCGAAAAACCGCCCTGCAAGGCGGTTTTTTTTCGTTTTCGAAACAGAAGATTACGCGCAGACCAAAAACGATCTCAAGAAGATCATCTTATTAATCAGATAAAATATTTCAAGATTTCAGTGCAATTTATCTCTTCAAATGTAGCACCTGAAGTCAGCCCCATACGATATAAGTTGTAATTCTCATGTTTGACAGCTTATCATCATTAAATTGAA   **++55<>AC;:::;;CBAAB5((.1-44255+++3)))(&&%&'12<>EGGF@=*&&&'+246699998:1111/////3//.--0521048;?CD=((((3234345558AIGGGMDKB6612334CDCA??@CCFC87.**))'(()+66*)))*89744)))))4/)('&&()(((((78889?87778<;:@<,+*''&'(,**+,--+'&&&'./7;=@A@BA@@7444*)-6667:<=@8854510100643.-+++,,-.....67?BC@E@A?;;;==;;0'&&%$$$%'(*+'())))..22-+*'&&&'+,/-))))-./+((('()*+0.+&%$$$%%&&%%%%')((()*.388;CFB@@@>@;8;?AD:99@@A?<:9887787')((()/,,&%%%$%%&&87:>::44))&&&&%%&798::.*)(*/-(((()888211//0,*))''((('(@?????BAB,,,,.?@@BCCBAB@A@DDB@@@:::::@@A;,&&%%%%&&''()-**''((((787--,'&&&'%&'():>>98111;=?>766-,3569:;;<<99899<;==>>>@==;<<=>BA21////38:;;:<<:66(''''*)))++/077;?AA<?CA?////02'&&&&&$$$$%'()*4/.236,'''&&')(()).56:CHC<;;>@=221/.1&%%%%&,---,,*,+)+--2>IOF::::;EHEDDB?999EGKJEEGJLFFKRFGFJACCBCCD>BE555568ECA>.733110116-,*(''''&%%%$%%%$$$%%''(+./7*))(+),**+1531011--+++59<@@@B@>55>00-,-/1-,&%%%%$$*002A>?D><;;...=EFFDB=<<<>BG854457AAAFAB@999:ADB...--2;;210('*.'&&)/42&%%%&&''''&*,*****,32688/++++-.8;<<7*()((**'&%%%%%&&%(),222355<,,,,,,2248<=CCEIHJCIGD?98C>A>=999;66678;=>=@=:9-::9<=J7>EJA?DD?;?><.-.-.99:;>EKMJJIKNKH{EG?GCEG74487<<AADDKIKDEE****+IA=;<===<=420-,-('(,,,89ABIB94522233744411)((**++(&&'(+,.6ADEEEEEKIHEFCHECA?>>>=EC@ABB?@8767212268<@><11111FCABAA???8231/1*))(2+'('&%$$$%$$%%%%(-231/15438@;;;;>=;9;7:?@FEABDC{LHHH@?@EEECDEGJ{?>><57645EDDC55>=<<@GB@?==>@AA?>?@;::97EBB?>>><>?+,6<<=?@A@CBCCC=<<<=A?=<<;;=?=>??AEAA@></./..../:=ABHK>><:5*)**:4-..-)'&%$$%%$%%$$$$%%%%'&%$&+012;<?>??BDC;:==@JMEDECEFGFMHIHFIF765222)('&&%%',,-/47DAE@?@@AE55544888@@CA>A?E{EA?9844234==99=?AHFEHK{EE:((''')3;D5??DEHFKFHFGEEGACAA?B=@DFDDBDAD<;;;;BA@77778E@??>:-)''*+&(**,+++,--.767689<<>CBCDDJZLJ{IEGHHDECBCBCECBD>>><=C@<<=7//-((&%###%&()*,,(()))045,(('&&(()4/+***,2?AA00000?<<??>BBBA@/ABGFB:>..-----7:0//((-...5667300+*+++3BBA?@AD<>=669<==GCHGIFF{BA>;::<=>C@A>==>>DCABEBDH620('&&%'067<=>=85'&%%''''&''(4+))))))-376)'$$('('&&&&'(''%%%%&&'&('&&&*/6:::<<=ABCFHE@/..(''&%%%%&''''''-//0//,,.+*,+,-58<?BCG;:::9311101..//004@'0<922;;?32248>?>?>@BAB216779744557?>>>=>==BEE@@?>?>>>@@>=;('''(2;;;ECGA@?>>?ABA;?????A??B@@>>?:9843)))'&'()))*77)&&%%),-../=?BDEBA@>>>>>?@?@>6>??55566:9;<>>?@AAA>000,++*()),B>:1/.-''&%%&%$$%&&'''())*)**-,--.//19:4+++1<;.....**-;=?DCCB@@@>>>=>>?@886((&&))%$$%%&'''*03677>@@???=:11///03@BBCDBCBC*)))*/--04<9789:+''(0921350,-...------((&&),.1200042899211963+)(''(:;A8***)))**.::<:9777:81,))(()((()+/);3222//..))'&*+4559?332223<?@A::32234KIJOGHFEHEE<1100012.-1700.//'*(%$%%%%&&$%%%%(**.4;B<:77766??:999;=?BGDFFDECBCGDBBBEDDDCIDA=;;778-,/>>=777>>99<=?D>@HBEB?111>>733333'&&())**,???76669;<=?11,,,,++&%%&&&%&&'()*(('&$$$%%'+,/CDDBCCDFDG{KGFHDFEGFFIFFGCCB?>?A?@B@B@?@@@B?ECECCEBBECCA?AAA@AEHKDRGGEGDDBB??;53334347:777.+*,-,,,/228:*(&%$$$#$%%((*++.000-)(+,0//00=<7@>=:755787;2122:7-,''0231/04:A>>=>::333---;<9,)(*+//3244367@<<<<=>?@EIB>=:<@>3333:DBEABC/.;9955<?6CE>?<9?@;7+**+,+,,2346E>@=>0/0//8889;A=B=ABFED:000123?A900CBDCCJJGM.-,,16>798:;7/-(&')7:4)((((../@@>@BA@?@EBDGDFPGBBAA@BA<;;>@544449=;;;89:;@><<<?@ADCCCFDGEDGIKJDFB;;::;AFA((('&&&('''%%%   NM:i:214    ms:i:4609   AS:i:4574   nn:i:0  tp:A:P  cm:i:315    s1:i:2073   s2:i:44 de:f:0.0601 rl:i:0  ru:i:0
dd79fada-ad4d-4023-b244-d7f54f9fd87a    0   pACBB_PL    4295    60  111S17M1I76M2I175M1I15M1I10M1D25M1D2M1D34M1I63M1D31M1D1M2D7M2I52M1I2M1I2M1D60M1I83M1I35M1I44M2I57M3I32M1I86M1I32M2D13M1I4M2I140M1D7M1I179M1D132M2D11M2D7M5D280M1D12M1I99M1D50M3D141M2D9M1D188M2D15M1D81M2I124M1D22M1D154M1I20M3I6M1D121M1I84M2I36M  *   0   0   TAATTCATCCTGAACTTATCTGAACCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATCGTCTCAGTGAGCCTTGAGACCTTCCACTTCTGCGTAAGGTCGCCTGGCAGATGAGACGATTCTCCTTACCACATCTGTGCGGTATTTCACACCGCATGTACTGGTTAACTTCAGGACCTGCGGCTCGGGCAGCGTTGGGTCACGGCCCCACGGGTGCGCATGATCGTGCTCCTGTCGTTGAGGACCCGGCTAGGCTGGCGAGGTTGCCTTACTGGTTAGCAGAATGAATCACCGATACGCGAGCGAACGTGAAGCGACTGCTGCTGCAAAACGTCTGCGACCTGAGCAACAACATGAATGGTCTTCGGTTTCCGTGTTTCGTAAAAGTCTGGAAACGCGGGAAGTCCCCACGATATACTGAAGTTGCCCGCAACGAAGTGGAGCCAACCGGTGATACCACGATACTATGAGCTGAGAGTCAACGCCGCAGGCGGCCTCATTTCTTATTCTGAGTTACAACAGTCCGCACCGCTGCCGGTAGCTCCTTCCGGTGGGCGCGGGGCATAATCGTCGCCCCGCACTTATGACTGTCTTCTTTATCATGCAACTCGTAGGACAGGTGCCGGCTTGTCGCCAACAGTCCCCCGGCCACGGGGCCTGCCACCATACCCACGCCGAAACAAGCGCCCTGCATTCATTATGTTCCGGATCTGCATCGCAGGATGCTGCTGGCTACCCTGTGGAACACCTACATCTGTATTAACGAAGCGCTAACCGTTTTTTATCAGGCTCTGGGAGGCAGAATAAATGATCGCTATCGTCAATTATTACCTCCACGGGGAGAGCCTGAGCAAACTGCAACCTCAGGCATTTGAGAAGCACACGGTCACACTGCTTCCGGTAGTCAATAAACCGGTAAAAAACCAGCAATAGACATAAGCGGCTATTTAACTGACCCTGCCCTGAACCGACGACCGGGTCGAATTTGCTTTCGAATTTCTGCCATTCATCCGCTTATTATCACTTATTCAGGCGTAGCAACCAGGCGTTTAAGGGCACCAATAACTGCCTTAAAAATTACGCCCGCTCCCACTGCCACTCATCGCAGTACTGTTGTAATTCGTTAAGCATTCTGCCGACATGGAAGCCATCACAAACGGCATGATGAACCTGAATCGCCAGCGGCATCAGCACCTTGTCGCCTTGCGTATAATATTTGCCCATGGTGAAAATGGGGCGAAAGAAGTTGTCCATATTGGCCACGTTTAAATCAAAACTGGTGAAACTCACCCAGGGATTGGCTGAGACGAAAAACATATTCTCAATAAACCCTTTAGGGAAATAGGCGAGGTTTTCACCGTAACACGCCACATCTTGCGAATATATGTGTAGAAACTGCCGGAAATCGTCGTGGTATTCCTCCAGAGCGATGAAAACGTTTCAGTTTGCTCATGGAAAACGGTGTAACAAGGGTGAACACTATCCCATATCACCAGCTCACCGTCTTTCATTGCCATACGGAACTCCGGATGAGCATTCATCAGGCGGGCAAATGTGAATGAGCCGGATTTGTGCTTATTTTTCTTTACGGTCTTTAAAAAGGCCGTAATATCCAGCTGAACGGTCTGGTTATAGGTACATTGAACAACTGACTGAAATGCCTCAAAATGTTCTTTACGATGCCATTGGGATATATCAACGGTGGTATATCCAGTGATTTTTTTCTCCATTTTAGCTTCCTTAGCTCCTGAAAATCTCGATAACTCAAAAAATACGCCCGGTAGTGATCTTATTTCATTATGGTGAAAGTTGGAACCTCTTACGTGCCGATCAACGTCTCATTTTCGCCAAAGTTGGCCCAGGGGCTTCCCGGTATCAGCAGGGACACCAGGATTTATTTATTCTGCGAAGTGATCTTCCGTCACGGGTATTTATTCGGCGCAAAGTGCGTCGGGTGATGCACCAACTTGCTGATTTAGTGTATGATGGTGTTTTTGAGGTGCTCCAGTGGCTGTTTCTATCAGCTGTCCCTCCTGTTCAGCTACTGACGGGGTGGTGCGTAACGGCAAAAGCACCGCCGGACATCAGCGCTAGCGGAGTGTATACTGGCTTACTATGTTGGCACTGATGAGGGTGTCAGTGAAGTGCTTCATGGCAGGAGAAAAAGGCTGCACCGGTGCGTCGGCAGAATATGTGATACAGGATATATTCCGCTTCCTCGCTCACTGACTCGCTACGCTCGGTCGTTCGACTGCGGCGAGCGGAAATGGCTTACGAACGGGGCGGAGATTTCCTGGAAGATGCCAGGAAGATACTTAACAGGGAAGTGAGAGGGCCGCGGCAAAGCCGTTTCCATAGGCTCCGCCCCCTGACAAGCATCACGAAATCTGACGCTCAAATCAGTGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTTCCCCCTGGCGGCTCCCTCGTGCGCTCTCCTGTTCCTGCCTTTCGGTTTACCGGTGTCATTCCGCTGTTATGGCCGCGTTTGTCTCATTCCACGCCTGACACTCAGTTCCGGGTGAGCAGTTCGCTTAAGCTGGACTGTATGCACGAACCCCCGTTCAGTCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGAAAGACATGCAAAAGCACCACTGGCAGCAGCCACTGGTAATTGATTTAGAGGAGTTAGTCTTGAAGTCATGCGCCGGTTAAGGCTAAACTGAAAGGGACAAGTTTTGGTGACTGCATAGCTCCTCAAGCCAGTTACCTCGGTTCAAAGAGTTGGTAGCTCAGGGAACCTTCGAAAAACCGCCCTGCAAGGCGGTTTTTTCGTTTTCAGAGCAAGAGATTACGCGCAGACCAAAACGATCTCAAGAGAGATCATCTTATTAATCAGATAAAATATTTCAAGATTTCAGTGCAATTTATCTCTTCAAATGTAGCACCTGAAGTCAGCCCCATACACGATATAAGTTGTAATTCTCATGTTTGACAGCTTA 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  NM:i:104    ms:i:5236   AS:i:5216   nn:i:0  tp:A:P  cm:i:376    s1:i:2394   s2:i:0  de:f:0.0281 SA:Z:pACBB_PL,1,+,1S73M2947S,4,2;   rl:i:0  ru:i:0
dd79fada-ad4d-4023-b244-d7f54f9fd87a    2048    pACBB_PL    1   4   1H73M2947H  *   0   0   AATTCATCCTGAACTTATCTGAACCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATCGTCTCAGTGAGCCT   =>???655>>?@@@?????5111111;<<>>>>60++++,34799;:89<???<<<<<=<<<<=/////;865   NM:i:2  ms:i:134    AS:i:134    nn:i:0  tp:A:P  cm:i:6  s1:i:43 s2:i:0  de:f:0.0274 SA:Z:pACBB_PL,4295,+,111S2910M1D,60,104;    rl:i:0  ru:i:0
