Question - WES/WGS data

[Pharmacogenetecist]

Question rather than a issue. Could you provide suggested method/code for running WES/ WGS BAM files please. Thank you.

[mourisl]

The command is about the same for WES/WGS. You provide the BAM file through the option "-b", and another file containing the gene coordinate information for the "-c" option. You can create the coordinate file following the Input/Output section in the readme file using a gene annotation GTF file, e.g. from gencode. You can also find the file in https://github.com/mourisl/T1K_manuscript_evaluation/blob/master/hlaidx_3_44_0.tar.gz, assuming you are genotyping HLAs. You can use "--preset hla" option when genotyping HLAs.

[Pharmacogenetecist]

The command is about the same for WES/WGS. You provide the BAM file through the option "-b", and another file containing the gene coordinate information for the "-c" option. You can create the coordinate file following the Input/Output section in the readme file using a gene annotation GTF file, e.g. from gencode. You can also find the file in https://github.com/mourisl/T1K_manuscript_evaluation/blob/master/hlaidx_3_44_0.tar.gz, assuming you are genotyping HLAs. You can use "--preset hla" option when genotyping HLAs.

Thank you, I am assuming you mean the following to create the input file for -c perl t1k-build.pl -o kiridx -d kiridx/kir.da -g gencode.gtf" to create "_{dna,rna}_coord.fa" file ?

Also, is the default not to call/genotype HLA? or is --preset hla required?

[mourisl]

Yes, that is the command.

The default is for the genes without ultra-high-quality reference sequences, such as KIRs. So the default parameter will allow more mismatches during alignment. The reference sequences in HLA are very comprehensive, and aggressive read alignments will confuse the genotyper and harm the accuracy. The --preset hla is required for genotyping HLAs. It is equivalent of using "-s 0.97" which filters read alignment with less than 97% alignment similarity.

[Pharmacogenetecist]

Yes, that is the command.

The default is for the genes without ultra-high-quality reference sequences, such as KIRs. So the default parameter will allow more mismatches during alignment. The reference sequences in HLA are very comprehensive, and aggressive read alignments will confuse the genotyper and harm the accuracy. The --preset hla is required for genotyping HLAs. It is equivalent of using "-s 0.97" which filters read alignment with less than 97% alignment similarity.

Thank you, Dumb question, in the perl t1k-build.pl -o kiridx -d kiridx/kir.da -g gencode.gtf I understand the -o is where the -c file will be outputted, but what is the -d for?

I ran the script and got files of 0kb in size

[mourisl]

It is the EMBL-ENA dat file containing the allele sequences. They will be downloaded and stored in the folder after running "perl t1k-build.pl -o hlaidx --download IPD-IMGT/HLA". I think the file name will be "hla.dat" or "KIR.dat".

[Pharmacogenetecist]

It is the EMBL-ENA dat file containing the allele sequences. They will be downloaded and stored in the folder after running "perl t1k-build.pl -o hlaidx --download IPD-IMGT/HLA". I think the file name will be "hla.dat" or "KIR.dat".

Thank you! okay, I created the .fa files, it is running now and I will update soon.

When I created the -c .fa input, I get a long string of txt as follows (below is just the last few lines):

Use of uninitialized value $cols[2] in string ne at /home/smaggo/T1K/AddGeneCoord.pl line 30, line 154358. Use of uninitialized value $cols[2] in string ne at /home/smaggo/T1K/AddGeneCoord.pl line 30, line 154359. Use of uninitialized value $cols[2] in string ne at /home/smaggo/T1K/AddGeneCoord.pl line 30, line 154361. Use of uninitialized value $cols[2] in string ne at /home/smaggo/T1K/AddGeneCoord.pl line 30, line 154362. Use of uninitialized value $cols[2] in string ne at /home/smaggo/T1K/AddGeneCoord.pl line 30, line 154364. Use of uninitialized value $cols[2] in string ne at /home/smaggo/T1K/AddGeneCoord.pl line 30, line 154365. Use of uninitialized value $cols[2] in string ne at /home/smaggo/T1K/AddGeneCoord.pl line 30, line 154366. Use of uninitialized value $cols[2] in string ne at /home/smaggo/T1K/AddGeneCoord.pl line 30, line 154367. Use of uninitialized value $cols[2] in string ne at /home/smaggo/T1K/AddGeneCoord.pl line 30, line 154369. Use of uninitialized value $cols[2] in string ne at /home/smaggo/T1K/AddGeneCoord.pl line 30, line 154370. Use of uninitialized value $cols[2] in string ne at /home/smaggo/T1K/AddGeneCoord.pl line 30, line 154372. Use of uninitialized value $cols[2] in string ne at /home/smaggo/T1K/AddGeneCoord.pl line 30, line 154373. Use of uninitialized value $cols[2] in string ne at /home/smaggo/T1K/AddGeneCoord.pl line 30, line 154374. Use of uninitialized value $cols[2] in string ne at /home/smaggo/T1K/AddGeneCoord.pl line 30, line 154375. Use of uninitialized value $cols[2] in string ne at /home/smaggo/T1K/AddGeneCoord.pl line 30, line 154376. Use of uninitialized value $cols[2] in string ne at /home/smaggo/T1K/AddGeneCoord.pl line 30, line 154377. Use of uninitialized value $cols[2] in string ne at /home/smaggo/T1K/AddGeneCoord.pl line 30, line 154379. Use of uninitialized value $cols[2] in string ne at /home/smaggo/T1K/AddGeneCoord.pl line 30, line 154380. Use of uninitialized value $cols[2] in string ne at /home/smaggo/T1K/AddGeneCoord.pl line 30, line 154381. Use of uninitialized value $cols[2] in string ne at /home/smaggo/T1K/AddGeneCoord.pl line 30, line 154382. Use of uninitialized value $cols[2] in string ne at /home/smaggo/T1K/AddGeneCoord.pl line 30, line 154383. Use of uninitialized value $cols[2] in string ne at /home/smaggo/T1K/AddGeneCoord.pl line 30, line 154384. Use of uninitialized value $cols[2] in string ne at /home/smaggo/T1K/AddGeneCoord.pl line 30, line 154386. Use of uninitialized value $cols[2] in string ne at /home/smaggo/T1K/AddGeneCoord.pl line 30, line 154387. Use of uninitialized value $cols[2] in string ne at /home/smaggo/T1K/AddGeneCoord.pl line 30, line 154388. Use of uninitialized value $cols[2] in string ne at /home/smaggo/T1K/AddGeneCoord.pl line 30, line 154390. Use of uninitialized value $cols[2] in string ne at /home/smaggo/T1K/AddGeneCoord.pl line 30, line 154391. Use of uninitialized value $cols[2] in string ne at /home/smaggo/T1K/AddGeneCoord.pl line 30, line 154392. Use of uninitialized value $cols[2] in string ne at /home/smaggo/T1K/AddGeneCoord.pl line 30, line 154393. Use of uninitialized value $cols[2] in string ne at /home/smaggo/T1K/AddGeneCoord.pl line 30, line 154395. Use of uninitialized value $cols[2] in string ne at /home/smaggo/T1K/AddGeneCoord.pl line 30, line 154396. Use of uninitialized value $cols[2] in string ne at /home/smaggo/T1K/AddGeneCoord.pl line 30, line 154397. Use of uninitialized value $cols[2] in string ne at /home/smaggo/T1K/AddGeneCoord.pl line 30, line 154399.

Edit, I ran a Bam file and only got data back for HLA-DQB1

[mourisl]

Could you please show me the first a few lines of the gtf file you used?

[Pharmacogenetecist]

I got the gtf file from here: ftp://ftp.ensembl.org/pub/release-75/gtf/homo_sapiens/Homo_sapiens.GRCh37.75.gtf.gz

!genome-build GRCh37.p13

!genome-version GRCh37

!genome-date 2009-02

!genome-build-accession NCBI:GCA_000001405.14

!genebuild-last-updated 2013-09

1 pseudogene gene 11869 14412 . + . gene_id "ENSG00000223972"; gene_name "DDX11L1"; gene_source "ensembl_havana"; gene_biotype "pseudogene"; 1 processed_transcript transcript 11869 14409 . + . gene_id "ENSG00000223972"; transcript_id "ENST00000456328"; gene_name "DDX11L1"; gene_source "ensembl_havana"; gene_biotype "pseudogene"; transcript_name "DDX11L1-002"; transcript_source "havana"; 1 processed_transcript exon 11869 12227 . + . gene_id "ENSG00000223972"; transcript_id "ENST00000456328"; exon_number "1"; gene_name "DDX11L1"; gene_source "ensembl_havana"; gene_biotype "pseudogene"; transcript_name "DDX11L1-002"; transcript_source "havana"; exon_id "ENSE00002234944"; 1 processed_transcript exon 12613 12721 . + . gene_id "ENSG00000223972"; transcript_id "ENST00000456328"; exon_number "2"; gene_name "DDX11L1"; gene_source "ensembl_havana"; gene_biotype "pseudogene"; transcript_name "DDX11L1-002"; transcript_source "havana"; exon_id "ENSE00003582793"; 1 processed_transcript exon 13221 14409 . + . gene_id "ENSG00000223972"; transcript_id "ENST00000456328"; exon_number "3"; gene_name "DDX11L1"; gene_source "ensembl_havana"; gene_biotype "pseudogene"; transcript_name "DDX11L1-002"; transcript_source "havana"; exon_id "ENSE00002312635";

[mourisl]

I tried the command with Homo_sapiens.GRCh37.75.gtf but did not observe the errors you mentioned. Could you please show me the exact command you used for t1k-build?