b01d11eb-419c-4cb1-bc5e-7752a1591d1c    16  pACBB_PL    3174    60  523S32M1D77M1I3M3I2M1D4M1D9M1I129M3D13M1D427M1D21M2D23M1D21M1D58M1D176M2I18M1D7M1D502M1I4M1D34M3I41M2I10M1D3M1D7M2I2M4D18M2D2M2D32M1D4M2I2M3I330M1I38M2D63M1D3M1I188M1D334M1I229M2I77M1I4M1D3M1I85M2I18M3D22M1D50M1D73M4I58M2D22M3D15M1D6M2I44M1D45M3I24M1I6M1D37M1D92M1D71M1D14M1D39M1I41M2D2M1I60M2I3M1I11M1D8M1I2M2I70M1D2M1D38M1D2M1D75M4S  *   0   0   TGGCGCCGGCTGGTTCGTATCGGTTTGTCAACCGGCAGCGCAACGGTACCGTCAGACAGCGCGGTGATTTCGAAGTCACCCAGCAGCATACGGTAGTAACCCGGCGCAGAGGTACGAACCTGCGGTGCCGCCGCAGCACGCGTGGGTAACGAAAACCATCTGCGCCGTGGTGCACAGACCTGCCAGCAGCAGAGAGGTTTTGGTCAGGTTACGCATCTGCTACTTTCCTGTGTGACTCTAGATTAAACAAAAATTATTTGTAGAGGCTGTTTCGTCCTCACGGACTCATCAGACCGGAAAGCACATCCGGTGACAGCTAAAGGCTAGCATTGTACCTAGGACTGAGCTAGCCATAAAAGGCCTTTTGAGACGATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGTCTACTTTCTAGTACATGCGGTAACGCACAGATGCGTAAGGAGAATCGTCTCACTTTGTGAAGAGACCTTACTTGTAAAGTGTAGCACTAAGGCGAAGATACAAAGAAGCCACGTTCGCTTTCCAGTCGGGTCTAGCAGTAGACCTTATCGAAAGCGGGCAGTGAGCGCAACGCAATCGTCTCGGCGGGCTCTGAGCATTTGCTCAAGCGGCCTTGGGCAATTGATTCAGTCAAGCTTAAAAGCCCGAGGCAAGCGCGGCTTAATTTCGCTCGCTCCGATCATTCTAAAAATTAGAAACACATTGAAAAACAATACCTTGAAGTCGGTTTTCAGACCTTGGCACAGCTGTTGCACTTTGTCCTGCGCTTTTCTAGAGAAAGAGGAGAAATAGCAGATGCGTAAAGGCGAAGAGCTGTTCACTGGTGTCGTCCCTATTCTGGTGGAACTGGATGGTGATGTCAACGGTCATAAGTTTTCCGTGCGTGGCGAGGGTGAAGGTGACGCAACTAATGGTAAACTGACGCTGAAGTTCATCTGTACTACTGGTAAACTGCCGGTACCTTGGCCGACTCTGGTAACGACGCTGACTTATGGTGTTCAGTGCTTTGCTCGTTATCCGGACCATATGAAGCAGCATGACTTCTTCAAGTCCGCCATGCCGGAAGGCTATGTGCAGGAACGCACGATTTCCTTTAAGGATGACGGCACGTACAAAACGCGTGCGGAAGTGAAATTTGAAGGCGATACCCTGGTAAACCGCATTGAGCTGAAAGGCATTGACTTTAAAGAAGACGGCAATATCCTGGGCCATAAGCTGAATACAATTTTAACAGCCACCGTTTACATCACCGCCGATAAACAAAAAATGGCATTAAAGCGAATTTAAAATTCGCCACAACGTGGAGGATGGCAGCGTGCAGCTGGCTGATCACTACCAGCAAACACTCCAATCGGTGATGGTCCTGTTCTGCCGCTAGACAATCACTATCTGAGCACGCAAAGCGTTCTGTCTAAAGATCCGAACGAGAAACGCGATCATATGGTTCTGCTGGAGTTCGTAACCGCAGCGGGCATCACGCATGGTATGGATGAACTGTACAAATAACTAATTTTCGGAAAAAAGGCCTCCCAAATCGGGGGCCTTTTTATTGATAACAAAATCACTCACTGAGACGATTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATGTACTAGTCACTTAGTGAGACGATTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATGTACTAGTTAACTTCAGGACCTGCAGCTCGGGCAGCGTTGGGTCCTGGCCACGGGTGCGCATGATCGTGCTCCTGTCGTTGAGGACCCGGCTAGGCTGGCGGGGTTGCCTTACTGGTTAGCAGAATGAATCACCGATACGCGAGCGAACGTGAAGCGACTGCTGCTGCAAAACGTCTGCGACCTGAGCAACAACATGAATGGTCTTCGGTTTCCGTGTTTCGTAAAGTCTGGAAACGCGGAAGTCCCCTACGTGCTGCTGAAGTTGCCCGCAACAGAGAGTGGAACCAACCGGTGATACCACGATACTATGACTGAGAGTCAACGCCATGAGCGGCCTCATTTCTTATTCTGAGTTACAACAGTCCGCACCAGCTGCCGGTAGCTCCTTCCGGTGGGCGCGGGGCATGACTATTATCGTCGCCGCACTTATGACTGTCTTCTTTATCATGCAACCTTCGTAGGACAGAGCGGCAGCCTGCCAGTCCCCCGGCCACGGGCTCACCATACCCACGCCGAAACAAGCGCCTGGCAAATTCAATAGCGGTCTCGATCTGCATCGCAGGATGCTGCTGGCTACCCTGTGGAACACCTACATCTGTATTAACGAAGCGCTAACCGTTTTTATCAGGCTCTGGGAGGCAGAATAAATGATCATATCGTCAATTATTACCTCCACGGGGAGAGCCTGAGCAAACTGGCCTCAGGCATTTGAGAAGCACACGGTCACACTGCTTCCGGTAGTCAATAAACCGGTAAACCAGCAATAGACATAAGCGGCTATTTAACGACCCTGCCCTGAACTGACGACCGGGTCGAATTTGCTTTCGAATTTCTGCCATTCATCCGCTTATTATCACTTATTCAGGCGTAGCAACCAGGCGTTTAAGGGCACCAATAACTGCCTTAAAAAATACGCCCCGCCCTGCCACTCATCGCAGTACTGTTGTAATTCATTAAGCATTCTGCCGACATGGTGCTCATCACAAACGGCATGATGAACCTGAATCGCCAGCGGCATCAGCACCTTGTCGCCTTGCGTATAATATTTGCCCATGGTGAAAATGGGGGCGAAGAAGTTGTCCATATTGGCCACGTTTAAATCAAAACTGGTGAAACTCACCCAGGGATTGGCTGAGACGAAAAACATATTCTCAATAAACCCTTTAGGAAATAGGCCAGGTTTTCACCGTAACACGCCACATCTTGCGAATATATGTGTAGAAACTGCCGGAAATCGTCGTGGTATTCACTCCAGAGCGATGAAAACGTTTCAGTTTGCTCATGGAAAACGGTGTAACAAGGGTGAACACTATCCCATATCACCAGCTCACTGTCTTTCATTGCCATACGGAACTCCGGATGAGCATTCATCAGGCGGGCAAGAATGTGAATAAAGGCCGGATAAAACTTGTGCTTATTTTTCTTTACGGTCTTTAAAAAGGCCGTAATATCCAGCTGAACGGTCTGGTTATAGGTACATTGAGCAACTGACTGAAATGCTCTCAAAATGTTCTTTACGATGCCATTGGGATATATCAACGGTGGTATATCCAGTGATTTTTTTCTCCATTTTAGCTTCCTTAGCTCCTGAAAATCTCGATAACTCAAAAAATACGCCCGGTAGTGATCTTATTTCATTATGGTGAAAGTTGGAACCTCTTACGTGCCGATCAACGTCTCATTTTCGCCAAAAGTTGGCCCAGGGCTTCCCGGTATCAACAGGGACACCAGGGGATTTATTTATTCTGCGAAGTGATCTTCCGTCACAGGTATTTATTCGGCGCAAAGTGCGTCGGGTGATGCTGCCAATCGCATCATTTTAGTGTATGATGGTGTTTTTGAGGTGCTCCAGTGGCTTCTGTTTCTATCAGCTGTCCCTCCTGTTCAGCTACTGACGGGGTGGTATGCGTAACGGCAAAAGCACCGGACATCAGCGCTAGCGGAGGTATACTGGCTTACTATGTTGGCACTGATGAGGGTGTCAGTGAAGTGCCTATGTGGCAGGAGAAAAAAGGCTGCACCGGTGCGTCAGCAGAATATGTGATACAGGATATATTCCGCTTCCTCGCTCTCTCACTGACTCGCTACGCTCGGTCGTTCGACTGCGGCGAGCGGAAATGGCTGGCGAACGGTGGAGATTTCCTGGAAGATGGAAAGATACTTAACAGGAAGTGCGAGAGGGCCGCGGCAAAGCCGTTTTTCCATAGGCTCCGCCCCCCGACAAGCATCACGAAATCTGACGCTCAAATCAGTGGTGGCGAAACCTGCCGACAGGACGAGGAAGATGCCCGTGCGTTTCCCTGGCGGCTCCCTCGTGCGCTCTCCTGTTCCTGCCTTCGGTTTACCGGTGTCATTCCGCTGTTATGGCCGCGTTTGTCTCATTCCACGCCTGACACTCAGTTCCGGGTAGGCAGTTCGCTCCAAGCTGACTGTATGCACGAACCCCCCGTTCAGTCTGACCGCTGCGCCTTATCCGGTAACTACCGTCTTGAGTCCAACCGGAAAGACATGCAAAGCACCACTGGCAGCAGCCACTGGTAATTGATTTAGAGGGAGTTAGTCTGGAAGTCATGCGCCGGTTAAGGCTAAACTGAGCGACAAGTTTTGGTGACTGCGCTCCTCCAAGCCAGTTACCTCGGTTCAAAGAGTTGGTAGCCTTCATGAGAACCTTCGAAAACCGCTCCGGTGCAAGGCGGTTTTTTCGTTTTCAGAGCAAGAGATTACGCGCAGACCAAAACGATCTCAAGAAGATCATCCACAATCAGATAAAATATTTCAAGATTTCAGTGCAATTTACTTTCAAATGTAGCACCTGAAGTCAGCCCCATACGATATAAGTTGTAATTCTCATGTTTGACAGCTTATCATCGATGAATT &2348333,*)'&'''((*('(**56<>:::9:?@76DDA@?===88889@@@BABA?@>==@@CCB<444@????=<;;:8:DBA@?777<=>?>?=;;>===;;<;2((('(+:77553//1.-//0><:54*)))0///,--'''')31:::>99888<<76644<<::::;;*****?>A????@>52=<>?>@<<==?=77><;;;;<>>;54445:89789::@A@A=<=;:;;=;<3@@8429:91.,'***./55//)((('(*,//1//0/----.28?::897778855446><=;;:9:::;::98<<=;:100;==>>;755556;<=<<<;455555556638989<?7:75577799;99999:88779::::::;;;=<:;44>>><6444311+,011:9974%&'(((&'%%%%%(**+,89999<<==<AB@@?<::9:;><;<;=>>@:::88932876:>;;7:<87(''&$$%')**&%%&&%$###$$$$%'(**///--.-+)/3;@>?>?>@>>=>=;<;<<<-+'&$$#$%')+,-0)9988888;8889:>>;;;;;;75)('&&'&&&*+-/1:;<<B>>=;9987888:;775..*))'''&&&$$$$$$%'(/0468:5'''''---::AA?>::::7550/00.---+*+++4::=?=<==4.,<?@>>>>;<;;;=:/0/..???@@:9885588878:;<?BDE<;:755./66444:9<:;:;;:883,-;;)((((;?CDC855568ACA<:7778=/++)(),,9@<865577;;999:;=<;<=@BB=<;;<;<;87779=<>AHDEA98778@CAABBB=<<==????=>><<=<<==;===<@?>??>==><<=>;:889::@:9999;;;;=<==>@>==>>?>>766:667747766<=;;87766779?0//0098878>==<98889<:9644677>@?A;:999<==66687888765<===9:=>?BCB????@ADFC?=;<;>==>D???<<98;;:878<<<<>=@>>>?<====CD?@7325)&'''(559>BCGBA>==<=>??>B=CDA?>><===BBA@@?====<?>@AEC@>=:9??B@EB@@?>@PGFEDIEB@B>>>@>===98794A<<8;??@@ACC@@AFC<982+))))()-79?EDFFEDC@@A?BBA=>AA=ACCEGJC@;::<CGF:::::;<GBDIDD-,,,,,?ACDGGGGEH:::::EGGEMHC@B;50'')))34*986779<(66KED?@CCG>?>>>@?A@AGMHIC@???BGK{HIA@>@222--/B>=988978@A:<?@600009:=DGIDGA2000/-++,,-*(((&%%%&4@BBHFIHFJGHDF{I9998?CFEKIHKIF>?>:::=<43'''''9/./CB?C@?;*)((((+.013212;=CDBFDCBBBCBDGEEFFGHCAAAACDRJJIOKREFDGHFCISKHI{IJHFDECEEAA9896+)1/..068==210/-*(()+7DB>ABB63325BA76777CLFGE8:7:1.,,++,/8888KFCA000//.0--8<<<?87888<<IHMEPCFPGJGIBCC<<<;))))*::>EFJMLGJ{IKS{{HKFCBDB<;54599@@AEBB;;;<<@@ADGIGKKMB;:312228?C767555999:@@@??D@?>>?CCEGGED<<;<<;?;7222DEDFBD==<<<EBADAA>@==>/////BABBDJGLFFHEEGB@?@>BFBA>?327677<>>?BDGFGDKKGHPIFDABEIFHHG{D@IFFD@=3/-)(()*258EGHEEICCBAAFA99:2++++,66:::::=<<>>=<=::>CC@>>7766;:::;<=@A>====@@=;.----B776555:BDA@;;;BAB>>@?===8755689<==::88;=@@???@?@A>>===>?A???>>@?>>>>?@C7546556::9778:+****33;;;=====1---/C=@>44444@@A@?ABA?;//.4/../,,,+,-,((%%%%$%%''))*.345>=>@?B99?>A@:4,,,../.(((,097-,,,ABA@@???<;;;;>@@B====:=5,+++6>?DEFC>--00..050,,,-/))****-,)(&''&'..3200//037<=<4+)***''))*4@??==+'>>??<BCBBBA@534/.---010+(('&&%$$&'''(-@ECD@DCCFFGEFG>:::9<E?=<:/.../BCJFEFFIDCBA@443335ACEF@>6656:86778:889;>;>><<?4/0..//,.AAFCEDEBFIBB77:{EIPQHGFGH>>>??MIB>:::;C>:32;?B=7*(&%&((()3556=88=>>FEIH{MJFDMD<<7744449;;<<.--,,/0067>A:;<<67776JCSDFGGFGCDG?87;<777?988>>?>K?HEKIKGEFGDCBAAA?<>?@B65556CKB{O{EFAERGQ;+B=:BEHMDJLEBAAADBD854434998877;;===<;/,,-?<A>3355464444:?>43333FGGC?@?@76777C..--/02//55:@;++,:>984778?@@BAHCFEEDDIKIMLGPIFGII{EC?@CC?4343346511221111.-+'''&&&&&0:<A>7EGEEEJENDCCBG76667HJFHFEGGFJJHIJGSUFCA?==BCD333333@CBCEFTNRGAFBFA66556;;=>:AB?>>::;;;@AA0///06@BDDEF=<<<----,--.>ADBFG777664777877776,,+++++<:>?<>?>>AB@??@>CB?><<<<>@@BBDF@FA<D8*)(%%(*9<<:8554111///10---.==EEFDEIFHEC<87HGKGIOGGIHA<<<441/-.---0078EEDFEB@65556;;;<;>@@@BNDB@@?45563677:;;<7775*))))////{JEFEDBFFFHFADB:853;<EA<<<<;BCB=000011<;443/''''(<4/0?:::::ACDIEJHDDB>>>?@CKFMGHEHEJKGDEAACA66/,-4((-./;=@EDEHBAAJDEDE{ECDEBBECBEB@@ACGF<==66;'''')-*451//B?@A54445{HKEDDDB>>>DB?<<>>77778?=45;>?B@--,.-+*++-++++,D@>)(('(6<<<431244@46AACABEDBD@B@AD@@AGOQDHDCE@><<<==CDAB?:899DFQ{GQC??<?D:IHDBAB?<55<<A=<===>23:655667=>DB.-,,+*-44==<=?>CDCBEDDEE89999:@EBC9:---.=@DEDEEE@>?AB<<?0**+3236:ECAFBGEFEDFEG@I;DDECA005:57644000155558;;40000001LK{T{HAMHHFA?A2+,,DHGFM{BE@@?;20*))****+CCD?DEAEIEF@54667@CCA;;;<JF2*'??><885532268:>40/,,,(''&&'&&&'*73=@3111=<BACDCCC;@6:?>;75&&&&&((((.0:851.-.00:,,,,=;;=<=84=A00000;>?@BI99899@8/(54/)(('(((*357756877A?@6+(447BA@>740744344++.---00334450/.,))))*-7553('')2?DB;:5666677788:86******38??BAD@AADFKHIE88889?>??@A@@??@CKBAAAABBB@BCBAB@@@A?>=:99:;>:::;86446.+*('')+(((()1---:;;<98898>>??<<4438664457988878999;22,)''''%&&$$&&'.=?@;8++/./1?<9&&&&')57=?<=88888.-31,*((((11,&))((*++-..322666BDD;9/+++-1565../8/-))))ABDD@>:98)))>:::222A??>?ABDECDDAB::9443202&%&%%''(/.-'&&&%%%'(-)&%&&&&'))610.,)69::>B@?@A<;;;;>IBBA78600889D?>><;;()<@>>CC@??=7111156AD<33334>?CBAFCI{FFDDCCHDB>=<>?EHE<<<<;EB@?CDFD=@<97797677<@@CC=<5+))(''''+43224446;;=><<:820002:=<1'&&'''(''&%%$$&&'(*6:66743*'%&&'*44449AA?9,,()-.;@AAB@=41,*&*+/66879999<==>EFGF@?>>?LHDECCB?9,+++3)*AB>??@>643430//.....;<@BCADSEEEBKC>AB4103222750-,--)'''*(++++.*))):333444;;>@@>0++******,+/5;><;998-'&&%%$&&&(;<;9.,++.23/777)((&&&&&&(4489====1/,,,-179:;ADDBA@AAEGEJFIE??;<BCCAA@@AAEBB@?=567777?>8422*(&&&&&,,,.5?>?=<>>>666==>>>?>==<==???AB??=889BCHE>>=987/(((()??A?@?:9889;>ADBE??@@??DC222/*****0ABE@A@B44444@BB@@BBAFGKIGEB: NM:i:147    ms:i:7261   AS:i:7228   nn:i:0  tp:A:P  cm:i:594    s1:i:3402   s2:i:63 de:f:0.0276 rl:i:0  ru:i:0