This GTF file also represents the HLA genes on chr6 and alternative contigs, and the AddGeneCoord script used the later one when building the reference sequences. As a result, many reads aligned to the HLA regions on chr6 will not be captured. I have fixed this issue and uploaded the new script to the github. You can pull the new update and give it a try.

[Pharmacogenetecist]

t1k-build.pl -o "/home/smaggo/T1K/" -d "/home/smaggo/T1K/hlaidx/hla.dat" -g "/home/smaggo/T1K/Homo_sapiens.GRCh37.75.gtf"

[mourisl]

The command looks right to me. Not sure why it throws those warning messages before. Can you give the new script a try and share the first a few lines of the t1k_ref_dna_coord.fa file? Thank you.

[Pharmacogenetecist]

The command looks right to me. Not sure why it throws those warning messages before. Can you give the new script a try and share the first a few lines of the t1k_ref_dna_coord.fa file? Thank you.

I will do that asap and get back to you, I am currently travelling, but should be able to get back to this later today or this week. Thank you for your help.

[Pharmacogenetecist]

The command looks right to me. Not sure why it throws those warning messages before. Can you give the new script a try and share the first a few lines of the t1k_ref_dna_coord.fa file? Thank you.

I downloaded the latest T1K master from Github and redid the above process of making a coord file.

t1k-build.pl -o /home/smaggo/T1K/T1Kmaster_05012023/REFFILES/ -d /home/smaggo/T1K/T1Kmaster_05012023/hlaidx/hla.dat -g /home/smaggo/T1K/Homo_sapiens.GRCh37.75.gtf

run-t1k -b CES_Clinical3.0.bam -f /home/smaggo/T1K/T1Kmaster_05012023/hlaidx/hla.dat -c /home/smaggo/T1K/T1Kmaster_05012023/REFFILES/_dna_coord.fa --preset hla -o /home/smaggo/T1K/T1Kmaster_05012023/OUTPUT/

[Mon May 1 10:57:41 2023] run-t1k begins. [Mon May 1 10:57:41 2023] SYSTEM CALL: /home/smaggo/T1K/T1Kmaster_05012023/bam-extractor -b Clinical3.0.bam -t 1 -f /home/smaggo/T1K/T1Kmaster_05012023/REFFILES/_dna_coord.fa -o /home/smaggo/T1K/T1Kmaster_05012023/OUTPUT/_candidate [Mon May 1 10:57:42 2023] Start to extract candidate reads from bam file. Unknown genome name: chrHSCHR6_MHC_QBL system /home/smaggo/T1K/T1Kmaster_05012023/bam-extractor -b Clinical3.0.bam -t 1 -f /home/smaggo/T1K/T1Kmaster_05012023/REFFILES/_dna_coord.fa -o /home/smaggo/T1K/T1Kmaster_05012023/OUTPUT/_candidate failed: 256 at /home/smaggo/T1K/T1Kmaster_05012023/run-t1k line 59.

HEAD of coord file:

HLA-A01:01:01:01 chr19 -1 -1 + TAAAGTCCGCACGCACCCACCGGGACTCAGATTCTCCCCAGACGCCGAGGATGGCCGTCATGGCGCCCCGAACCCTCCTCCTGCTACTCTCGGGGGCCCTGGCCCTGACCCAGACCTGGGCGGGTGAGTGCGGGGTCGGGAGGGAAACCGCCTCTGCGGGGAGAAGCAAGGGGCCCTCCTGGCGGGGGCGCAGGACCGGGGGAGCCGCGCCGGGAGGAGGGTCGGGCAGGTCTCAGCCACTGCTCGCCCCCAGGCTCCCACTCCATGAGGTATTTCTTCACATCCGTGTCCCGGCCCGGCCGCGGGGAGCCCCGCTTCATCGCCGTGGGCTACGTGGACGACACGCAGTTCGTGCGGTTCGACAGCGACGCCGCGAGCCAGAAGATGGAGCCGCGGGCGCCGTGGATAGAGCAGGAGGGGCCGGAGTATTGGGACCAGGAGACACGGAATATGAAGGCCCACTCACAGACTGACCGAGCGAACCTGGGGACCCTGCGCGGCTACTACAACCAGAGCGAGGACGGTGAGTGACCCCGGCCCGGGGCGCAGGTCACGACCCCTCATCCCCCACGGACGGGCCAGGTCGCCCACAGTCTCCGGGTCCGAGATCCACCCCGAAGCCGCGGGACTCCGAGACCCTTGTCCCGGGAGAGGCCCAGGCGCCTTTACCCGGTTTCATTTTCAGTTTAGGCCAAAAATCCCCCCGGGTTGGTCGGGGCGGGGCGGGGCTCGGGGGACTGGGCTGACCGCGGGGTCGGGGCCAGGTTCTCACACCATCCAGATAATGTATGGCTGCGACGTGGGGCCGGACGGGCGCTTCCTCCGCGGGTACCGGCAGGACGCCTACGACGGCAAGGATTACATCGCCCTGAACGAGGACCTGCGCTCTTGGACCGCGGCGGACATGGCAGCTCAGATCACCAAGCGCAAGTGGGAGGCGGTCCATGCGGCGGAGCAGCGGAGAGTCTACCTGGAGGGCCGGTGCGTGGACGGGCTCCGCAGATACCTGGAGAACGGGAAGGAGACGCTGCAGCGCACGGGTACCAGGGGCCACGGGGCGCCTCCCTGATCGCCTATAGATCTCCCGGGCTGGCCTCCCACAAGGAGGGGAGACAATTGGGACCAACACTAGAATATCACCCTCCCTCTGGTCCTGAGGGAGAGGAATCCTCCTGGGTTTCCAGATCCTGTACCAGAGAGTGACTCTGAGGTTCCGCCCTGCTCTCTGACACAATTAAGGNAGGTCAGGACCAGAAGTCGCTGTTCCCTTCTCAGGGAATAGAAGATTATCCCAGGTGCCTGTGTCCAGGCTGGTGTCTGGGTTCTGTGCTCTCTTCCCCATCCCGGGTGTCCTGTCCATTCTCAAGATGGCCACATGCGTGCTGGTGGAGTGTCCCATGACAGATGCAAAATGCCTGAATTTTCTGACTCTTCCCGTCAGACCCCCCCAAGACACATATGACCCACCACCCCATCTCTGACCATGAGGCCACCCTGAGGTGCTGGGCCCTGGGCTTCTACCCTGCGGAGATCACACTGACCTGGCAGCGGGATGGGGAGGACCAGACCCAGGACACGGAGCTCGTGGAGACCAGGCCTGCAGGGGATGGAACCTTCCAGAAGTGGGCGGCTGTGGTGGTGCCTTCTGGAGAGGAGCAGAGATACACCTGCCATGTGCAGCATGAGGGTCTGCCCAAGCCCCTCACCCTGAGATGGGGTAAGGAGGGAGATGGGGGTGTCATGTCTCTTAGGGAAAGCAGGAGCCTCTCTGGAGACCTTTAGCAGGGTCAGGGCCCCTCACCTTCCCCTCTTTTCCCAGAGCTGTCTTCCCAGCCCACCATCCCCATCGTGGGCATCATTGCTGGCCTGGTTCTCCTTGGAGCTGTGATCACTGGAGCTGTGGTCGCTGCCGTGATGTGGAGGAGGAAGAGCTCAGGTGGAGAAGGGGTGAAGGGTGGGGTCTGAGATTTCTTGTCTCACTGAGGGTTCCAAGCCCCAGCTAGAAATGTGCCCTGTCTCATTACTGGGAAGCACCTTCCACAATCATGGGCCGACCCAGCCTGGGCCCTGTGTGCCAGCACTTACTCTTTTGTAAAGCACCTGTTAAAATGAAGGACAGATTTATCACCTTGATTANGGAAGGTCCCTGAGGACAGACCTCAGGAGGGCTATTGGTCCAGGACCCACACCTGCTTTCTTCATGTTTCCTGATCCCGCCCTGGGTCTGCAGTCACACATTTCTGGAAACTTCTCTGGGGTCCAAGACTAGGAGGTTCCTCTAGGACCTTAAGGCCCTGGCTCCTTTCTGGTATCTCACAGGACATTTTCTTCCCACAGATAGAAAAGGAGGGAGTTACACTCAGGCTGCAAGTAAGTATGAAGGAGGCTGATGCCTGAGGTCCTTGGGATATTGTGTTTGGGAGCCCATGGGGGAGCTCACCCACCCCACAATTCCTCCTCTAGCCACATCTTCTGTGGGATCTGACCAGGTTCTGTTTTTGTTCTACCCCAGGCAGTGACAGTGCCCAGGGCTCTGATGTGTCTCTCACAGCTTGTAAAGGTGAGAGCTTGGAGGGCCTGATGTGTGTTGGGTGTTGGGTGGAACAGTGGACACAGCTGTGCTATGGGGTTTCTTTGCGTTGGATGTATTGAGCATGCGATGGGCTGTTTAAGGTGTGACCCCTCACTGTGATGGATATGAATTTGTTCATGAATATTTTTTTCTATAGTGTGAGACAGCTGCCTTGTGTGGGACTGAGAGGCAAGAGTTGTTCCTGCCCTTCC HLA-A01:01:01:02N chr19 -1 -1 + TAAAGTCCGCACGCACCCACCGGGACTCAGATTCTCCCCAGACGCCGAGGATGGCCGTCATGGCGCCCCGAACCCTCCTCCTGCTACTCTCGGGGGCCCTGGCCCTGACCCAGACCTGGGCGGGTGAGTGCGGGGTCGGGAGGGAAACCGCCTCTGCGGGGAGAAGCAAGGGGCCCTCCTGGCGGGGGCGCAGGACCGGGGGAGCCGCGCCGGGAGGAGGGTCGGGCAGGTCTCAGCCACTGCTCGCCCCCAGGCTCCCACTCCATGAGGTATTTCTTCACATCCGTGTCCCGGCCCGGCCGCGGGGAGCCCCGCTTCATCGCCGTGGGCTACGTGGACGACACGCAGTTCGTGCGGTTCGACAGCGACGCCGCGAGCCAGAAGATGGAGCCGCGGGCGCCGTGGATAGAGCAGGAGGGGCCGGAGTATTGGGACCAGGAGACACGGAATATGAAGGCCCACTCACAGACTGACCGAGCGAACCTGGGGACCCTGCGCGGCTACTACAACCAGAGCGAGGACGGTGACCCCGGCCCGGGGCGCAGGTCACGACCCCTCATCCCCCACGGACGGGCCAGGTCGCCCACAGTCTCCGGGTCCGAGATCCACCCCGAAGCCGCGGGACTCCGAGACCCTTGTCCCGGGAGAGGCCCAGGCGCCTTTACCCGGTTTCATTTTCAGTTTAGGCCAAAAATCCCCCCGGGTTGGTCGGGGCGGGGCGGGGCTCGGGGGACTGGGCTGACCGCGGGGTCGGGGCCAGGTTCTCACACCATCCAGATAATGTATGGCTGCGACGTGGGGCCGGACGGGCGCTTCCTCCGCGGGTACCGGCAGGACGCCTACGACGGCAAGGATTACATCGCCCTGAACGAGGACCTGCGCTCTTGGACCGCGGCGGACATGGCAGCTCAGATCACCAAGCGCAAGTGGGAGGCGGTCCATGCGGCGGAGCAGCGGAGAGTCTACCTGGAGGGCCGGTGCGTGGACGGGCTCCGCAGATACCTGGAGAACGGGAAGGAGACGCTGCAGCGCACGGGTACCAGGGGCCACGGGGCGCCTCCCTGATCGCCTATAGATCTCCCGGGCTGGCCTCCCACAAGGAGGGGAGACAATTGGGACCAACACTAGAATATCACCCTCCCTCTGGTCCTGAGGGAGAGGAATCCTCCTGGGTTTCCAGATCCTGTACCAGAGAGTGACTCTGAGGTTCCGCCCTGCTCTCTGACACAATTAAGGNAGGTCAGGACCAGAAGTCGCTGTTCCCTTCTCAGGGAATAGAAGATTATCCCAGGTGCCTGTGTCCAGGCTGGTGTCTGGGTTCTGTGCTCTCTTCCCCATCCCGGGTGTCCTGTCCATTCTCAAGATGGCCACATGCGTGCTGGTGGAGTGTCCCATGACAGATGCAAAATGCCTGAATTTTCTGACTCTTCCCGTCAGACCCCCCCAAGACACATATGACCCACCACCCCATCTCTGACCATGAGGCCACCCTGAGGTGCTGGGCCCTGGGCTTCTACCCTGCGGAGATCACACTGACCTGGCAGCGGGATGGGGAGGACCAGACCCAGGACACGGAGCTCGTGGAGACCAGGCCTGCAGGGGATGGAACCTTCCAGAAGTGGGCGGCTGTGGTGGTGCCTTCTGGAGAGGAGCAGAGATACACCTGCCATGTGCAGCATGAGGGTCTGCCCAAGCCCCTCACCCTGAGATGGGGTAAGGAGGGAGATGGGGGTGTCATGTCTCTTAGGGAAAGCAGGAGCCTCTCTGGAGACCTTTAGCAGGGTCAGGGCCCCTCACCTTCCCCTCTTTTCCCAGAGCTGTCTTCCCAGCCCACCATCCCCATCGTGGGCATCATTGCTGGCCTGGTTCTCCTTGGAGCTGTGATCACTGGAGCTGTGGTCGCTGCCGTGATGTGGAGGAGGAAGAGCTCAGGTGGAGAAGGGGTGAAGGGTGGGGTCTGAGATTTCTTGTCTCACTGAGGGTTCCAAGCCCCAGCTAGAAATGTGCCCTGTCTCATTACTGGGAAGCACCTTCCACAATCATGGGCCGACCCAGCCTGGGCCCTGTGTGCCAGCACTTACTCTTTTGTAAAGCACCTGTTAAAATGAAGGACAGATTTATCACCTTGATTANGGAAGGTCCCTGAGGACAGACCTCAGGAGGGCTATTGGTCCAGGACCCACACCTGCTTTCTTCATGTTTCCTGATCCCGCCCTGGGTCTGCAGTCACACATTTCTGGAAACTTCTCTGGGGTCCAAGACTAGGAGGTTCCTCTAGGACCTTAAGGCCCTGGCTCCTTTCTGGTATCTCACAGGACATTTTCTTCCCACAGATAGAAAAGGAGGGAGTTACACTCAGGCTGCAAGTAAGTATGAAGGAGGCTGATGCCTGAGGTCCTTGGGATATTGTGTTTGGGAGCCCATGGGGGAGCTCACCCACCCCACAATTCCTCCTCTAGCCACATCTTCTGTGGGATCTGACCAGGTTCTGTTTTTGTTCTACCCCAGGCAGTGACAGTGCCCAGGGCTCTGATGTGTCTCTCACAGCTTGTAAAGGTGAGAGCTTGGAGGGCCTGATGTGTGTTGGGTGTTGGGTGGAACAGTGGACACAGCTGTGCTATGGGGTTTCTTTGCGTTGGATGTATTGAGCATGCGATGGGCTGTTTAAGGTGTGACCCCTCACTGTGATGGATATGAATTTGTTCATGAATATTTTTTTCTATAGTGTGAGACAGCTGCCTTGTGTGGGACTGAGAGGCAAGAGTTGTTCCTGCCCTTCC HLA-A01:01:01:03 chr19 -1 -1 + TAAAGTCCGCACGCACCCACCGGGACTCAGATTCTCCCCAGACGCCGAGGATGGCCGTCATGGCGCCCCGAACCCTCCTCCTGCTACTCTCGGGGGCCCTGGCCCTGACCCAGACCTGGGCGGGTGAGTGCGGGGTCGGGAGGGAAACCGCCTCTGCGGGGAGAAGCAAGGGGCCCTCCTGGCGGGGGCGCAGGACCGGGGGAGCCGCGCGGGGAGGAGGGTCGGGCAGGTCTCAGCCACTGCTCGCCCCCAGGCTCCCACTCCATGAGGTATTTCTTCACATCCGTGTCCCGGCCCGGCCGCGGGGAGCCCCGCTTCATCGCCGTGGGCTACGTGGACGACACGCAGTTCGTGCGGTTCGACAGCGACGCCGCGAGCCAGAAGATGGAGCCGCGGGCGCCGTGGATAGAGCAGGAGGGGCCGGAGTATTGGGACCAGGAGACACGGAATATGAAGGCCCACTCACAGACTGACCGAGCGAACCTGGGGACCCTGCGCGGCTACTACAACCAGAGCGAGGACGGTGAGTGACCCCGGCCCGGGGCGCAGGTCACGACCCCTCATCCCCCACGGACGGGCCAGGTCGCCCACAGTCTCCGGGTCCGAGATCCACCCCGAAGCCGCGGGACTCCGAGACCCTTGTCCCGGGAGAGGCCCAGGCGCCTTTACCCGGTTTCATTTTCAGTTTAGGCCAAAAATCCCCCCGGGTTGGTCGGGGCGGGGCGGGGCTCGGGGGACTGGGCTGACCGCGGGGTCGGGGCCAGGTTCTCACACCATCCAGATAATGTATGGCTGCGACGTGGGGCCGGACGGGCGCTTCCTCCGCGGGTACCGGCAGGACGCCTACGACGGCAAGGATTACATCGCCCTGAACGAGGACCTGCGCTCTTGGACCGCGGCGGACATGGCAGCTCAGATCACCAAGCGCAAGTGGGAGGCGGTCCATGCGGCGGAGCAGCGGAGAGTCTACCTGGAGGGCCGGTGCGTGGACGGGCTCCGCAGATACCTGGAGAACGGGAAGGAGACGCTGCAGCGCACGGGTACCAGGGGCCACGGGGCGCCTCCCTGATCGCCTATAGATCTCCCGGGCTGGCCTCCCACAAGGAGGGGAGACAATTGGGACCAACACTAGAATATCACCCTCCCTCTGGTCCTGAGGGAGAGGAATCCTCCTGGGTTTCCAGATCCTGTACCAGAGAGTGACTCTGAGGTTCCGCCCTGCTCTCTGACACAATTAAGGNAGGTCAGGACCAGAAGTCGCTGTTCCCTTCTCAGGGAATAGAAGATTATCCCAGGTGCCTGTGTCCAGGCTGGTGTCTGGGTTCTGTGCTCTCTTCCCCATCCCGGGTGTCCTGTCCATTCTCAAGATGGCCACATGCGTGCTGGTGGAGTGTCCCATGACAGATGCAAAATGCCTGAATTTTCTGACTCTTCCCGTCAGACCCCCCCAAGACACATATGACCCACCACCCCATCTCTGACCATGAGGCCACCCTGAGGTGCTGGGCCCTGGGCTTCTACCCTGCGGAGATCACACTGACCTGGCAGCGGGATGGGGAGGACCAGACCCAGGACACGGAGCTCGTGGAGACCAGGCCTGCAGGGGATGGAACCTTCCAGAAGTGGGCGGCTGTGGTGGTGCCTTCTGGAGAGGAGCAGAGATACACCTGCCATGTGCAGCATGAGGGTCTGCCCAAGCCCCTCACCCTGAGATGGGGTAAGGAGGGAGATGGGGGTGTCATGTCTCTTAGGGAAAGCAGGAGCCTCTCTGGAGACCTTTAGCAGGGTCAGGGCCCCTCACCTTCCCCTCTTTTCCCAGAGCTGTCTTCCCAGCCCACCATCCCCATCGTGGGCATCATTGCTGGCCTGGTTCTCCTTGGAGCTGTGATCACTGGAGCTGTGGTCGCTGCCGTGATGTGGAGGAGGAAGAGCTCAGGTGGAGAAGGGGTGAAGGGTGGGGTCTGAGATTTCTTGTCTCACTGAGGGTTCCAAGCCCCAGCTAGAAATGTGCCCTGTCTCATTACTGGGAAGCACCTTCCACAATCATGGGCCGACCCAGCCTGGGCCCTGTGTGCCAGCACTTACTCTTTTGTAAAGCACCTGTTAAAATGAAGGACAGATTTATCACCTTGATTANGGAAGGTCCCTGAGGACAGACCTCAGGAGGGCTATTGGTCCAGGACCCACACCTGCTTTCTTCATGTTTCCTGATCCCGCCCTGGGTCTGCAGTCACACATTTCTGGAAACTTCTCTGGGGTCCAAGACTAGGAGGTTCCTCTAGGACCTTAAGGCCCTGGCTCCTTTCTGGTATCTCACAGGACATTTTCTTCCCACAGATAGAAAAGGAGGGAGTTACACTCAGGCTGCAAGTAAGTATGAAGGAGGCTGATGCCTGAGGTCCTTGGGATATTGTGTTTGGGAGCCCATGGGGGAGCTCACCCACCCCACAATTCCTCCTCTAGCCACATCTTCTGTGGGATCTGACCAGGTTCTGTTTTTGTTCTACCCCAGGCAGTGACAGTGCCCAGGGCTCTGATGTGTCTCTCACAGCTTGTAAAGGTGAGAGCTTGGAGGGCCTGATGTGTGTTGGGTGTTGGGTGGAACAGTGGACACAGCTGTGCTATGGGGTTTCTTTGCGTTGGATGTATTGAGCATGCGATGGGCTGTTTAAGGTGTGACCCCTCACTGTGATGGATATGAATTTGTTCATGAATATTTTTTTCTATAGTGTGAGACAGCTGCCTTGTGTGGGACTGAGAGGCAAGAGTTGTTCCTGCCCTTCC HLA-A01:01:01:04 chr19 -1 -1 + TAAAGTCCGCACGCACCCACCGGGACTCAGATTCTCCCCAGACGCCGAGGATGGCCGTCATGGCGCCCCGAACCCTCCTCCTGCTACTCTCGGGGGCCCTGGCCCTGACCCAGACCTGGGCGGGTGAGTGCGGGGTCGGGAGGGAAACCGCCTCTGCGGGGAGAAGCAAGGGGCCCTCCTGGCGGGGGCGCAGGACCGGGGGAGCCGCGCCGGGAGGAGGGTCGGGCAGGTCTCAGCCACTGCTCGCCCCCAGGCTCCCACTCCATGAGGTATTTCTTCACATCCGTGTCCCGGCCCGGCCGCGGGGAGCCCCGCTTCATCGCCGTGGGCTACGTGGACGACACGCAGTTCGTGCGGTTCGACAGCGACGCCGCGAGCCAGAAGATGGAGCCGCGGGCGCCGTGGATAGAGCAGGAGGGGCCGGAGTATTGGGACCAGGAGACACGGAATATGAAGGCCCACTCACAGACTGACCGAGCGAACCTGGGGACCCTGCGCGGCTACTACAACCAGAGCGAGGACGGTGAGTGACCCCGGCCCGGGGCGCAGGTCACGACCCCTCATCCCCCACGGACGGGCCAGGTCGCCCACAGTCTCCGGGTCCGAGATCCACCCCGAAGCCGCGGGACTCCGAGACCCTTGTCCCGGGAGAGGCCCAGGCGCCTTTACCCGGTTTCATTTTCAGTTTAGGCCAAAAATCCCCCCGGGTTGGTCGGGGCGGGGCGGGGCTCGGGGGACTGGGCTGACCGCGGGGTCGGGGCCAGGTTCTCACACCATCCAGATAATGTATGGCTGCGACGTGGGGCCGGACGGGCGCTTCCTCCGCGGGTACCGGCAGGACGCCTACGACGGCAAGGATTACATCGCCCTGAACGAGGACCTGCGCTCTTGGACCGCGGCGGACATGGCAGCTCAGATCACCAAGCGCAAGTGGGAGGCGGTCCATGCGGCGGAGCAGCGGAGAGTCTACCTGGAGGGCCGGTGCGTGGACGGGCTCCGCAGATACCTGGAGAACGGGAAGGAGACGCTGCAGCGCACGGGTACCAGGGGCCACGGGGCGCCTCCCTGATCGCCTATAGATCTCCCGGGCTGGCCTCCCACAAGGAGGGGAGACAATTGGGACCAACACTAGAATATCACCCTCCCTCTGGTCCTGAGGGAGAGGAATCCTCCTGGGTTTCCAGATCCTGTACCAGAGAGTGACTCTGAGGTTCCGCCCTGCTCTCTGACACAATTAAGGNAGGTCAGGACCAGAAGTCGCTGTTCCCTTCTCAGGGAATAGAAGATTATCCCAGGTGCCTGTGTCCAGGCTGGTGTCTGGGTTCTGTGCTCTCTTCCCCATCCCGGGTGTCCTGTCCATTCTCAAGATGGCCACATGCGTGCTGGTGGAGTGTCCCATGACAGATGCAAAATGCCTGAATTTTCTGACTCTTCCCGTCAGACCCCCCCAAGACACATATGACCCACCACCCCATCTCTGACCATGAGGCCACCCTGAGGTGCTGGGCCCTGGGCTTCTACCCTGCGGAGATCACACTGACCTGGCAGCGGGATGGGGAGGACCAGACCCAGGACACGGAGCTCGTGGAGACCAGGCCTGCAGGGGATGGAACCTTCCAGAAGTGGGCGGCTGTGGTGGTGCCTTCTGGAGAGGAGCAGAGATACACCTGCCATGTGCAGCATGAGGGTCTGCCCAAGCCCCTCACCCTGAGATGGGGTAAGGAGGGAGATGGGGGTGTCATGTCTCTTAGGGAAAGCAGGAGCCTCTCTGGAGACCTTTAGCAGGGTCAGGGCCCCTCACCTTCCCCTCTTTTCCCAGAGCTGTCTTCCCAGCCCACCATCCCCATCGTGGGCATCATTGCTGGCCTGGTTCTCCTTGGAGCTGTGATCACTGGAGCTGTGGTCGCTGCCGTGATGTGGAGGAGGAAGAGCTCAGGTGGAGAAGGGGTGAAGGGTGGGGTCTGAGATTTCTTGTCTCACTGAGGGTTCCAAGCCCCAGCTAGAAATGTGCCCTGTCTCATTACTGGGAAGCACCTTCCACAATCATGGGCCGACCCAGCCTGGGCCCTGTGTGCCAGCACTTACTCTTTTGTAAAGCACCTGTTAAAATGAAGGACAGATTTATCACCTTGATTANGGAAGGTCCCTGAGGACAGACCTCAGGAGGGCTATTGGTCCAGGACCCACACCTGCTTTCTTCATGTTTCCTGATCCCGCCCTGGGTCTGCAGTCACACATTTCTGGAAACTTCTCTGGGGTCCAAGACTAGGAGGTTCCTCTAGGACCTTAAGGCCCTGGCTCCTTTCTGGTATCTCACAGGACATTTTCTTCCCACAGATAGAAAAGGAGGGAGTTACACTCAGGCTGCAAGTAAGTATGAAGGAGGCTGATGCCTGAGGTCCTTGGGATATTGTGTTTGGGAGCCCATGGGGGAGCTCACCCACCCCACAATTCCTCCTCTAGCCACATCTTCTGTGGGATCTGACCAGGTTCTGTTTTTGTTCTACCCCAGGCAGTGACAGTGCCCAGGGCTCTGATGTGTCTCTCACAGCTTGTAAAGGTGAGAGCTTGGAGGGCCTGATGTGTGTTGGGTGTTGGGTGGAACAGTGGACACAGCTGTGCTATGGGGTTTCTTTGCGTTGGATGTATTGAGCATGCGATGGGCTGTTTAAGGTGTGACCCCTCACTGTGATGGATATGAATTTGTTCATGAATATTTTTTTCTATAGTGTGAGACAGCTGCCTTGTGTGGGACTGAGAGGCAAGAGTTGTTCCTGCCCTTCC HLA-A*01:01:01:05 chr19 -1 -1 + TAAAGTCCGCACGCACCCACCGGGACTCAGATTCTCCCCAGACGCCGAGGATGGCCGTCATGGCGCCCCGAACCCTCCTCCTGCTACTCTCGGGGGCCCTGGCCCTGACCCAGACCTGGGCGGGTGAGTGCGGGGTCGGGAGGGAAACCGCCTCTGCGGGGAGAAGCAAGGGGCCCTCCTGGCGGGGGCGCAGGACCGGGGGAGCCGCGCCGGGAGGAGGGTCGGGCAGGTCTCAGCCACTGCTCGCCCCCAGGCTCCCACTCCATGAGGTATTTCTTCACATCCGTGTCCCGGCCCGGCCGCGGGGAGCCCCGCTTCATCGCCGTGGGCTACGTGGACGACACGCAGTTCGTGCGGTTCGACAGCGACGCCGCGAGCCAGAAGATGGAGCCGCGGGCGCCGTGGATAGAGCAGGAGGGGCCGGAGTATTGGGACCAGGAGACACGGAATATGAAGGCCCACTCACAGACTGACCGAGCGAACCTGGGGACCCTGCGCGGCTACTACAACCAGAGCGAGGACGGTGAGTGACCCCGGCCCGGGGCGCAGGTCACGACCCCTCATCCCCCACGGACGGGCCAGGTCGCCCACAGTCTCCGGGTCCGAGATCCGCCCCGAAGCCGCGGGACCCCGAGACCCTTGCCCCGGGAGAGGCCCAGGCGCCTTTACCCGGTTTCATTTTCAGTTTAGGCCAAAAATCCCCCCGGGTTGGTCGGGGCGGGGCGGGGCTCGGGGGACTGGGCTGACCGCGGGGTCGGGGCCAGGTTCTCACACCATCCAGATAATGTATGGCTGCGACGTGGGGCCGGACGGGCGCTTCCTCCGCGGGTACCGGCAGGACGCCTACGACGGCAAGGATTACATCGCCCTGAACGAGGACCTGCGCTCTTGGACCGCGGCGGACATGGCAGCTCAGATCACCAAGCGCAAGTGGGAGGCGGTCCATGCGGCGGAGCAGCGGAGAGTCTACCTGGAGGGCCGGTGCGTGGACGGGCTCCGCAGATACCTGGAGAACGGGAAGGAGACGCTGCAGCGCACGGGTACCAGGGGCCACGGGGCGCCTCCCTGATCGCCTATAGATCTCCCGGGCTGGCCTCCCACAAGGAGGGGAGACAATTGGGACCAACACTAGAATATCACCCTCCCTCTGGTCCTGAGGGAGAGGAATCCTCCTGGGTTTCCAGATCCTGTACCAGAGAGTGACTCTGAGGTTCCGCCCTGCTCTCTGACACAATTAAGGNAGGTCAGGACCAGAAGTCGCTGTTCCCTTCTCAGGGAATAGAAGATTATCCCAGGTGCCTGTGTCCAGGCTGGTGTCTGGGTTCTGTGCTCTCTTCCCCATCCCGGGTGTCCTGTCCATTCTCAAGATGGCCACATGCGTGCTGGTGGAGTGTCCCATGACAGATGCAAAATGCCTGAATTTTCTGACTCTTCCCGTCAGACCCCCCCAAGACACATATGACCCACCACCCCATCTCTGACCATGAGGCCACCCTGAGGTGCTGGGCCCTGGGCTTCTACCCTGCGGAGATCACACTGACCTGGCAGCGGGATGGGGAGGACCAGACCCAGGACACGGAGCTCGTGGAGACCAGGCCTGCAGGGGATGGAACCTTCCAGAAGTGGGCGGCTGTGGTGGTGCCTTCTGGAGAGGAGCAGAGATACACCTGCCATGTGCAGCATGAGGGTCTGCCCAAGCCCCTCACCCTGAGATGGGGTAAGGAGGGAGATGGGGGTGTCATGTCTCTTAGGGAAAGCAGGAGCCTCTCTGGAGACCTTTAGCAGGGTCAGGGCCCCTCACCTTCCCCTCTTTTCCCAGAGCTGTCTTCCCAGCCCACCATCCCCATCGTGGGCATCATTGCTGGCCTGGTTCTCCTTGGAGCTGTGATCACTGGAGCTGTGGTCGCTGCCGTGATGTGGAGGAGGAAGAGCTCAGGTGGAGAAGGGGTGAAGGGTGGGGTCTGAGATTTCTTGTCTCACTGAGGGTTCCAAGCCCCAGCTAGAAATGTGCCCTGTCTCATTACTGGGAAGCACCTTCCACAATCATGGGCCGACCCAGCCTGGGCCCTGTGTGCCAGCACTTACTCTTTTGTAAAGCACCTGTTAAAATGAAGGACAGATTTATCACCTTGATTANGGAAGGTCCCTGAGGACAGACCTCAGGAGGGCTATTGGTCCAGGACCCACACCTGCTTTCTTCATGTTTCCTGATCCCGCCCTGGGTCTGCAGTCACACATTTCTGGAAACTTCTCTGGGGTCCAAGACTAGGAGGTTCCTCTAGGACCTTAAGGCCCTGGCTCCTTTCTGGTATCTCACAGGACATTTTCTTCCCACAGATAGAAAAGGAGGGAGTTACACTCAGGCTGCAAGTAAGTATGAAGGAGGCTGATGCCTGAGGTCCTTGGGATATTGTGTTTGGGAGCCCATGGGGGAGCTCACCCACCCCACAATTCCTCCTCTAGCCACATCTTCTGTGGGATCTGACCAGGTTCTGTTTTTGTTCTACCCCAGGCAGTGACAGTGCCCAGGGCTCTGATGTGTCTCTCACAGCTTGTAAAGGTGAGAGCTTGGAGGGCCTGATGTGTGTTGGGTGTTGGGTGGAACAGTGGACACAGCTGTGCTATGGGGTTTCTTTGCGTTGGATGTATTGAGCATGCGATGGGCTGTTTAAGGTGTGACCCCTCACTGTGATGGATATGAATTTGTTCATGAATATTTTTTTCTATAGTGTGAGACAGCTGCCTTGTGTGGGACTGAGAGGCAAGAGTTGTTCCTGCCCTTCC