3.5 Convert to bam files

bamfile = merged_samfile.replace("sam", "bam")

exestr = f"samtools view -bS -e 'mapq >= 25' {merged_samfile} > {bamfile}"
#print(exestr)
!udocker --allow-root run -v=/content/drive/MyDrive/design_build:/design_build gatk bash -c "{exestr}"
 
 ****************************************************************************** 
 *                                                                            * 
 *               STARTING 43258f40-d39f-316f-8a89-594ce69950cc                * 
 *                                                                            * 
 ****************************************************************************** 
 executing: bash

3.6 Sorting and Indexing

sortedfile = bamfile.replace(".bam", "-sort.bam").replace("/bam/", "/sort/")

exestr1 = f"samtools sort {bamfile} -o {sortedfile}"
print(exestr1)
!udocker --allow-root run -v=/content/drive/MyDrive/design_build:/design_build gatk bash -c "{exestr1}"

exestr2 = f"samtools index {sortedfile}"
print(exestr2)
!udocker --allow-root run -v=/content/drive/MyDrive/design_build:/design_build gatk bash -c "{exestr2}"
samtools sort /design_build/bam/FAT01036_pass_barcode01_5eb57f2c_2b75e8f5_merged.bam -o /design_build/sort/FAT01036_pass_barcode01_5eb57f2c_2b75e8f5_merged-sort.bam
 
 ****************************************************************************** 
 *                                                                            * 
 *               STARTING 43258f40-d39f-316f-8a89-594ce69950cc                * 
 *                                                                            * 
 ****************************************************************************** 
 executing: bash
samtools index /design_build/sort/FAT01036_pass_barcode01_5eb57f2c_2b75e8f5_merged-sort.bam
 
 ****************************************************************************** 
 *                                                                            * 
 *               STARTING 43258f40-d39f-316f-8a89-594ce69950cc                * 
 *                                                                            * 
 ****************************************************************************** 
 executing: bash
!ls -al sort
total 29746
-rw------- 1 root root 30458496 Jun  5 03:21 FAT01036_pass_barcode01_5eb57f2c_2b75e8f5_merged-sort.bam
-rw------- 1 root root       96 Jun  5 03:21 FAT01036_pass_barcode01_5eb57f2c_2b75e8f5_merged-sort.bam.bai

3.6.1 Extracting consensus

  • The lastest version of samtools_V1.16 allows you to generate a consensus from a SAM, BAM or CRAM file based on the contents of the alignment records.
consensusfile = sortedfile.replace("-sort", "-consensus").replace("bam", "fasta")

exestr1 = f"samtools consensus {sortedfile} -o {consensusfile}"
print(exestr1)
#!udocker --allow-root run -v=/content/drive/MyDrive/design_build:/design_build gatk bash -c "{exestr1}"
!udocker --allow-root run -v=/content/drive/MyDrive/design_build:/design_build gatk bash -c "samtools --help"
samtools consensus /design_build/sort/FAT01036_pass_barcode01_5eb57f2c_2b75e8f5_merged-sort.bam -o /design_build/sort/FAT01036_pass_barcode01_5eb57f2c_2b75e8f5_merged-consensus.fasta
 
 ****************************************************************************** 
 *                                                                            * 
 *               STARTING 43258f40-d39f-316f-8a89-594ce69950cc                * 
 *                                                                            * 
 ****************************************************************************** 
 executing: bash

Program: samtools (Tools for alignments in the SAM format)
Version: 1.13 (using htslib 1.13+ds)

Usage:   samtools <command> [options]

Commands:
  -- Indexing
     dict           create a sequence dictionary file
     faidx          index/extract FASTA
     fqidx          index/extract FASTQ
     index          index alignment

  -- Editing
     calmd          recalculate MD/NM tags and '=' bases
     fixmate        fix mate information
     reheader       replace BAM header
     targetcut      cut fosmid regions (for fosmid pool only)
     addreplacerg   adds or replaces RG tags
     markdup        mark duplicates
     ampliconclip   clip oligos from the end of reads

  -- File operations
     collate        shuffle and group alignments by name
     cat            concatenate BAMs
     merge          merge sorted alignments
     mpileup        multi-way pileup
     sort           sort alignment file
     split          splits a file by read group
     quickcheck     quickly check if SAM/BAM/CRAM file appears intact
     fastq          converts a BAM to a FASTQ
     fasta          converts a BAM to a FASTA
     import         Converts FASTA or FASTQ files to SAM/BAM/CRAM

  -- Statistics
     bedcov         read depth per BED region
     coverage       alignment depth and percent coverage
     depth          compute the depth
     flagstat       simple stats
     idxstats       BAM index stats
     phase          phase heterozygotes
     stats          generate stats (former bamcheck)
     ampliconstats  generate amplicon specific stats

  -- Viewing
     flags          explain BAM flags
     tview          text alignment viewer
     view           SAM<->BAM<->CRAM conversion
     depad          convert padded BAM to unpadded BAM

  -- Misc
     help [cmd]     display this help message or help for [cmd]
     version        detailed version information
from Bio import SeqIO

cons_seq = SeqIO.read(consensusfile.replace("/design_build/", ""), "fasta")
print(cons_seq)
ID: pACBB_PL
Name: pACBB_PL
Description: pACBB_PL
Number of features: 0
Seq('AATTCATCCTGAACTTATCTAGACCCGACTGGAAAGCGGGCAGTGAGCGCAACG...ATG')

3.7 Statistics

logfile =  "/design_build/info/mapping_stats.log"

exestr1 = f"samtools flagstat {sortedfile} >> {logfile}"
print(exestr1)
!udocker --allow-root run -v=/content/drive/MyDrive/design_build:/design_build gatk bash -c "{exestr1}"
samtools flagstat /design_build/sort/FAT01036_pass_barcode01_5eb57f2c_2b75e8f5_merged-sort.bam >> /design_build/info/mapping_stats.log
 
 ****************************************************************************** 
 *                                                                            * 
 *               STARTING 43258f40-d39f-316f-8a89-594ce69950cc                * 
 *                                                                            * 
 ****************************************************************************** 
 executing: bash
!cat info/mapping_stats.log
9167 + 0 in total (QC-passed reads + QC-failed reads)
7370 + 0 primary
0 + 0 secondary
1797 + 0 supplementary
0 + 0 duplicates
0 + 0 primary duplicates
9167 + 0 mapped (100.00% : N/A)
7370 + 0 primary mapped (100.00% : N/A)
0 + 0 paired in sequencing
0 + 0 read1
0 + 0 read2
0 + 0 properly paired (N/A : N/A)
0 + 0 with itself and mate mapped
0 + 0 singletons (N/A : N/A)
0 + 0 with mate mapped to a different chr
0 + 0 with mate mapped to a different chr (mapQ>=5)

3.8 Create bed file

!pip install pyfaidx
Collecting pyfaidx
  Downloading pyfaidx-0.8.1.1-py3-none-any.whl (28 kB)
Requirement already satisfied: setuptools in /usr/local/lib/python3.10/dist-packages (from pyfaidx) (67.7.2)
Requirement already satisfied: importlib-metadata in /usr/local/lib/python3.10/dist-packages (from pyfaidx) (7.1.0)
Requirement already satisfied: zipp>=0.5 in /usr/local/lib/python3.10/dist-packages (from importlib-metadata->pyfaidx) (3.19.0)
Installing collected packages: pyfaidx
Successfully installed pyfaidx-0.8.1.1
!faidx --transform bed pACBB_PL.fasta > pACBB_PL.bed
cat pACBB_PL.bed
pACBB_PL    0   7214

3.9 Mapped reads

mapped_readfile =  "/design_build/info/mapped_reads.txt"
bedfile = "/design_build/pACBB_PL.bed"

exestr1 = f"bedtools intersect -a {bedfile} -b {sortedfile} -wo > {mapped_readfile}"
print(exestr1)
!udocker --allow-root run -v=/content/drive/MyDrive/design_build:/design_build gatk bash -c "{exestr1}"
bedtools intersect -a /design_build/pACBB_PL.bed -b /design_build/sort/FAT01036_pass_barcode01_5eb57f2c_2b75e8f5_merged-sort.bam -wo > /design_build/info/mapped_reads.txt
 
 ****************************************************************************** 
 *                                                                            * 
 *               STARTING 43258f40-d39f-316f-8a89-594ce69950cc                * 
 *                                                                            * 
 ****************************************************************************** 
 executing: bash
!ls -alh info
total 716K
-rw------- 1 root root 715K Jun  5 02:54 mapped_reads.txt
-rw------- 1 root root  466 Jun  5 02:43 mapping_stats.log
!tail -n 30 info/mapped_reads.txt
pACBB_PL    0   7214    pACBB_PL    6294    7208    0dc5bff4-7df0-499e-9d95-dd2baffa55c3    60  -   914
pACBB_PL    0   7214    pACBB_PL    6301    6523    a2b4db66-56e1-4686-9ad8-72293ff0d6b7    41  +   222
pACBB_PL    0   7214    pACBB_PL    6303    7214    df6cad8b-0549-4ffe-a3b3-ecff002e99e4    60  -   911
pACBB_PL    0   7214    pACBB_PL    6317    7214    26d14fbc-e519-4a72-84f8-d76ef205922c    60  +   897
pACBB_PL    0   7214    pACBB_PL    6382    7214    b1f0d4d9-33f4-4158-8575-e84b372d1018    60  -   832
pACBB_PL    0   7214    pACBB_PL    6395    7208    30b29f15-7289-4db9-b132-33eeccb06226    60  -   813
pACBB_PL    0   7214    pACBB_PL    6396    7214    53f8526e-a6b1-41fb-bd47-51b1df2f98f4    60  -   818
pACBB_PL    0   7214    pACBB_PL    6423    7214    359f8cad-8740-49a4-95d8-306ab7db7161    60  +   791
pACBB_PL    0   7214    pACBB_PL    6498    7214    1619964a-fe7c-4bf8-b178-5ecfc1add0dd    60  -   716
pACBB_PL    0   7214    pACBB_PL    6509    7214    d14e1bc8-7880-4624-936e-9e863d59c956    60  -   705
pACBB_PL    0   7214    pACBB_PL    6536    7214    7e4bf642-5ba7-41fc-8f69-b00053b25fe7    60  +   678
pACBB_PL    0   7214    pACBB_PL    6618    7207    c644b959-dee1-4415-a022-6fb84a1f22d8    60  +   589
pACBB_PL    0   7214    pACBB_PL    6683    7214    69a08152-4449-4dbb-8aa9-f3d30a1d3575    60  -   531
pACBB_PL    0   7214    pACBB_PL    6689    7191    9f7ee30b-459d-4c49-84dc-51824c1732e7    60  -   502
pACBB_PL    0   7214    pACBB_PL    6697    7214    46869993-9b25-419c-bd2c-401a7b50379f    60  -   517
pACBB_PL    0   7214    pACBB_PL    6716    7214    fede98b9-20cc-48d1-a7cd-7261866f7d7e    60  -   498
pACBB_PL    0   7214    pACBB_PL    6722    7214    be5f5927-9b58-4f84-9803-9cb7be7077a3    27  -   492
pACBB_PL    0   7214    pACBB_PL    6769    7214    4488f591-b10b-4dfc-a32d-e22243ded2cf    60  -   445
pACBB_PL    0   7214    pACBB_PL    6984    7214    896713b6-9b3b-4958-8882-bfd6d5b72136    60  -   230
pACBB_PL    0   7214    pACBB_PL    6996    7214    9eb6cdae-e08f-43c0-8ae6-172f1ac5aca9    60  -   218
pACBB_PL    0   7214    pACBB_PL    6998    7214    f15b6fc8-4734-4c1a-abb0-e8842936aa6b    60  +   216
pACBB_PL    0   7214    pACBB_PL    7012    7199    9eb6cdae-e08f-43c0-8ae6-172f1ac5aca9    60  +   187
pACBB_PL    0   7214    pACBB_PL    7040    7208    f4abe4b1-cfcb-4d02-8009-92298f8da641    60  -   168
pACBB_PL    0   7214    pACBB_PL    7053    7214    0e187fe1-6f4f-4fc0-bd15-1bb756b85374    60  -   161
pACBB_PL    0   7214    pACBB_PL    7068    7214    05140fb8-f24b-4478-8159-505cdb96dd45    60  -   146
pACBB_PL    0   7214    pACBB_PL    7071    7214    83cdbaf0-f78e-4f0e-9a85-7bc1ec673eae    60  -   143
pACBB_PL    0   7214    pACBB_PL    7082    7214    196231fa-b3df-4b33-9bd5-9edf232cfe48    60  -   132
pACBB_PL    0   7214    pACBB_PL    7093    7214    454fdd00-183f-44e4-9aa2-f3aa12a01b04    52  +   121
pACBB_PL    0   7214    pACBB_PL    7099    7214    89a4caf9-be0a-49c0-9a93-636cbdd4e8eb    60  -   115
pACBB_PL    0   7214    pACBB_PL    7100    7199    137279f0-bb3a-400f-b39e-492bade3acd6    31  +   99
!cat info/mapped_reads.txt | wc -l
9167

3.10 Fully mapped reads

full_mapped_readfile =  "/design_build/info/full_mapped_reads.txt"

exestr1 = f"bedtools intersect -f 1 -a {bedfile} -b {sortedfile} -wo > {full_mapped_readfile}"
print(exestr1)
!udocker --allow-root run -v=/content/drive/MyDrive/design_build:/design_build gatk bash -c "{exestr1}"
bedtools intersect -f 1 -a /design_build/pACBB_PL.bed -b /design_build/sort/FAT01036_pass_barcode01_5eb57f2c_2b75e8f5_merged-sort.bam -wo > /design_build/info/full_mapped_reads.txt
 
 ****************************************************************************** 
 *                                                                            * 
 *               STARTING 43258f40-d39f-316f-8a89-594ce69950cc                * 
 *                                                                            * 
 ****************************************************************************** 
 executing: bash
!head -n 30 info/full_mapped_reads.txt
pACBB_PL    0   7214    pACBB_PL    0   7214    bb6e8f07-a2c0-4485-b535-6c41c3fed79b    60  +   7214
pACBB_PL    0   7214    pACBB_PL    0   7214    0a4649e3-f378-479f-ba8f-17d64ccbda45    60  +   7214
pACBB_PL    0   7214    pACBB_PL    0   7214    ce483c40-c300-4f24-888e-84af48a82d63    60  +   7214
pACBB_PL    0   7214    pACBB_PL    0   7214    d7da0817-e6f7-4bf1-92ed-8ee8708a51fb    60  +   7214
pACBB_PL    0   7214    pACBB_PL    0   7214    611aedb8-f793-47ea-ae2d-0c6c40dbe8c1    60  +   7214
pACBB_PL    0   7214    pACBB_PL    0   7214    f1c401d1-432a-43b7-9167-5c08212fa6a9    60  +   7214
pACBB_PL    0   7214    pACBB_PL    0   7214    d32cac23-1c9c-4e74-89dd-0d8f7f5aba99    60  +   7214
pACBB_PL    0   7214    pACBB_PL    0   7214    6e2227a1-3b3b-4857-bb40-971e206e9bde    60  +   7214
pACBB_PL    0   7214    pACBB_PL    0   7214    c020cc11-7bd1-474d-a0a8-549afd6e2c96    60  +   7214
pACBB_PL    0   7214    pACBB_PL    0   7214    147a33ab-78b8-4e69-96dc-4a732945b69a    60  +   7214
pACBB_PL    0   7214    pACBB_PL    0   7214    b7ef7c70-f67f-46eb-8bea-0c8ebe3c49c8    60  +   7214
pACBB_PL    0   7214    pACBB_PL    0   7214    5e1de09f-9e8b-4339-bb5c-8ae9aeb79c14    60  +   7214
pACBB_PL    0   7214    pACBB_PL    0   7214    979d5ee6-afab-4142-ac2f-de37ecee859e    60  +   7214
pACBB_PL    0   7214    pACBB_PL    0   7214    c746207e-cbdb-4704-ab60-cd1ce22db5ae    60  +   7214
pACBB_PL    0   7214    pACBB_PL    0   7214    cb0ef94c-0dc2-4e2e-9c76-f240ee0aea7d    60  +   7214
pACBB_PL    0   7214    pACBB_PL    0   7214    f54079d5-0608-4f5e-8685-236cf889f065    60  +   7214
pACBB_PL    0   7214    pACBB_PL    0   7214    06d5e1b1-fc9f-4b90-b98d-553c5cd5126d    60  +   7214
pACBB_PL    0   7214    pACBB_PL    0   7214    8c3b4f0b-2da5-4324-a5d2-a6a29c6106f8    60  +   7214
pACBB_PL    0   7214    pACBB_PL    0   7214    d03eeed8-6f6b-47a2-9507-39edf23d2ee2    60  +   7214
pACBB_PL    0   7214    pACBB_PL    0   7214    f569902d-888f-4e2b-9b42-2d37b9e788c0    60  +   7214
pACBB_PL    0   7214    pACBB_PL    0   7214    7d396b1b-fccf-471c-a6ca-b866e200f272    60  +   7214
pACBB_PL    0   7214    pACBB_PL    0   7214    fada2c2b-9feb-4d9f-9c6c-67eec7e7503d    60  +   7214
pACBB_PL    0   7214    pACBB_PL    0   7214    1914effa-f564-4396-a2ac-2bc30e04b6e4    60  +   7214
pACBB_PL    0   7214    pACBB_PL    0   7214    66fa0c26-3b75-46c6-9225-24129275fe80    60  +   7214
pACBB_PL    0   7214    pACBB_PL    0   7214    985f513e-622f-4424-8526-f92f886992ac    60  +   7214
pACBB_PL    0   7214    pACBB_PL    0   7214    8d94fdc7-6955-4dc5-a287-3e455560aca5    60  +   7214
pACBB_PL    0   7214    pACBB_PL    0   7214    20bed882-779b-40e7-ad52-a87478df5ccc    60  +   7214
pACBB_PL    0   7214    pACBB_PL    0   7214    fc9d4f91-2040-43d1-b3bf-f803c28fc9a8    60  +   7214
pACBB_PL    0   7214    pACBB_PL    0   7214    23b5b625-f23f-4b46-97e2-5baece318f9f    60  +   7214
pACBB_PL    0   7214    pACBB_PL    0   7214    233f6d97-8818-4a99-8fb1-c0c841dccfa8    60  +   7214
!cat info/full_mapped_reads.txt | wc -l
1795

3.11 Variant Calling

  • Creates a sequence dictionary for a reference sequence. This tool creates a sequence dictionary file (with “.dict” extension) from a reference sequence provided in FASTA format, which is required by many processing and analysis tools. The output file contains a header but no SAMRecords, and the header contains only sequence records. (gatk)

  • https://www.melbournebioinformatics.org.au/tutorials/tutorials/variant_calling_gatk1/variant_calling_gatk1/#2-mark-duplicate-reads

dict_file = reference_filename.replace(".fasta", ".dict")

exestr = f"gatk CreateSequenceDictionary -R {reference_filename} -O {dict_file}"
print(exestr)
!udocker --allow-root run -v=/content/drive/MyDrive/design_build:/design_build gatk bash -c "{exestr}"
gatk CreateSequenceDictionary -R /design_build/pACBB_PL.fasta -O /design_build/pACBB_PL.dict
 
 ****************************************************************************** 
 *                                                                            * 
 *               STARTING 43258f40-d39f-316f-8a89-594ce69950cc                * 
 *                                                                            * 
 ****************************************************************************** 
 executing: bash
Using GATK jar /gatk/gatk-package-4.5.0.0-local.jar
Running:
    java -Dsamjdk.use_async_io_read_samtools=false -Dsamjdk.use_async_io_write_samtools=true -Dsamjdk.use_async_io_write_tribble=false -Dsamjdk.compression_level=2 -jar /gatk/gatk-package-4.5.0.0-local.jar CreateSequenceDictionary -R /design_build/pACBB_PL.fasta -O /design_build/pACBB_PL.dict
03:06:50.937 INFO  NativeLibraryLoader - Loading libgkl_compression.so from jar:file:/gatk/gatk-package-4.5.0.0-local.jar!/com/intel/gkl/native/libgkl_compression.so
[Wed Jun 05 03:06:51 GMT 2024] CreateSequenceDictionary --OUTPUT /design_build/pACBB_PL.dict --REFERENCE /design_build/pACBB_PL.fasta --TRUNCATE_NAMES_AT_WHITESPACE true --NUM_SEQUENCES 2147483647 --VERBOSITY INFO --QUIET false --VALIDATION_STRINGENCY STRICT --COMPRESSION_LEVEL 2 --MAX_RECORDS_IN_RAM 500000 --CREATE_INDEX false --CREATE_MD5_FILE false --help false --version false --showHidden false --USE_JDK_DEFLATER false --USE_JDK_INFLATER false
[Wed Jun 05 03:06:51 GMT 2024] Executing as root@adbb3a1816f8 on Linux 6.1.85+ amd64; OpenJDK 64-Bit Server VM 17.0.9+9-Ubuntu-122.04; Deflater: Intel; Inflater: Intel; Provider GCS is available; Picard version: Version:4.5.0.0
[Wed Jun 05 03:06:51 GMT 2024] picard.sam.CreateSequenceDictionary done. Elapsed time: 0.01 minutes.
Runtime.totalMemory()=113246208
Tool returned:
0
!cat pACBB_PL.dict
@HD VN:1.6
@SQ SN:pACBB_PL LN:7214 M5:592a62e277441af177eea5aef27995a7 UR:file:/design_build/pACBB_PL.fasta
  • “java.lang.IllegalStateException: the sample list cannot be null or empty”
vcf_file = reference_filename.replace("fasta", "vcf")
withrg_file = sortedfile.replace("-sort", "-sort-withrg")
rgsm = "pACBB-PL"

exestr = f"gatk --java-options '-Xmx10g' AddOrReplaceReadGroups -I {sortedfile} -O {withrg_file} --SORT_ORDER coordinate --RGLB bar --RGPL ont --RGPU 'run barcode' --RGSM {rgsm}"
#print(withrg_file)
!udocker --allow-root run -v=/content/drive/MyDrive/design_build:/design_build gatk bash -c "{exestr}"
 
 ****************************************************************************** 
 *                                                                            * 
 *               STARTING 43258f40-d39f-316f-8a89-594ce69950cc                * 
 *                                                                            * 
 ****************************************************************************** 
 executing: bash
Using GATK jar /gatk/gatk-package-4.5.0.0-local.jar
Running:
    java -Dsamjdk.use_async_io_read_samtools=false -Dsamjdk.use_async_io_write_samtools=true -Dsamjdk.use_async_io_write_tribble=false -Dsamjdk.compression_level=2 -Xmx10g -jar /gatk/gatk-package-4.5.0.0-local.jar AddOrReplaceReadGroups -I /design_build/sort/FAT01036_pass_barcode01_5eb57f2c_2b75e8f5_merged-sort.bam -O /design_build/sort/FAT01036_pass_barcode01_5eb57f2c_2b75e8f5_merged-sort-withrg.bam --SORT_ORDER coordinate --RGLB bar --RGPL ont --RGPU run barcode --RGSM pACBB-PL
03:22:24.085 INFO  NativeLibraryLoader - Loading libgkl_compression.so from jar:file:/gatk/gatk-package-4.5.0.0-local.jar!/com/intel/gkl/native/libgkl_compression.so
[Wed Jun 05 03:22:24 GMT 2024] AddOrReplaceReadGroups --INPUT /design_build/sort/FAT01036_pass_barcode01_5eb57f2c_2b75e8f5_merged-sort.bam --OUTPUT /design_build/sort/FAT01036_pass_barcode01_5eb57f2c_2b75e8f5_merged-sort-withrg.bam --SORT_ORDER coordinate --RGLB bar --RGPL ont --RGPU run barcode --RGSM pACBB-PL --RGID 1 --VERBOSITY INFO --QUIET false --VALIDATION_STRINGENCY STRICT --COMPRESSION_LEVEL 2 --MAX_RECORDS_IN_RAM 500000 --CREATE_INDEX false --CREATE_MD5_FILE false --help false --version false --showHidden false --USE_JDK_DEFLATER false --USE_JDK_INFLATER false
[Wed Jun 05 03:22:24 GMT 2024] Executing as root@adbb3a1816f8 on Linux 6.1.85+ amd64; OpenJDK 64-Bit Server VM 17.0.9+9-Ubuntu-122.04; Deflater: Intel; Inflater: Intel; Provider GCS is available; Picard version: Version:4.5.0.0
INFO    2024-06-05 03:22:24 AddOrReplaceReadGroups  Created read-group ID=1 PL=ont LB=bar SM=pACBB-PL

[Wed Jun 05 03:22:27 GMT 2024] picard.sam.AddOrReplaceReadGroups done. Elapsed time: 0.06 minutes.
Runtime.totalMemory()=419430400
Tool returned:
0
!ls -alh sort
total 60M
-rw------- 1 root root 30M Jun  5 03:21 FAT01036_pass_barcode01_5eb57f2c_2b75e8f5_merged-sort.bam
-rw------- 1 root root  96 Jun  5 03:21 FAT01036_pass_barcode01_5eb57f2c_2b75e8f5_merged-sort.bam.bai
-rw------- 1 root root 31M Jun  5 03:22 FAT01036_pass_barcode01_5eb57f2c_2b75e8f5_merged-sort-withrg.bam
  • indexing for the new sorted bamfile
exestr = f"samtools index {withrg_file}"
!udocker --allow-root run -v=/content/drive/MyDrive/design_build:/design_build gatk bash -c "{exestr}"
 
 ****************************************************************************** 
 *                                                                            * 
 *               STARTING 43258f40-d39f-316f-8a89-594ce69950cc                * 
 *                                                                            * 
 ****************************************************************************** 
 executing: bash
!ls -alh sort
total 60M
-rw------- 1 root root 30M Jun  5 03:21 FAT01036_pass_barcode01_5eb57f2c_2b75e8f5_merged-sort.bam
-rw------- 1 root root  96 Jun  5 03:21 FAT01036_pass_barcode01_5eb57f2c_2b75e8f5_merged-sort.bam.bai
-rw------- 1 root root 31M Jun  5 03:22 FAT01036_pass_barcode01_5eb57f2c_2b75e8f5_merged-sort-withrg.bam
-rw------- 1 root root  96 Jun  5 03:23 FAT01036_pass_barcode01_5eb57f2c_2b75e8f5_merged-sort-withrg.bam.bai
  • Variant calling (it will take >10min)

vcf_file = reference_filename.replace("fasta", "vcf")

exestr = f"gatk --java-options '-Xmx10g' HaplotypeCaller -I {withrg_file} -R {reference_filename} -O {vcf_file}"
#print(exestr)
!udocker --allow-root run -v=/content/drive/MyDrive/design_build:/design_build gatk bash -c "{exestr}"
 
 ****************************************************************************** 
 *                                                                            * 
 *               STARTING 43258f40-d39f-316f-8a89-594ce69950cc                * 
 *                                                                            * 
 ****************************************************************************** 
 executing: bash
Using GATK jar /gatk/gatk-package-4.5.0.0-local.jar
Running:
    java -Dsamjdk.use_async_io_read_samtools=false -Dsamjdk.use_async_io_write_samtools=true -Dsamjdk.use_async_io_write_tribble=false -Dsamjdk.compression_level=2 -Xmx10g -jar /gatk/gatk-package-4.5.0.0-local.jar HaplotypeCaller -I /design_build/sort/FAT01036_pass_barcode01_5eb57f2c_2b75e8f5_merged-sort-withrg.bam -R /design_build/pACBB_PL.fasta -O /design_build/pACBB_PL.vcf
03:27:30.109 INFO  NativeLibraryLoader - Loading libgkl_compression.so from jar:file:/gatk/gatk-package-4.5.0.0-local.jar!/com/intel/gkl/native/libgkl_compression.so
03:27:30.406 INFO  HaplotypeCaller - ------------------------------------------------------------
03:27:30.411 INFO  HaplotypeCaller - The Genome Analysis Toolkit (GATK) v4.5.0.0
03:27:30.411 INFO  HaplotypeCaller - For support and documentation go to https://software.broadinstitute.org/gatk/
03:27:30.412 INFO  HaplotypeCaller - Executing as root@adbb3a1816f8 on Linux v6.1.85+ amd64
03:27:30.412 INFO  HaplotypeCaller - Java runtime: OpenJDK 64-Bit Server VM v17.0.9+9-Ubuntu-122.04
03:27:30.412 INFO  HaplotypeCaller - Start Date/Time: June 5, 2024 at 3:27:30 AM GMT
03:27:30.413 INFO  HaplotypeCaller - ------------------------------------------------------------
03:27:30.413 INFO  HaplotypeCaller - ------------------------------------------------------------
03:27:30.415 INFO  HaplotypeCaller - HTSJDK Version: 4.1.0
03:27:30.415 INFO  HaplotypeCaller - Picard Version: 3.1.1
03:27:30.416 INFO  HaplotypeCaller - Built for Spark Version: 3.5.0
03:27:30.417 INFO  HaplotypeCaller - HTSJDK Defaults.COMPRESSION_LEVEL : 2
03:27:30.417 INFO  HaplotypeCaller - HTSJDK Defaults.USE_ASYNC_IO_READ_FOR_SAMTOOLS : false
03:27:30.418 INFO  HaplotypeCaller - HTSJDK Defaults.USE_ASYNC_IO_WRITE_FOR_SAMTOOLS : true
03:27:30.419 INFO  HaplotypeCaller - HTSJDK Defaults.USE_ASYNC_IO_WRITE_FOR_TRIBBLE : false
03:27:30.419 INFO  HaplotypeCaller - Deflater: IntelDeflater
03:27:30.420 INFO  HaplotypeCaller - Inflater: IntelInflater
03:27:30.420 INFO  HaplotypeCaller - GCS max retries/reopens: 20
03:27:30.420 INFO  HaplotypeCaller - Requester pays: disabled
03:27:30.421 INFO  HaplotypeCaller - Initializing engine
03:27:30.801 INFO  HaplotypeCaller - Done initializing engine
03:27:30.853 INFO  NativeLibraryLoader - Loading libgkl_utils.so from jar:file:/gatk/gatk-package-4.5.0.0-local.jar!/com/intel/gkl/native/libgkl_utils.so
03:27:30.860 INFO  NativeLibraryLoader - Loading libgkl_smithwaterman.so from jar:file:/gatk/gatk-package-4.5.0.0-local.jar!/com/intel/gkl/native/libgkl_smithwaterman.so
03:27:30.865 INFO  SmithWatermanAligner - Using AVX accelerated SmithWaterman implementation
03:27:30.876 INFO  HaplotypeCallerEngine - Disabling physical phasing, which is supported only for reference-model confidence output
03:27:30.905 INFO  NativeLibraryLoader - Loading libgkl_pairhmm_omp.so from jar:file:/gatk/gatk-package-4.5.0.0-local.jar!/com/intel/gkl/native/libgkl_pairhmm_omp.so
03:27:30.930 INFO  IntelPairHmm - Flush-to-zero (FTZ) is enabled when running PairHMM
03:27:30.931 INFO  IntelPairHmm - Available threads: 2
03:27:30.932 INFO  IntelPairHmm - Requested threads: 4
03:27:30.932 WARN  IntelPairHmm - Using 2 available threads, but 4 were requested
03:27:30.932 INFO  PairHMM - Using the OpenMP multi-threaded AVX-accelerated native PairHMM implementation
03:27:30.992 INFO  ProgressMeter - Starting traversal
03:27:30.992 INFO  ProgressMeter -        Current Locus  Elapsed Minutes     Regions Processed   Regions/Minute
03:27:39.199 WARN  InbreedingCoeff - InbreedingCoeff will not be calculated at position pACBB_PL:77 and possibly subsequent; at least 10 samples must have called genotypes
03:28:56.467 INFO  ProgressMeter -        pACBB_PL:1002              1.4                    10              7.0
03:32:13.771 INFO  ProgressMeter -        pACBB_PL:3014              4.7                    20              4.2
03:36:17.536 INFO  ProgressMeter -        pACBB_PL:4670              8.8                    30              3.4
03:42:35.816 INFO  ProgressMeter -        pACBB_PL:6309             15.1                    40              2.7
03:44:46.102 INFO  HaplotypeCaller - 0 read(s) filtered by: MappingQualityReadFilter 
0 read(s) filtered by: MappingQualityAvailableReadFilter 
0 read(s) filtered by: MappedReadFilter 
0 read(s) filtered by: NotSecondaryAlignmentReadFilter 
0 read(s) filtered by: NotDuplicateReadFilter 
0 read(s) filtered by: PassesVendorQualityCheckReadFilter 
0 read(s) filtered by: NonZeroReferenceLengthAlignmentReadFilter 
1 read(s) filtered by: GoodCigarReadFilter 
0 read(s) filtered by: WellformedReadFilter 
1 total reads filtered out of 9167 reads processed
03:44:46.102 INFO  ProgressMeter -        pACBB_PL:6309             17.3                    46              2.7
03:44:46.103 INFO  ProgressMeter - Traversal complete. Processed 46 total regions in 17.3 minutes.
03:44:46.122 INFO  VectorLoglessPairHMM - Time spent in setup for JNI call : 0.168806741
03:44:46.123 INFO  PairHMM - Total compute time in PairHMM computeLogLikelihoods() : 982.7120195350001
03:44:46.123 INFO  SmithWatermanAligner - Total compute time in native Smith-Waterman : 2.93 sec
03:44:46.125 INFO  HaplotypeCaller - Shutting down engine
[June 5, 2024 at 3:44:46 AM GMT] org.broadinstitute.hellbender.tools.walkers.haplotypecaller.HaplotypeCaller done. Elapsed time: 17.27 minutes.
Runtime.totalMemory()=1501560832
!ls -alh
total 37M
drwx------ 2 root root 4.0K Jun  5 02:26 bam
-rw------- 1 root root  19M Jun  5 01:53 FAT01036_pass_barcode01_5eb57f2c_2b75e8f5_0.fastq.gz
-rw------- 1 root root  19M Jun  5 01:53 FAT01036_pass_barcode01_5eb57f2c_2b75e8f5_1.fastq.gz
drwx------ 2 root root 4.0K Jun  5 02:56 info
drwx------ 2 root root 4.0K Jun  5 02:14 .ipynb_checkpoints
-rw------- 1 root root   16 Jun  5 02:53 pACBB_PL.bed
-rw------- 1 root root  108 Jun  5 03:06 pACBB_PL.dict
-rw------- 1 root root 7.2K Jun  5 01:56 pACBB_PL.fasta
-rw------- 1 root root   23 Jun  5 02:53 pACBB_PL.fasta.fai
-rw------- 1 root root  15K Jun  5 01:53 pACBB-PL-from_EcoRI_hs.gb
-rw------- 1 root root  22K Jun  5 03:44 pACBB_PL.vcf
-rw------- 1 root root  285 Jun  5 03:44 pACBB_PL.vcf.idx
drwx------ 2 root root 4.0K Jun  5 02:24 sam
drwx------ 2 root root 4.0K Jun  5 03:23 sort
!cat pACBB_PL.vcf
##fileformat=VCFv4.2
##FILTER=<ID=LowQual,Description="Low quality">
##FORMAT=<ID=AD,Number=R,Type=Integer,Description="Allelic depths for the ref and alt alleles in the order listed">
##FORMAT=<ID=DP,Number=1,Type=Integer,Description="Approximate read depth (reads with MQ=255 or with bad mates are filtered)">
##FORMAT=<ID=GQ,Number=1,Type=Integer,Description="Genotype Quality">
##FORMAT=<ID=GT,Number=1,Type=String,Description="Genotype">
##FORMAT=<ID=PL,Number=G,Type=Integer,Description="Normalized, Phred-scaled likelihoods for genotypes as defined in the VCF specification">
##GATKCommandLine=<ID=HaplotypeCaller,CommandLine="HaplotypeCaller --output /design_build/pACBB_PL.vcf --input /design_build/sort/FAT01036_pass_barcode01_5eb57f2c_2b75e8f5_merged-sort-withrg.bam --reference /design_build/pACBB_PL.fasta --use-posteriors-to-calculate-qual false --dont-use-dragstr-priors false --use-new-qual-calculator true --annotate-with-num-discovered-alleles false --heterozygosity 0.001 --indel-heterozygosity 1.25E-4 --heterozygosity-stdev 0.01 --standard-min-confidence-threshold-for-calling 30.0 --max-alternate-alleles 6 --max-genotype-count 1024 --sample-ploidy 2 --num-reference-samples-if-no-call 0 --genotype-assignment-method USE_PLS_TO_ASSIGN --contamination-fraction-to-filter 0.0 --output-mode EMIT_VARIANTS_ONLY --all-site-pls false --flow-likelihood-parallel-threads 0 --flow-likelihood-optimized-comp false --trim-to-haplotype true --exact-matching false --flow-use-t0-tag false --flow-probability-threshold 0.003 --flow-remove-non-single-base-pair-indels false --flow-remove-one-zero-probs false --flow-quantization-bins 121 --flow-fill-empty-bins-value 0.001 --flow-symmetric-indel-probs false --flow-report-insertion-or-deletion false --flow-disallow-probs-larger-than-call false --flow-lump-probs false --flow-retain-max-n-probs-base-format false --flow-probability-scaling-factor 10 --flow-order-cycle-length 4 --keep-boundary-flows false --gvcf-gq-bands 1 --gvcf-gq-bands 2 --gvcf-gq-bands 3 --gvcf-gq-bands 4 --gvcf-gq-bands 5 --gvcf-gq-bands 6 --gvcf-gq-bands 7 --gvcf-gq-bands 8 --gvcf-gq-bands 9 --gvcf-gq-bands 10 --gvcf-gq-bands 11 --gvcf-gq-bands 12 --gvcf-gq-bands 13 --gvcf-gq-bands 14 --gvcf-gq-bands 15 --gvcf-gq-bands 16 --gvcf-gq-bands 17 --gvcf-gq-bands 18 --gvcf-gq-bands 19 --gvcf-gq-bands 20 --gvcf-gq-bands 21 --gvcf-gq-bands 22 --gvcf-gq-bands 23 --gvcf-gq-bands 24 --gvcf-gq-bands 25 --gvcf-gq-bands 26 --gvcf-gq-bands 27 --gvcf-gq-bands 28 --gvcf-gq-bands 29 --gvcf-gq-bands 30 --gvcf-gq-bands 31 --gvcf-gq-bands 32 --gvcf-gq-bands 33 --gvcf-gq-bands 34 --gvcf-gq-bands 35 --gvcf-gq-bands 36 --gvcf-gq-bands 37 --gvcf-gq-bands 38 --gvcf-gq-bands 39 --gvcf-gq-bands 40 --gvcf-gq-bands 41 --gvcf-gq-bands 42 --gvcf-gq-bands 43 --gvcf-gq-bands 44 --gvcf-gq-bands 45 --gvcf-gq-bands 46 --gvcf-gq-bands 47 --gvcf-gq-bands 48 --gvcf-gq-bands 49 --gvcf-gq-bands 50 --gvcf-gq-bands 51 --gvcf-gq-bands 52 --gvcf-gq-bands 53 --gvcf-gq-bands 54 --gvcf-gq-bands 55 --gvcf-gq-bands 56 --gvcf-gq-bands 57 --gvcf-gq-bands 58 --gvcf-gq-bands 59 --gvcf-gq-bands 60 --gvcf-gq-bands 70 --gvcf-gq-bands 80 --gvcf-gq-bands 90 --gvcf-gq-bands 99 --floor-blocks false --indel-size-to-eliminate-in-ref-model 10 --disable-optimizations false --dragen-mode false --dragen-378-concordance-mode false --flow-mode NONE --apply-bqd false --apply-frd false --disable-spanning-event-genotyping false --transform-dragen-mapping-quality false --mapping-quality-threshold-for-genotyping 20 --max-effective-depth-adjustment-for-frd 0 --just-determine-active-regions false --dont-genotype false --do-not-run-physical-phasing false --do-not-correct-overlapping-quality false --use-filtered-reads-for-annotations false --use-flow-aligner-for-stepwise-hc-filtering false --adaptive-pruning false --do-not-recover-dangling-branches false --recover-dangling-heads false --kmer-size 10 --kmer-size 25 --dont-increase-kmer-sizes-for-cycles false --allow-non-unique-kmers-in-ref false --num-pruning-samples 1 --min-dangling-branch-length 4 --recover-all-dangling-branches false --max-num-haplotypes-in-population 128 --min-pruning 2 --adaptive-pruning-initial-error-rate 0.001 --pruning-lod-threshold 2.302585092994046 --pruning-seeding-lod-threshold 9.210340371976184 --max-unpruned-variants 100 --linked-de-bruijn-graph false --disable-artificial-haplotype-recovery false --enable-legacy-graph-cycle-detection false --debug-assembly false --debug-graph-transformations false --capture-assembly-failure-bam false --num-matching-bases-in-dangling-end-to-recover -1 --error-correction-log-odds -Infinity --error-correct-reads false --kmer-length-for-read-error-correction 25 --min-observations-for-kmer-to-be-solid 20 --likelihood-calculation-engine PairHMM --base-quality-score-threshold 18 --dragstr-het-hom-ratio 2 --dont-use-dragstr-pair-hmm-scores false --pair-hmm-gap-continuation-penalty 10 --expected-mismatch-rate-for-read-disqualification 0.02 --pair-hmm-implementation FASTEST_AVAILABLE --pcr-indel-model CONSERVATIVE --phred-scaled-global-read-mismapping-rate 45 --disable-symmetric-hmm-normalizing false --disable-cap-base-qualities-to-map-quality false --enable-dynamic-read-disqualification-for-genotyping false --dynamic-read-disqualification-threshold 1.0 --native-pair-hmm-threads 4 --native-pair-hmm-use-double-precision false --flow-hmm-engine-min-indel-adjust 6 --flow-hmm-engine-flat-insertion-penatly 45 --flow-hmm-engine-flat-deletion-penatly 45 --pileup-detection false --use-pdhmm false --use-pdhmm-overlap-optimization false --make-determined-haps-from-pd-code false --print-pileupcalling-status false --fallback-gga-if-pdhmm-fails true --pileup-detection-enable-indel-pileup-calling false --pileup-detection-active-region-phred-threshold 0.0 --num-artificial-haplotypes-to-add-per-allele 5 --artifical-haplotype-filtering-kmer-size 10 --pileup-detection-snp-alt-threshold 0.1 --pileup-detection-indel-alt-threshold 0.1 --pileup-detection-absolute-alt-depth 0.0 --pileup-detection-snp-adjacent-to-assembled-indel-range 5 --pileup-detection-snp-basequality-filter 12 --pileup-detection-bad-read-tolerance 0.0 --pileup-detection-proper-pair-read-badness true --pileup-detection-edit-distance-read-badness-threshold 0.08 --pileup-detection-chimeric-read-badness true --pileup-detection-template-mean-badness-threshold 0.0 --pileup-detection-template-std-badness-threshold 0.0 --pileup-detection-filter-assembly-alt-bad-read-tolerance 0.0 --pileup-detection-edit-distance-read-badness-for-assembly-filtering-threshold 0.12 --bam-writer-type CALLED_HAPLOTYPES --dont-use-soft-clipped-bases false --override-fragment-softclip-check false --min-base-quality-score 10 --smith-waterman FASTEST_AVAILABLE --emit-ref-confidence NONE --max-mnp-distance 0 --force-call-filtered-alleles false --reference-model-deletion-quality 30 --soft-clip-low-quality-ends false --allele-informative-reads-overlap-margin 2 --smith-waterman-dangling-end-match-value 25 --smith-waterman-dangling-end-mismatch-penalty -50 --smith-waterman-dangling-end-gap-open-penalty -110 --smith-waterman-dangling-end-gap-extend-penalty -6 --smith-waterman-haplotype-to-reference-match-value 200 --smith-waterman-haplotype-to-reference-mismatch-penalty -150 --smith-waterman-haplotype-to-reference-gap-open-penalty -260 --smith-waterman-haplotype-to-reference-gap-extend-penalty -11 --smith-waterman-read-to-haplotype-match-value 10 --smith-waterman-read-to-haplotype-mismatch-penalty -15 --smith-waterman-read-to-haplotype-gap-open-penalty -30 --smith-waterman-read-to-haplotype-gap-extend-penalty -5 --flow-assembly-collapse-hmer-size 0 --flow-assembly-collapse-partial-mode false --flow-filter-alleles false --flow-filter-alleles-qual-threshold 30.0 --flow-filter-alleles-sor-threshold 3.0 --flow-filter-lone-alleles false --flow-filter-alleles-debug-graphs false --min-assembly-region-size 50 --max-assembly-region-size 300 --active-probability-threshold 0.002 --max-prob-propagation-distance 50 --force-active false --assembly-region-padding 100 --padding-around-indels 75 --padding-around-snps 20 --padding-around-strs 75 --max-extension-into-assembly-region-padding-legacy 25 --max-reads-per-alignment-start 50 --enable-legacy-assembly-region-trimming false --interval-set-rule UNION --interval-padding 0 --interval-exclusion-padding 0 --interval-merging-rule ALL --read-validation-stringency SILENT --seconds-between-progress-updates 10.0 --disable-sequence-dictionary-validation false --create-output-bam-index true --create-output-bam-md5 false --create-output-variant-index true --create-output-variant-md5 false --max-variants-per-shard 0 --lenient false --add-output-sam-program-record true --add-output-vcf-command-line true --cloud-prefetch-buffer 40 --cloud-index-prefetch-buffer -1 --disable-bam-index-caching false --sites-only-vcf-output false --help false --version false --showHidden false --verbosity INFO --QUIET false --use-jdk-deflater false --use-jdk-inflater false --gcs-max-retries 20 --gcs-project-for-requester-pays  --disable-tool-default-read-filters false --minimum-mapping-quality 20 --disable-tool-default-annotations false --enable-all-annotations false --allow-old-rms-mapping-quality-annotation-data false",Version="4.5.0.0",Date="June 5, 2024 at 3:27:30 AM GMT">
##INFO=<ID=AC,Number=A,Type=Integer,Description="Allele count in genotypes, for each ALT allele, in the same order as listed">
##INFO=<ID=AF,Number=A,Type=Float,Description="Allele Frequency, for each ALT allele, in the same order as listed">
##INFO=<ID=AN,Number=1,Type=Integer,Description="Total number of alleles in called genotypes">
##INFO=<ID=BaseQRankSum,Number=1,Type=Float,Description="Z-score from Wilcoxon rank sum test of Alt Vs. Ref base qualities">
##INFO=<ID=DP,Number=1,Type=Integer,Description="Approximate read depth; some reads may have been filtered">
##INFO=<ID=ExcessHet,Number=1,Type=Float,Description="Phred-scaled p-value for exact test of excess heterozygosity">
##INFO=<ID=FS,Number=1,Type=Float,Description="Phred-scaled p-value using Fisher's exact test to detect strand bias">
##INFO=<ID=InbreedingCoeff,Number=1,Type=Float,Description="Inbreeding coefficient as estimated from the genotype likelihoods per-sample when compared against the Hardy-Weinberg expectation">
##INFO=<ID=MLEAC,Number=A,Type=Integer,Description="Maximum likelihood expectation (MLE) for the allele counts (not necessarily the same as the AC), for each ALT allele, in the same order as listed">
##INFO=<ID=MLEAF,Number=A,Type=Float,Description="Maximum likelihood expectation (MLE) for the allele frequency (not necessarily the same as the AF), for each ALT allele, in the same order as listed">
##INFO=<ID=MQ,Number=1,Type=Float,Description="RMS Mapping Quality">
##INFO=<ID=MQRankSum,Number=1,Type=Float,Description="Z-score From Wilcoxon rank sum test of Alt vs. Ref read mapping qualities">
##INFO=<ID=QD,Number=1,Type=Float,Description="Variant Confidence/Quality by Depth">
##INFO=<ID=ReadPosRankSum,Number=1,Type=Float,Description="Z-score from Wilcoxon rank sum test of Alt vs. Ref read position bias">
##INFO=<ID=SOR,Number=1,Type=Float,Description="Symmetric Odds Ratio of 2x2 contingency table to detect strand bias">
##contig=<ID=pACBB_PL,length=7214>
##source=HaplotypeCaller
#CHROM  POS ID  REF ALT QUAL    FILTER  INFO    FORMAT  pACBB-PL
pACBB_PL    77  .   T   G   1325.06 .   AC=2;AF=1.00;AN=2;DP=37;ExcessHet=0.0000;FS=0.000;MLEAC=2;MLEAF=1.00;MQ=59.92;QD=34.72;SOR=3.086    GT:AD:DP:GQ:PL  1/1:0,37:37:99:1339,110,0
pACBB_PL    132 .   G   GATTTTGTT   90.60   .   AC=1;AF=0.500;AN=2;BaseQRankSum=1.645;DP=7;ExcessHet=0.0000;FS=0.000;MLEAC=1;MLEAF=0.500;MQ=60.00;MQRankSum=0.000;QD=18.12;ReadPosRankSum=1.645;SOR=1.022   GT:AD:DP:GQ:PL  0/1:2,3:5:40:98,0,40
pACBB_PL    134 .   C   T   112.63  .   AC=1;AF=0.500;AN=2;BaseQRankSum=1.150;DP=7;ExcessHet=0.0000;FS=0.000;MLEAC=1;MLEAF=0.500;MQ=60.00;MQRankSum=0.000;QD=22.53;ReadPosRankSum=1.150;SOR=1.022   GT:AD:DP:GQ:PL  0/1:2,3:5:40:120,0,40
pACBB_PL    135 .   C   A   32.64   .   AC=1;AF=0.500;AN=2;BaseQRankSum=1.150;DP=7;ExcessHet=0.0000;FS=0.000;MLEAC=1;MLEAF=0.500;MQ=60.00;MQRankSum=0.000;QD=6.53;ReadPosRankSum=-1.150;SOR=0.446   GT:AD:DP:GQ:PL  0/1:3,2:5:40:40,0,120
pACBB_PL    136 .   G   A   115.64  .   AC=1;AF=0.500;AN=2;BaseQRankSum=-1.150;DP=4;ExcessHet=0.0000;FS=0.000;MLEAC=1;MLEAF=0.500;MQ=60.00;MQRankSum=0.000;QD=28.91;ReadPosRankSum=1.150;SOR=1.609  GT:AD:DP:GQ:PL  0/1:1,3:4:33:123,0,33
pACBB_PL    138 .   G   T   115.64  .   AC=1;AF=0.500;AN=2;BaseQRankSum=-1.150;DP=4;ExcessHet=0.0000;FS=0.000;MLEAC=1;MLEAF=0.500;MQ=60.00;MQRankSum=0.000;QD=28.91;ReadPosRankSum=1.150;SOR=1.609  GT:AD:DP:GQ:PL  0/1:1,3:4:33:123,0,33
pACBB_PL    149 .   G   GGCCCC  51.98   .   AC=1;AF=0.500;AN=2;BaseQRankSum=-0.674;DP=4;ExcessHet=0.0000;FS=0.000;MLEAC=1;MLEAF=0.500;MQ=60.00;MQRankSum=0.000;QD=12.99;ReadPosRankSum=0.674;SOR=0.693  GT:AD:DP:GQ:PL  0/1:1,3:4:33:82,0,33
pACBB_PL    153 .   AAGC    A   70.60   .   AC=1;AF=0.500;AN=2;BaseQRankSum=1.383;DP=4;ExcessHet=0.0000;FS=0.000;MLEAC=1;MLEAF=0.500;MQ=60.00;MQRankSum=0.000;QD=17.65;ReadPosRankSum=1.383;SOR=0.693   GT:AD:DP:GQ:PL  0/1:2,2:4:43:78,0,43
pACBB_PL    157 .   T   TTTG    105.60  .   AC=1;AF=0.500;AN=2;BaseQRankSum=0.967;DP=4;ExcessHet=0.0000;FS=0.000;MLEAC=1;MLEAF=0.500;MQ=60.00;MQRankSum=0.000;QD=26.40;ReadPosRankSum=0.967;SOR=1.179   GT:AD:DP:GQ:PL  0/1:1,3:4:33:113,0,33
pACBB_PL    160 .   A   AGGCCTTTTT  83.60   .   AC=1;AF=0.500;AN=2;BaseQRankSum=0.967;DP=4;ExcessHet=0.0000;FS=0.000;MLEAC=1;MLEAF=0.500;MQ=60.00;MQRankSum=0.000;QD=20.90;ReadPosRankSum=0.967;SOR=1.179   GT:AD:DP:GQ:PL  0/1:1,3:4:33:91,0,33
pACBB_PL    162 .   C   CGAAAA  115.60  .   AC=1;AF=0.500;AN=2;BaseQRankSum=0.319;DP=4;ExcessHet=0.0000;FS=0.000;MLEAC=1;MLEAF=0.500;MQ=60.00;MQRankSum=0.000;QD=28.90;ReadPosRankSum=1.150;SOR=1.609   GT:AD:DP:GQ:PL  0/1:1,3:4:33:123,0,33
pACBB_PL    1161    .   T   TTAA    70.60   .   AC=1;AF=0.500;AN=2;BaseQRankSum=0.674;DP=5;ExcessHet=0.0000;FS=0.000;MLEAC=1;MLEAF=0.500;MQ=60.00;MQRankSum=0.000;QD=17.65;ReadPosRankSum=-0.431;SOR=0.105  GT:AD:DP:GQ:PL  0/1:2,2:4:78:78,0,78
pACBB_PL    1181    .   C   T   115.64  .   AC=1;AF=0.500;AN=2;BaseQRankSum=-1.150;DP=4;ExcessHet=0.0000;FS=0.000;MLEAC=1;MLEAF=0.500;MQ=60.00;MQRankSum=0.000;QD=28.91;ReadPosRankSum=-1.150;SOR=0.446 GT:AD:DP:GQ:PL  0/1:1,3:4:33:123,0,33
pACBB_PL    1204    .   G   A   115.64  .   AC=1;AF=0.500;AN=2;BaseQRankSum=-1.150;DP=4;ExcessHet=0.0000;FS=0.000;MLEAC=1;MLEAF=0.500;MQ=60.00;MQRankSum=0.000;QD=28.91;ReadPosRankSum=-1.150;SOR=0.446 GT:AD:DP:GQ:PL  0/1:1,3:4:33:123,0,33
pACBB_PL    1206    .   C   A   268.01  .   AC=2;AF=1.00;AN=2;DP=11;ExcessHet=0.0000;FS=0.000;MLEAC=2;MLEAF=1.00;MQ=60.00;QD=30.50;SOR=1.609    GT:AD:DP:GQ:PL  1/1:0,7:7:21:282,21,0
pACBB_PL    1213    .   CT  C   92.60   .   AC=1;AF=0.500;AN=2;BaseQRankSum=-2.071;DP=13;ExcessHet=0.0000;FS=2.616;MLEAC=1;MLEAF=0.500;MQ=60.00;MQRankSum=0.000;QD=7.72;ReadPosRankSum=-0.409;SOR=0.169 GT:AD:DP:GQ:PL  0/1:8,4:12:99:100,0,261
pACBB_PL    1326    .   ATTTTGTTAT  A   53.60   .   AC=1;AF=0.500;AN=2;BaseQRankSum=-2.287;DP=15;ExcessHet=0.0000;FS=0.000;MLEAC=1;MLEAF=0.500;MQ=60.00;MQRankSum=0.000;QD=5.36;ReadPosRankSum=-0.253;SOR=1.609 GT:AD:DP:GQ:PL  0/1:8,2:10:61:61,0,329
pACBB_PL    1339    .   T   G   49.64   .   AC=1;AF=0.500;AN=2;BaseQRankSum=0.114;DP=11;ExcessHet=0.0000;FS=0.000;MLEAC=1;MLEAF=0.500;MQ=60.00;MQRankSum=0.000;QD=4.51;ReadPosRankSum=-0.447;SOR=1.371  GT:AD:DP:GQ:PL  0/1:9,2:11:57:57,0,372
pACBB_PL    1347    .   GC  G   102.36  .   AC=1;AF=0.500;AN=2;BaseQRankSum=-0.405;DP=11;ExcessHet=0.0000;FS=3.680;MLEAC=1;MLEAF=0.500;MQ=60.00;MQRankSum=0.000;QD=11.37;ReadPosRankSum=0.272;SOR=0.039 GT:AD:DP:GQ:PL  0/1:4,5:9:94:110,0,94
pACBB_PL    1355    .   A   AAGC    52.54   .   AC=1;AF=0.500;AN=2;BaseQRankSum=-1.501;DP=11;ExcessHet=0.0000;FS=0.000;MLEAC=1;MLEAF=0.500;MQ=60.00;MQRankSum=0.000;QD=5.25;ReadPosRankSum=-0.493;SOR=1.609 GT:AD:DP:GQ:PL  0/1:8,2:10:60:60,0,330
pACBB_PL    1356    .   TTTG    T   52.54   .   AC=1;AF=0.500;AN=2;BaseQRankSum=-1.701;DP=11;ExcessHet=0.0000;FS=0.000;MLEAC=1;MLEAF=0.500;MQ=60.00;MQRankSum=0.000;QD=5.25;ReadPosRankSum=-0.253;SOR=1.609 GT:AD:DP:GQ:PL  0/1:8,2:10:60:60,0,330
pACBB_PL    1362    .   AGGCCTTTTT  A   52.54   .   AC=1;AF=0.500;AN=2;BaseQRankSum=0.493;DP=11;ExcessHet=0.0000;FS=0.000;MLEAC=1;MLEAF=0.500;MQ=60.00;MQRankSum=0.000;QD=5.25;ReadPosRankSum=-0.493;SOR=1.609  GT:AD:DP:GQ:PL  0/1:8,2:10:60:60,0,330
pACBB_PL    1373    .   CGAAAA  C   49.60   .   AC=1;AF=0.500;AN=2;BaseQRankSum=0.114;DP=11;ExcessHet=0.0000;FS=0.000;MLEAC=1;MLEAF=0.500;MQ=60.00;MQRankSum=0.000;QD=4.51;ReadPosRankSum=-0.447;SOR=1.371  GT:AD:DP:GQ:PL  0/1:9,2:11:57:57,0,372
pACBB_PL    1399    .   AT  A   93.60   .   AC=1;AF=0.500;AN=2;BaseQRankSum=0.328;DP=12;ExcessHet=0.0000;FS=3.310;MLEAC=1;MLEAF=0.500;MQ=60.00;MQRankSum=0.000;QD=7.80;ReadPosRankSum=-0.816;SOR=2.303  GT:AD:DP:GQ:PL  0/1:6,6:12:99:101,0,149
pACBB_PL    1989    .   C   G   35.48   .   AC=2;AF=1.00;AN=2;DP=1;ExcessHet=0.0000;FS=0.000;MLEAC=1;MLEAF=0.500;MQ=60.00;QD=29.86;SOR=1.609    GT:AD:DP:GQ:PL  1/1:0,1:1:3:45,3,0
pACBB_PL    2418    .   T   C   554.06  .   AC=2;AF=1.00;AN=2;DP=14;ExcessHet=0.0000;FS=0.000;MLEAC=2;MLEAF=1.00;MQ=60.00;QD=26.67;SOR=1.765    GT:AD:DP:GQ:PL  1/1:0,14:14:42:568,42,0
pACBB_PL    3341    .   TA  T   48.60   .   AC=1;AF=0.500;AN=2;BaseQRankSum=0.967;DP=3;ExcessHet=0.0000;FS=0.000;MLEAC=1;MLEAF=0.500;MQ=60.00;MQRankSum=0.000;QD=16.20;ReadPosRankSum=0.000;SOR=1.179   GT:AD:DP:GQ:PL  0/1:1,2:3:24:56,0,24
pACBB_PL    3432    .   GATT    G   115.61  .   AC=1;AF=0.500;AN=2;DP=4;ExcessHet=0.0000;FS=0.000;MLEAC=1;MLEAF=0.500;MQ=60.00;QD=28.90;SOR=2.833   GT:AD:DP:GQ:PL  0/1:1,3:4:32:123,0,32
pACBB_PL    3448    .   TA  T   166.07  .   AC=2;AF=1.00;AN=2;DP=5;ExcessHet=0.0000;FS=0.000;MLEAC=2;MLEAF=1.00;MQ=60.00;QD=27.35;SOR=2.833 GT:AD:DP:GQ:PL  1/1:0,4:4:12:180,12,0
pACBB_PL    4165    .   CAAAT   C   35.44   .   AC=2;AF=1.00;AN=2;DP=1;ExcessHet=0.0000;FS=0.000;MLEAC=1;MLEAF=0.500;MQ=60.00;QD=32.29;SOR=1.609    GT:AD:DP:GQ:PL  1/1:0,1:1:3:45,3,0
pACBB_PL    4176    .   TTTTCG  T   35.44   .   AC=2;AF=1.00;AN=2;DP=1;ExcessHet=0.0000;FS=0.000;MLEAC=1;MLEAF=0.500;MQ=60.00;QD=29.42;SOR=1.609    GT:AD:DP:GQ:PL  1/1:0,1:1:3:45,3,0
pACBB_PL    4182    .   AAAAAG  A   35.43   .   AC=2;AF=1.00;AN=2;DP=1;ExcessHet=0.0000;FS=0.000;MLEAC=1;MLEAF=0.500;MQ=60.00;QD=28.13;SOR=1.609    GT:AD:DP:GQ:PL  1/1:0,1:1:3:45,3,0
pACBB_PL    4190    .   C   G   35.48   .   AC=2;AF=1.00;AN=2;DP=1;ExcessHet=0.0000;FS=0.000;MLEAC=1;MLEAF=0.500;MQ=60.00;QD=34.81;SOR=1.609    GT:AD:DP:GQ:PL  1/1:0,1:1:3:45,3,0
pACBB_PL    4191    .   T   TGA 35.43   .   AC=2;AF=1.00;AN=2;DP=1;ExcessHet=0.0000;FS=0.000;MLEAC=1;MLEAF=0.500;MQ=60.00;QD=28.71;SOR=1.609    GT:AD:DP:GQ:PL  1/1:0,1:1:3:45,3,0
pACBB_PL    4193    .   C   CGGCG   35.43   .   AC=2;AF=1.00;AN=2;DP=1;ExcessHet=0.0000;FS=0.000;MLEAC=1;MLEAF=0.500;MQ=60.00;QD=26.97;SOR=1.609    GT:AD:DP:GQ:PL  1/1:0,1:1:3:45,3,0
pACBB_PL    4194    .   CAA C   35.43   .   AC=2;AF=1.00;AN=2;DP=1;ExcessHet=0.0000;FS=0.000;MLEAC=1;MLEAF=0.500;MQ=60.00;QD=26.69;SOR=1.609    GT:AD:DP:GQ:PL  1/1:0,1:1:3:45,3,0
pACBB_PL    4201    .   GGGGGCC G   35.44   .   AC=2;AF=1.00;AN=2;DP=1;ExcessHet=0.0000;FS=0.000;MLEAC=1;MLEAF=0.500;MQ=60.00;QD=29.32;SOR=1.609    GT:AD:DP:GQ:PL  1/1:0,1:1:3:45,3,0
pACBB_PL    4208    .   T   TCACGC  35.43   .   AC=2;AF=1.00;AN=2;DP=1;ExcessHet=0.0000;FS=0.000;MLEAC=1;MLEAF=0.500;MQ=60.00;QD=32.77;SOR=1.609    GT:AD:DP:GQ:PL  1/1:0,1:1:3:45,3,0
pACBB_PL    4210    .   T   A   35.47   .   AC=2;AF=1.00;AN=2;DP=1;ExcessHet=0.0000;FS=0.000;MLEAC=1;MLEAF=0.500;MQ=60.00;QD=35.30;SOR=1.609    GT:AD:DP:GQ:PL  1/1:0,1:1:3:45,3,0
pACBB_PL    4214    .   A   G   35.48   .   AC=2;AF=1.00;AN=2;DP=1;ExcessHet=0.0000;FS=0.000;MLEAC=1;MLEAF=0.500;MQ=60.00;QD=33.58;SOR=1.609    GT:AD:DP:GQ:PL  1/1:0,1:1:3:45,3,0
pACBB_PL    4217    .   GATAACAA    G   35.43   .   AC=2;AF=1.00;AN=2;DP=2;ExcessHet=0.0000;FS=0.000;MLEAC=1;MLEAF=0.500;MQ=60.00;QD=36.61;SOR=1.609    GT:AD:DP:GQ:PL  1/1:0,1:1:3:45,3,0
pACBB_PL    4226    .   A   G   35.47   .   AC=2;AF=1.00;AN=2;DP=2;ExcessHet=0.0000;FS=0.000;MLEAC=1;MLEAF=0.500;MQ=60.00;QD=32.10;SOR=1.609    GT:AD:DP:GQ:PL  1/1:0,1:1:3:45,3,0
pACBB_PL    4714    .   GT  G   3062.03 .   AC=2;AF=1.00;AN=2;BaseQRankSum=1.671;DP=77;ExcessHet=0.0000;FS=3.813;MLEAC=2;MLEAF=1.00;MQ=60.00;MQRankSum=0.000;QD=29.96;ReadPosRankSum=0.180;SOR=0.840    GT:AD:DP:GQ:PL  1/1:1,76:77:99:3076,225,0
pACBB_PL    5211    .   C   CA  1291.03 .   AC=2;AF=1.00;AN=2;DP=29;ExcessHet=0.0000;FS=0.000;MLEAC=2;MLEAF=1.00;MQ=60.00;QD=28.82;SOR=2.226    GT:AD:DP:GQ:PL  1/1:0,29:29:87:1305,87,0
pACBB_PL    5243    .   TA  T   101.60  .   AC=1;AF=0.500;AN=2;BaseQRankSum=1.681;DP=30;ExcessHet=0.0000;FS=8.368;MLEAC=1;MLEAF=0.500;MQ=60.00;MQRankSum=0.000;QD=3.76;ReadPosRankSum=0.183;SOR=2.833   GT:AD:DP:GQ:PL  0/1:20,7:27:99:109,0,572
pACBB_PL    5326    .   G   A   1336.06 .   AC=2;AF=1.00;AN=2;DP=30;ExcessHet=0.0000;FS=0.000;MLEAC=2;MLEAF=1.00;MQ=60.00;QD=34.83;SOR=2.303    GT:AD:DP:GQ:PL  1/1:0,30:30:90:1350,90,0
pACBB_PL    5403    .   C   T   1595.06 .   AC=2;AF=1.00;AN=2;DP=44;ExcessHet=0.0000;FS=0.000;MLEAC=2;MLEAF=1.00;MQ=60.00;QD=30.94;SOR=1.127    GT:AD:DP:GQ:PL  1/1:0,44:44:99:1915,132,0
pACBB_PL    5897    .   AT  A   204.60  .   AC=1;AF=0.500;AN=2;BaseQRankSum=-1.194;DP=17;ExcessHet=0.0000;FS=0.000;MLEAC=1;MLEAF=0.500;MQ=60.00;MQRankSum=0.000;QD=12.04;ReadPosRankSum=-0.876;SOR=0.693    GT:AD:DP:GQ:PL  0/1:8,9:17:99:212,0,228
pACBB_PL    6347    .   GA  G   424.60  .   AC=1;AF=0.500;AN=2;BaseQRankSum=0.727;DP=46;ExcessHet=0.0000;FS=0.000;MLEAC=1;MLEAF=0.500;MQ=60.00;MQRankSum=0.000;QD=9.23;ReadPosRankSum=-1.381;SOR=0.576  GT:AD:DP:GQ:PL  0/1:28,18:46:99:432,0,709
pACBB_PL    6553    .   GC  G   54.61   .   AC=1;AF=0.500;AN=2;BaseQRankSum=0.000;DP=4;ExcessHet=0.0000;FS=0.000;MLEAC=1;MLEAF=0.500;MQ=60.00;MQRankSum=0.000;QD=13.65;ReadPosRankSum=0.967;SOR=1.179   GT:AD:DP:GQ:PL  0/1:1,3:4:20:62,0,20
pACBB_PL    6635    .   T   TC  90.61   .   AC=1;AF=0.500;AN=2;BaseQRankSum=1.150;DP=4;ExcessHet=0.0000;FS=0.000;MLEAC=1;MLEAF=0.500;MQ=60.00;MQRankSum=0.000;QD=22.65;ReadPosRankSum=1.150;SOR=1.609   GT:AD:DP:GQ:PL  0/1:1,3:4:21:98,0,21
pACBB_PL    6782    .   AC  A   633.60  .   AC=1;AF=0.500;AN=2;BaseQRankSum=-1.318;DP=67;ExcessHet=0.0000;FS=7.513;MLEAC=1;MLEAF=0.500;MQ=60.00;MQRankSum=0.000;QD=9.46;ReadPosRankSum=-0.145;SOR=1.780 GT:AD:DP:GQ:PL  0/1:38,29:67:99:641,0,1013

3.12 Filtering and Analysis

  • Variant Quality Score Recalibration or the VQSR strategy.
output_table = vcf_file.replace("vcf", "csv")

exestr = f"gatk VariantsToTable -R {reference_filename} -V {vcf_file} -F CHROM -F POS -F FILTER -F TYPE -GF AD -GF DP --show-filtered -O {output_table}"
#print(exestr)
!udocker --allow-root run -v=/content/drive/MyDrive/design_build:/design_build gatk bash -c "{exestr}"
 
 ****************************************************************************** 
 *                                                                            * 
 *               STARTING 43258f40-d39f-316f-8a89-594ce69950cc                * 
 *                                                                            * 
 ****************************************************************************** 
 executing: bash
Using GATK jar /gatk/gatk-package-4.5.0.0-local.jar
Running:
    java -Dsamjdk.use_async_io_read_samtools=false -Dsamjdk.use_async_io_write_samtools=true -Dsamjdk.use_async_io_write_tribble=false -Dsamjdk.compression_level=2 -jar /gatk/gatk-package-4.5.0.0-local.jar VariantsToTable -R /design_build/pACBB_PL.fasta -V /design_build/pACBB_PL.vcf -F CHROM -F POS -F FILTER -F TYPE -GF AD -GF DP --show-filtered -O /design_build/pACBB_PL.csv
03:45:37.746 INFO  NativeLibraryLoader - Loading libgkl_compression.so from jar:file:/gatk/gatk-package-4.5.0.0-local.jar!/com/intel/gkl/native/libgkl_compression.so
03:45:38.175 INFO  VariantsToTable - ------------------------------------------------------------
03:45:38.180 INFO  VariantsToTable - The Genome Analysis Toolkit (GATK) v4.5.0.0
03:45:38.180 INFO  VariantsToTable - For support and documentation go to https://software.broadinstitute.org/gatk/
03:45:38.182 INFO  VariantsToTable - Executing as root@adbb3a1816f8 on Linux v6.1.85+ amd64
03:45:38.182 INFO  VariantsToTable - Java runtime: OpenJDK 64-Bit Server VM v17.0.9+9-Ubuntu-122.04
03:45:38.183 INFO  VariantsToTable - Start Date/Time: June 5, 2024 at 3:45:37 AM GMT
03:45:38.183 INFO  VariantsToTable - ------------------------------------------------------------
03:45:38.183 INFO  VariantsToTable - ------------------------------------------------------------
03:45:38.185 INFO  VariantsToTable - HTSJDK Version: 4.1.0
03:45:38.185 INFO  VariantsToTable - Picard Version: 3.1.1
03:45:38.185 INFO  VariantsToTable - Built for Spark Version: 3.5.0
03:45:38.186 INFO  VariantsToTable - HTSJDK Defaults.COMPRESSION_LEVEL : 2
03:45:38.186 INFO  VariantsToTable - HTSJDK Defaults.USE_ASYNC_IO_READ_FOR_SAMTOOLS : false
03:45:38.186 INFO  VariantsToTable - HTSJDK Defaults.USE_ASYNC_IO_WRITE_FOR_SAMTOOLS : true
03:45:38.187 INFO  VariantsToTable - HTSJDK Defaults.USE_ASYNC_IO_WRITE_FOR_TRIBBLE : false
03:45:38.187 INFO  VariantsToTable - Deflater: IntelDeflater
03:45:38.187 INFO  VariantsToTable - Inflater: IntelInflater
03:45:38.188 INFO  VariantsToTable - GCS max retries/reopens: 20
03:45:38.188 INFO  VariantsToTable - Requester pays: disabled
03:45:38.189 INFO  VariantsToTable - Initializing engine
03:45:38.727 INFO  FeatureManager - Using codec VCFCodec to read file file:///design_build/pACBB_PL.vcf
03:45:38.814 INFO  VariantsToTable - Done initializing engine
03:45:38.826 WARN  VariantsToTable - Allele-specific fields will only be split if splitting multi-allelic variants is specified (`--split-multi-allelic` or `-SMA`
03:45:38.827 INFO  ProgressMeter - Starting traversal
03:45:38.828 INFO  ProgressMeter -        Current Locus  Elapsed Minutes    Variants Processed  Variants/Minute
03:45:38.896 INFO  ProgressMeter -             unmapped              0.0                    52          47272.7
03:45:38.896 INFO  ProgressMeter - Traversal complete. Processed 52 total variants in 0.0 minutes.
03:45:38.897 INFO  VariantsToTable - Shutting down engine
[June 5, 2024 at 3:45:38 AM GMT] org.broadinstitute.hellbender.tools.walkers.variantutils.VariantsToTable done. Elapsed time: 0.02 minutes.
Runtime.totalMemory()=312475648
!ls -alh
total 37M
drwx------ 2 root root 4.0K Jun  5 02:26 bam
-rw------- 1 root root  19M Jun  5 01:53 FAT01036_pass_barcode01_5eb57f2c_2b75e8f5_0.fastq.gz
-rw------- 1 root root  19M Jun  5 01:53 FAT01036_pass_barcode01_5eb57f2c_2b75e8f5_1.fastq.gz
drwx------ 2 root root 4.0K Jun  5 02:56 info
drwx------ 2 root root 4.0K Jun  5 02:14 .ipynb_checkpoints
-rw------- 1 root root   16 Jun  5 02:53 pACBB_PL.bed
-rw------- 1 root root 1.7K Jun  5 03:45 pACBB_PL.csv
-rw------- 1 root root  108 Jun  5 03:06 pACBB_PL.dict
-rw------- 1 root root 7.2K Jun  5 01:56 pACBB_PL.fasta
-rw------- 1 root root   23 Jun  5 02:53 pACBB_PL.fasta.fai
-rw------- 1 root root  15K Jun  5 01:53 pACBB-PL-from_EcoRI_hs.gb
-rw------- 1 root root  22K Jun  5 03:44 pACBB_PL.vcf
-rw------- 1 root root  285 Jun  5 03:44 pACBB_PL.vcf.idx
drwx------ 2 root root 4.0K Jun  5 02:24 sam
drwx------ 2 root root 4.0K Jun  5 03:23 sort
import pandas as pd

data = pd.read_csv("pACBB_PL.csv", sep='\t')
data
CHROM POS FILTER TYPE pACBB-PL.AD pACBB-PL.DP
0 pACBB_PL 77 PASS SNP 0,37 37
1 pACBB_PL 132 PASS INDEL 2,3 5
2 pACBB_PL 134 PASS SNP 2,3 5
3 pACBB_PL 135 PASS SNP 3,2 5
4 pACBB_PL 136 PASS SNP 1,3 4
5 pACBB_PL 138 PASS SNP 1,3 4
6 pACBB_PL 149 PASS INDEL 1,3 4
7 pACBB_PL 153 PASS INDEL 2,2 4
8 pACBB_PL 157 PASS INDEL 1,3 4
9 pACBB_PL 160 PASS INDEL 1,3 4
10 pACBB_PL 162 PASS INDEL 1,3 4
11 pACBB_PL 1161 PASS INDEL 2,2 4
12 pACBB_PL 1181 PASS SNP 1,3 4
13 pACBB_PL 1204 PASS SNP 1,3 4
14 pACBB_PL 1206 PASS SNP 0,7 7
15 pACBB_PL 1213 PASS INDEL 8,4 12
16 pACBB_PL 1326 PASS INDEL 8,2 10
17 pACBB_PL 1339 PASS SNP 9,2 11
18 pACBB_PL 1347 PASS INDEL 4,5 9
19 pACBB_PL 1355 PASS INDEL 8,2 10
20 pACBB_PL 1356 PASS INDEL 8,2 10
21 pACBB_PL 1362 PASS INDEL 8,2 10
22 pACBB_PL 1373 PASS INDEL 9,2 11
23 pACBB_PL 1399 PASS INDEL 6,6 12
24 pACBB_PL 1989 PASS SNP 0,1 1
25 pACBB_PL 2418 PASS SNP 0,14 14
26 pACBB_PL 3341 PASS INDEL 1,2 3
27 pACBB_PL 3432 PASS INDEL 1,3 4
28 pACBB_PL 3448 PASS INDEL 0,4 4
29 pACBB_PL 4165 PASS INDEL 0,1 1
30 pACBB_PL 4176 PASS INDEL 0,1 1
31 pACBB_PL 4182 PASS INDEL 0,1 1
32 pACBB_PL 4190 PASS SNP 0,1 1
33 pACBB_PL 4191 PASS INDEL 0,1 1
34 pACBB_PL 4193 PASS INDEL 0,1 1
35 pACBB_PL 4194 PASS INDEL 0,1 1
36 pACBB_PL 4201 PASS INDEL 0,1 1
37 pACBB_PL 4208 PASS INDEL 0,1 1
38 pACBB_PL 4210 PASS SNP 0,1 1
39 pACBB_PL 4214 PASS SNP 0,1 1
40 pACBB_PL 4217 PASS INDEL 0,1 1
41 pACBB_PL 4226 PASS SNP 0,1 1
42 pACBB_PL 4714 PASS INDEL 1,76 77
43 pACBB_PL 5211 PASS INDEL 0,29 29
44 pACBB_PL 5243 PASS INDEL 20,7 27
45 pACBB_PL 5326 PASS SNP 0,30 30
46 pACBB_PL 5403 PASS SNP 0,44 44
47 pACBB_PL 5897 PASS INDEL 8,9 17
48 pACBB_PL 6347 PASS INDEL 28,18 46
49 pACBB_PL 6553 PASS INDEL 1,3 4
50 pACBB_PL 6635 PASS INDEL 1,3 4
51 pACBB_PL 6782 PASS INDEL 38,29 67
  • https://igv.org/