[mourisl]

It's strange that the header of the gene coordinate should be on chr6. The t1k-build script should output the files to the REFILES/REFILES_XXX.fa , does the file /home/smaggo/T1K/T1Kmaster_05012023/REFFILES/REFILES_dna_coord.fa exist with correct headers? Thank you.

[Pharmacogenetecist]

It's strange that the header of the gene coordinate should be on chr6. The t1k-build script should output the files to the REFILES/REFILES_XXX.fa , does the file /home/smaggo/T1K/T1Kmaster_05012023/REFFILES/REFILES_dna_coord.fa exist with correct headers? Thank you.

Apologies, here is the new coord file generated by your update:

HLA-A01:01:01:01 chr6 29909037 29913661 + TAAAGTCCGCACGCACCCACCGGGACTCAGATTCTCCCCAGACGCCGAGGATGGCCGTCATGGCGCCCCGAACCCTCCTCCTGCTACTCTCGGGGGCCCTGGCCCTGACCCAGACCTGGGCGGGTGAGTGCGGGGTCGGGAGGGAAACCGCCTCTGCGGGGAGAAGCAAGGGGCCCTCCTGGCGGGGGCGCAGGACCGGGGGAGCCGCGCCGGGAGGAGGGTCGGGCAGGTCTCAGCCACTGCTCGCCCCCAGGCTCCCACTCCATGAGGTATTTCTTCACATCCGTGTCCCGGCCCGGCCGCGGGGAGCCCCGCTTCATCGCCGTGGGCTACGTGGACGACACGCAGTTCGTGCGGTTCGACAGCGACGCCGCGAGCCAGAAGATGGAGCCGCGGGCGCCGTGGATAGAGCAGGAGGGGCCGGAGTATTGGGACCAGGAGACACGGAATATGAAGGCCCACTCACAGACTGACCGAGCGAACCTGGGGACCCTGCGCGGCTACTACAACCAGAGCGAGGACGGTGAGTGACCCCGGCCCGGGGCGCAGGTCACGACCCCTCATCCCCCACGGACGGGCCAGGTCGCCCACAGTCTCCGGGTCCGAGATCCACCCCGAAGCCGCGGGACTCCGAGACCCTTGTCCCGGGAGAGGCCCAGGCGCCTTTACCCGGTTTCATTTTCAGTTTAGGCCAAAAATCCCCCCGGGTTGGTCGGGGCGGGGCGGGGCTCGGGGGACTGGGCTGACCGCGGGGTCGGGGCCAGGTTCTCACACCATCCAGATAATGTATGGCTGCGACGTGGGGCCGGACGGGCGCTTCCTCCGCGGGTACCGGCAGGACGCCTACGACGGCAAGGATTACATCGCCCTGAACGAGGACCTGCGCTCTTGGACCGCGGCGGACATGGCAGCTCAGATCACCAAGCGCAAGTGGGAGGCGGTCCATGCGGCGGAGCAGCGGAGAGTCTACCTGGAGGGCCGGTGCGTGGACGGGCTCCGCAGATACCTGGAGAACGGGAAGGAGACGCTGCAGCGCACGGGTACCAGGGGCCACGGGGCGCCTCCCTGATCGCCTATAGATCTCCCGGGCTGGCCTCCCACAAGGAGGGGAGACAATTGGGACCAACACTAGAATATCACCCTCCCTCTGGTCCTGAGGGAGAGGAATCCTCCTGGGTTTCCAGATCCTGTACCAGAGAGTGACTCTGAGGTTCCGCCCTGCTCTCTGACACAATTAAGGNAGGTCAGGACCAGAAGTCGCTGTTCCCTTCTCAGGGAATAGAAGATTATCCCAGGTGCCTGTGTCCAGGCTGGTGTCTGGGTTCTGTGCTCTCTTCCCCATCCCGGGTGTCCTGTCCATTCTCAAGATGGCCACATGCGTGCTGGTGGAGTGTCCCATGACAGATGCAAAATGCCTGAATTTTCTGACTCTTCCCGTCAGACCCCCCCAAGACACATATGACCCACCACCCCATCTCTGACCATGAGGCCACCCTGAGGTGCTGGGCCCTGGGCTTCTACCCTGCGGAGATCACACTGACCTGGCAGCGGGATGGGGAGGACCAGACCCAGGACACGGAGCTCGTGGAGACCAGGCCTGCAGGGGATGGAACCTTCCAGAAGTGGGCGGCTGTGGTGGTGCCTTCTGGAGAGGAGCAGAGATACACCTGCCATGTGCAGCATGAGGGTCTGCCCAAGCCCCTCACCCTGAGATGGGGTAAGGAGGGAGATGGGGGTGTCATGTCTCTTAGGGAAAGCAGGAGCCTCTCTGGAGACCTTTAGCAGGGTCAGGGCCCCTCACCTTCCCCTCTTTTCCCAGAGCTGTCTTCCCAGCCCACCATCCCCATCGTGGGCATCATTGCTGGCCTGGTTCTCCTTGGAGCTGTGATCACTGGAGCTGTGGTCGCTGCCGTGATGTGGAGGAGGAAGAGCTCAGGTGGAGAAGGGGTGAAGGGTGGGGTCTGAGATTTCTTGTCTCACTGAGGGTTCCAAGCCCCAGCTAGAAATGTGCCCTGTCTCATTACTGGGAAGCACCTTCCACAATCATGGGCCGACCCAGCCTGGGCCCTGTGTGCCAGCACTTACTCTTTTGTAAAGCACCTGTTAAAATGAAGGACAGATTTATCACCTTGATTANGGAAGGTCCCTGAGGACAGACCTCAGGAGGGCTATTGGTCCAGGACCCACACCTGCTTTCTTCATGTTTCCTGATCCCGCCCTGGGTCTGCAGTCACACATTTCTGGAAACTTCTCTGGGGTCCAAGACTAGGAGGTTCCTCTAGGACCTTAAGGCCCTGGCTCCTTTCTGGTATCTCACAGGACATTTTCTTCCCACAGATAGAAAAGGAGGGAGTTACACTCAGGCTGCAAGTAAGTATGAAGGAGGCTGATGCCTGAGGTCCTTGGGATATTGTGTTTGGGAGCCCATGGGGGAGCTCACCCACCCCACAATTCCTCCTCTAGCCACATCTTCTGTGGGATCTGACCAGGTTCTGTTTTTGTTCTACCCCAGGCAGTGACAGTGCCCAGGGCTCTGATGTGTCTCTCACAGCTTGTAAAGGTGAGAGCTTGGAGGGCCTGATGTGTGTTGGGTGTTGGGTGGAACAGTGGACACAGCTGTGCTATGGGGTTTCTTTGCGTTGGATGTATTGAGCATGCGATGGGCTGTTTAAGGTGTGACCCCTCACTGTGATGGATATGAATTTGTTCATGAATATTTTTTTCTATAGTGTGAGACAGCTGCCTTGTGTGGGACTGAGAGGCAAGAGTTGTTCCTGCCCTTCC HLA-A01:01:01:02N chr6 29909037 29913661 + TAAAGTCCGCACGCACCCACCGGGACTCAGATTCTCCCCAGACGCCGAGGATGGCCGTCATGGCGCCCCGAACCCTCCTCCTGCTACTCTCGGGGGCCCTGGCCCTGACCCAGACCTGGGCGGGTGAGTGCGGGGTCGGGAGGGAAACCGCCTCTGCGGGGAGAAGCAAGGGGCCCTCCTGGCGGGGGCGCAGGACCGGGGGAGCCGCGCCGGGAGGAGGGTCGGGCAGGTCTCAGCCACTGCTCGCCCCCAGGCTCCCACTCCATGAGGTATTTCTTCACATCCGTGTCCCGGCCCGGCCGCGGGGAGCCCCGCTTCATCGCCGTGGGCTACGTGGACGACACGCAGTTCGTGCGGTTCGACAGCGACGCCGCGAGCCAGAAGATGGAGCCGCGGGCGCCGTGGATAGAGCAGGAGGGGCCGGAGTATTGGGACCAGGAGACACGGAATATGAAGGCCCACTCACAGACTGACCGAGCGAACCTGGGGACCCTGCGCGGCTACTACAACCAGAGCGAGGACGGTGACCCCGGCCCGGGGCGCAGGTCACGACCCCTCATCCCCCACGGACGGGCCAGGTCGCCCACAGTCTCCGGGTCCGAGATCCACCCCGAAGCCGCGGGACTCCGAGACCCTTGTCCCGGGAGAGGCCCAGGCGCCTTTACCCGGTTTCATTTTCAGTTTAGGCCAAAAATCCCCCCGGGTTGGTCGGGGCGGGGCGGGGCTCGGGGGACTGGGCTGACCGCGGGGTCGGGGCCAGGTTCTCACACCATCCAGATAATGTATGGCTGCGACGTGGGGCCGGACGGGCGCTTCCTCCGCGGGTACCGGCAGGACGCCTACGACGGCAAGGATTACATCGCCCTGAACGAGGACCTGCGCTCTTGGACCGCGGCGGACATGGCAGCTCAGATCACCAAGCGCAAGTGGGAGGCGGTCCATGCGGCGGAGCAGCGGAGAGTCTACCTGGAGGGCCGGTGCGTGGACGGGCTCCGCAGATACCTGGAGAACGGGAAGGAGACGCTGCAGCGCACGGGTACCAGGGGCCACGGGGCGCCTCCCTGATCGCCTATAGATCTCCCGGGCTGGCCTCCCACAAGGAGGGGAGACAATTGGGACCAACACTAGAATATCACCCTCCCTCTGGTCCTGAGGGAGAGGAATCCTCCTGGGTTTCCAGATCCTGTACCAGAGAGTGACTCTGAGGTTCCGCCCTGCTCTCTGACACAATTAAGGNAGGTCAGGACCAGAAGTCGCTGTTCCCTTCTCAGGGAATAGAAGATTATCCCAGGTGCCTGTGTCCAGGCTGGTGTCTGGGTTCTGTGCTCTCTTCCCCATCCCGGGTGTCCTGTCCATTCTCAAGATGGCCACATGCGTGCTGGTGGAGTGTCCCATGACAGATGCAAAATGCCTGAATTTTCTGACTCTTCCCGTCAGACCCCCCCAAGACACATATGACCCACCACCCCATCTCTGACCATGAGGCCACCCTGAGGTGCTGGGCCCTGGGCTTCTACCCTGCGGAGATCACACTGACCTGGCAGCGGGATGGGGAGGACCAGACCCAGGACACGGAGCTCGTGGAGACCAGGCCTGCAGGGGATGGAACCTTCCAGAAGTGGGCGGCTGTGGTGGTGCCTTCTGGAGAGGAGCAGAGATACACCTGCCATGTGCAGCATGAGGGTCTGCCCAAGCCCCTCACCCTGAGATGGGGTAAGGAGGGAGATGGGGGTGTCATGTCTCTTAGGGAAAGCAGGAGCCTCTCTGGAGACCTTTAGCAGGGTCAGGGCCCCTCACCTTCCCCTCTTTTCCCAGAGCTGTCTTCCCAGCCCACCATCCCCATCGTGGGCATCATTGCTGGCCTGGTTCTCCTTGGAGCTGTGATCACTGGAGCTGTGGTCGCTGCCGTGATGTGGAGGAGGAAGAGCTCAGGTGGAGAAGGGGTGAAGGGTGGGGTCTGAGATTTCTTGTCTCACTGAGGGTTCCAAGCCCCAGCTAGAAATGTGCCCTGTCTCATTACTGGGAAGCACCTTCCACAATCATGGGCCGACCCAGCCTGGGCCCTGTGTGCCAGCACTTACTCTTTTGTAAAGCACCTGTTAAAATGAAGGACAGATTTATCACCTTGATTANGGAAGGTCCCTGAGGACAGACCTCAGGAGGGCTATTGGTCCAGGACCCACACCTGCTTTCTTCATGTTTCCTGATCCCGCCCTGGGTCTGCAGTCACACATTTCTGGAAACTTCTCTGGGGTCCAAGACTAGGAGGTTCCTCTAGGACCTTAAGGCCCTGGCTCCTTTCTGGTATCTCACAGGACATTTTCTTCCCACAGATAGAAAAGGAGGGAGTTACACTCAGGCTGCAAGTAAGTATGAAGGAGGCTGATGCCTGAGGTCCTTGGGATATTGTGTTTGGGAGCCCATGGGGGAGCTCACCCACCCCACAATTCCTCCTCTAGCCACATCTTCTGTGGGATCTGACCAGGTTCTGTTTTTGTTCTACCCCAGGCAGTGACAGTGCCCAGGGCTCTGATGTGTCTCTCACAGCTTGTAAAGGTGAGAGCTTGGAGGGCCTGATGTGTGTTGGGTGTTGGGTGGAACAGTGGACACAGCTGTGCTATGGGGTTTCTTTGCGTTGGATGTATTGAGCATGCGATGGGCTGTTTAAGGTGTGACCCCTCACTGTGATGGATATGAATTTGTTCATGAATATTTTTTTCTATAGTGTGAGACAGCTGCCTTGTGTGGGACTGAGAGGCAAGAGTTGTTCCTGCCCTTCC HLA-A01:01:01:03 chr6 29909037 29913661 + TAAAGTCCGCACGCACCCACCGGGACTCAGATTCTCCCCAGACGCCGAGGATGGCCGTCATGGCGCCCCGAACCCTCCTCCTGCTACTCTCGGGGGCCCTGGCCCTGACCCAGACCTGGGCGGGTGAGTGCGGGGTCGGGAGGGAAACCGCCTCTGCGGGGAGAAGCAAGGGGCCCTCCTGGCGGGGGCGCAGGACCGGGGGAGCCGCGCGGGGAGGAGGGTCGGGCAGGTCTCAGCCACTGCTCGCCCCCAGGCTCCCACTCCATGAGGTATTTCTTCACATCCGTGTCCCGGCCCGGCCGCGGGGAGCCCCGCTTCATCGCCGTGGGCTACGTGGACGACACGCAGTTCGTGCGGTTCGACAGCGACGCCGCGAGCCAGAAGATGGAGCCGCGGGCGCCGTGGATAGAGCAGGAGGGGCCGGAGTATTGGGACCAGGAGACACGGAATATGAAGGCCCACTCACAGACTGACCGAGCGAACCTGGGGACCCTGCGCGGCTACTACAACCAGAGCGAGGACGGTGAGTGACCCCGGCCCGGGGCGCAGGTCACGACCCCTCATCCCCCACGGACGGGCCAGGTCGCCCACAGTCTCCGGGTCCGAGATCCACCCCGAAGCCGCGGGACTCCGAGACCCTTGTCCCGGGAGAGGCCCAGGCGCCTTTACCCGGTTTCATTTTCAGTTTAGGCCAAAAATCCCCCCGGGTTGGTCGGGGCGGGGCGGGGCTCGGGGGACTGGGCTGACCGCGGGGTCGGGGCCAGGTTCTCACACCATCCAGATAATGTATGGCTGCGACGTGGGGCCGGACGGGCGCTTCCTCCGCGGGTACCGGCAGGACGCCTACGACGGCAAGGATTACATCGCCCTGAACGAGGACCTGCGCTCTTGGACCGCGGCGGACATGGCAGCTCAGATCACCAAGCGCAAGTGGGAGGCGGTCCATGCGGCGGAGCAGCGGAGAGTCTACCTGGAGGGCCGGTGCGTGGACGGGCTCCGCAGATACCTGGAGAACGGGAAGGAGACGCTGCAGCGCACGGGTACCAGGGGCCACGGGGCGCCTCCCTGATCGCCTATAGATCTCCCGGGCTGGCCTCCCACAAGGAGGGGAGACAATTGGGACCAACACTAGAATATCACCCTCCCTCTGGTCCTGAGGGAGAGGAATCCTCCTGGGTTTCCAGATCCTGTACCAGAGAGTGACTCTGAGGTTCCGCCCTGCTCTCTGACACAATTAAGGNAGGTCAGGACCAGAAGTCGCTGTTCCCTTCTCAGGGAATAGAAGATTATCCCAGGTGCCTGTGTCCAGGCTGGTGTCTGGGTTCTGTGCTCTCTTCCCCATCCCGGGTGTCCTGTCCATTCTCAAGATGGCCACATGCGTGCTGGTGGAGTGTCCCATGACAGATGCAAAATGCCTGAATTTTCTGACTCTTCCCGTCAGACCCCCCCAAGACACATATGACCCACCACCCCATCTCTGACCATGAGGCCACCCTGAGGTGCTGGGCCCTGGGCTTCTACCCTGCGGAGATCACACTGACCTGGCAGCGGGATGGGGAGGACCAGACCCAGGACACGGAGCTCGTGGAGACCAGGCCTGCAGGGGATGGAACCTTCCAGAAGTGGGCGGCTGTGGTGGTGCCTTCTGGAGAGGAGCAGAGATACACCTGCCATGTGCAGCATGAGGGTCTGCCCAAGCCCCTCACCCTGAGATGGGGTAAGGAGGGAGATGGGGGTGTCATGTCTCTTAGGGAAAGCAGGAGCCTCTCTGGAGACCTTTAGCAGGGTCAGGGCCCCTCACCTTCCCCTCTTTTCCCAGAGCTGTCTTCCCAGCCCACCATCCCCATCGTGGGCATCATTGCTGGCCTGGTTCTCCTTGGAGCTGTGATCACTGGAGCTGTGGTCGCTGCCGTGATGTGGAGGAGGAAGAGCTCAGGTGGAGAAGGGGTGAAGGGTGGGGTCTGAGATTTCTTGTCTCACTGAGGGTTCCAAGCCCCAGCTAGAAATGTGCCCTGTCTCATTACTGGGAAGCACCTTCCACAATCATGGGCCGACCCAGCCTGGGCCCTGTGTGCCAGCACTTACTCTTTTGTAAAGCACCTGTTAAAATGAAGGACAGATTTATCACCTTGATTANGGAAGGTCCCTGAGGACAGACCTCAGGAGGGCTATTGGTCCAGGACCCACACCTGCTTTCTTCATGTTTCCTGATCCCGCCCTGGGTCTGCAGTCACACATTTCTGGAAACTTCTCTGGGGTCCAAGACTAGGAGGTTCCTCTAGGACCTTAAGGCCCTGGCTCCTTTCTGGTATCTCACAGGACATTTTCTTCCCACAGATAGAAAAGGAGGGAGTTACACTCAGGCTGCAAGTAAGTATGAAGGAGGCTGATGCCTGAGGTCCTTGGGATATTGTGTTTGGGAGCCCATGGGGGAGCTCACCCACCCCACAATTCCTCCTCTAGCCACATCTTCTGTGGGATCTGACCAGGTTCTGTTTTTGTTCTACCCCAGGCAGTGACAGTGCCCAGGGCTCTGATGTGTCTCTCACAGCTTGTAAAGGTGAGAGCTTGGAGGGCCTGATGTGTGTTGGGTGTTGGGTGGAACAGTGGACACAGCTGTGCTATGGGGTTTCTTTGCGTTGGATGTATTGAGCATGCGATGGGCTGTTTAAGGTGTGACCCCTCACTGTGATGGATATGAATTTGTTCATGAATATTTTTTTCTATAGTGTGAGACAGCTGCCTTGTGTGGGACTGAGAGGCAAGAGTTGTTCCTGCCCTTCC HLA-A01:01:01:04 chr6 29909037 29913661 + TAAAGTCCGCACGCACCCACCGGGACTCAGATTCTCCCCAGACGCCGAGGATGGCCGTCATGGCGCCCCGAACCCTCCTCCTGCTACTCTCGGGGGCCCTGGCCCTGACCCAGACCTGGGCGGGTGAGTGCGGGGTCGGGAGGGAAACCGCCTCTGCGGGGAGAAGCAAGGGGCCCTCCTGGCGGGGGCGCAGGACCGGGGGAGCCGCGCCGGGAGGAGGGTCGGGCAGGTCTCAGCCACTGCTCGCCCCCAGGCTCCCACTCCATGAGGTATTTCTTCACATCCGTGTCCCGGCCCGGCCGCGGGGAGCCCCGCTTCATCGCCGTGGGCTACGTGGACGACACGCAGTTCGTGCGGTTCGACAGCGACGCCGCGAGCCAGAAGATGGAGCCGCGGGCGCCGTGGATAGAGCAGGAGGGGCCGGAGTATTGGGACCAGGAGACACGGAATATGAAGGCCCACTCACAGACTGACCGAGCGAACCTGGGGACCCTGCGCGGCTACTACAACCAGAGCGAGGACGGTGAGTGACCCCGGCCCGGGGCGCAGGTCACGACCCCTCATCCCCCACGGACGGGCCAGGTCGCCCACAGTCTCCGGGTCCGAGATCCACCCCGAAGCCGCGGGACTCCGAGACCCTTGTCCCGGGAGAGGCCCAGGCGCCTTTACCCGGTTTCATTTTCAGTTTAGGCCAAAAATCCCCCCGGGTTGGTCGGGGCGGGGCGGGGCTCGGGGGACTGGGCTGACCGCGGGGTCGGGGCCAGGTTCTCACACCATCCAGATAATGTATGGCTGCGACGTGGGGCCGGACGGGCGCTTCCTCCGCGGGTACCGGCAGGACGCCTACGACGGCAAGGATTACATCGCCCTGAACGAGGACCTGCGCTCTTGGACCGCGGCGGACATGGCAGCTCAGATCACCAAGCGCAAGTGGGAGGCGGTCCATGCGGCGGAGCAGCGGAGAGTCTACCTGGAGGGCCGGTGCGTGGACGGGCTCCGCAGATACCTGGAGAACGGGAAGGAGACGCTGCAGCGCACGGGTACCAGGGGCCACGGGGCGCCTCCCTGATCGCCTATAGATCTCCCGGGCTGGCCTCCCACAAGGAGGGGAGACAATTGGGACCAACACTAGAATATCACCCTCCCTCTGGTCCTGAGGGAGAGGAATCCTCCTGGGTTTCCAGATCCTGTACCAGAGAGTGACTCTGAGGTTCCGCCCTGCTCTCTGACACAATTAAGGNAGGTCAGGACCAGAAGTCGCTGTTCCCTTCTCAGGGAATAGAAGATTATCCCAGGTGCCTGTGTCCAGGCTGGTGTCTGGGTTCTGTGCTCTCTTCCCCATCCCGGGTGTCCTGTCCATTCTCAAGATGGCCACATGCGTGCTGGTGGAGTGTCCCATGACAGATGCAAAATGCCTGAATTTTCTGACTCTTCCCGTCAGACCCCCCCAAGACACATATGACCCACCACCCCATCTCTGACCATGAGGCCACCCTGAGGTGCTGGGCCCTGGGCTTCTACCCTGCGGAGATCACACTGACCTGGCAGCGGGATGGGGAGGACCAGACCCAGGACACGGAGCTCGTGGAGACCAGGCCTGCAGGGGATGGAACCTTCCAGAAGTGGGCGGCTGTGGTGGTGCCTTCTGGAGAGGAGCAGAGATACACCTGCCATGTGCAGCATGAGGGTCTGCCCAAGCCCCTCACCCTGAGATGGGGTAAGGAGGGAGATGGGGGTGTCATGTCTCTTAGGGAAAGCAGGAGCCTCTCTGGAGACCTTTAGCAGGGTCAGGGCCCCTCACCTTCCCCTCTTTTCCCAGAGCTGTCTTCCCAGCCCACCATCCCCATCGTGGGCATCATTGCTGGCCTGGTTCTCCTTGGAGCTGTGATCACTGGAGCTGTGGTCGCTGCCGTGATGTGGAGGAGGAAGAGCTCAGGTGGAGAAGGGGTGAAGGGTGGGGTCTGAGATTTCTTGTCTCACTGAGGGTTCCAAGCCCCAGCTAGAAATGTGCCCTGTCTCATTACTGGGAAGCACCTTCCACAATCATGGGCCGACCCAGCCTGGGCCCTGTGTGCCAGCACTTACTCTTTTGTAAAGCACCTGTTAAAATGAAGGACAGATTTATCACCTTGATTANGGAAGGTCCCTGAGGACAGACCTCAGGAGGGCTATTGGTCCAGGACCCACACCTGCTTTCTTCATGTTTCCTGATCCCGCCCTGGGTCTGCAGTCACACATTTCTGGAAACTTCTCTGGGGTCCAAGACTAGGAGGTTCCTCTAGGACCTTAAGGCCCTGGCTCCTTTCTGGTATCTCACAGGACATTTTCTTCCCACAGATAGAAAAGGAGGGAGTTACACTCAGGCTGCAAGTAAGTATGAAGGAGGCTGATGCCTGAGGTCCTTGGGATATTGTGTTTGGGAGCCCATGGGGGAGCTCACCCACCCCACAATTCCTCCTCTAGCCACATCTTCTGTGGGATCTGACCAGGTTCTGTTTTTGTTCTACCCCAGGCAGTGACAGTGCCCAGGGCTCTGATGTGTCTCTCACAGCTTGTAAAGGTGAGAGCTTGGAGGGCCTGATGTGTGTTGGGTGTTGGGTGGAACAGTGGACACAGCTGTGCTATGGGGTTTCTTTGCGTTGGATGTATTGAGCATGCGATGGGCTGTTTAAGGTGTGACCCCTCACTGTGATGGATATGAATTTGTTCATGAATATTTTTTTCTATAGTGTGAGACAGCTGCCTTGTGTGGGACTGAGAGGCAAGAGTTGTTCCTGCCCTTCC HLA-A*01:01:01:05 chr6 29909037 29913661 + TAAAGTCCGCACGCACCCACCGGGACTCAGATTCTCCCCAGACGCCGAGGATGGCCGTCATGGCGCCCCGAACCCTCCTCCTGCTACTCTCGGGGGCCCTGGCCCTGACCCAGACCTGGGCGGGTGAGTGCGGGGTCGGGAGGGAAACCGCCTCTGCGGGGAGAAGCAAGGGGCCCTCCTGGCGGGGGCGCAGGACCGGGGGAGCCGCGCCGGGAGGAGGGTCGGGCAGGTCTCAGCCACTGCTCGCCCCCAGGCTCCCACTCCATGAGGTATTTCTTCACATCCGTGTCCCGGCCCGGCCGCGGGGAGCCCCGCTTCATCGCCGTGGGCTACGTGGACGACACGCAGTTCGTGCGGTTCGACAGCGACGCCGCGAGCCAGAAGATGGAGCCGCGGGCGCCGTGGATAGAGCAGGAGGGGCCGGAGTATTGGGACCAGGAGACACGGAATATGAAGGCCCACTCACAGACTGACCGAGCGAACCTGGGGACCCTGCGCGGCTACTACAACCAGAGCGAGGACGGTGAGTGACCCCGGCCCGGGGCGCAGGTCACGACCCCTCATCCCCCACGGACGGGCCAGGTCGCCCACAGTCTCCGGGTCCGAGATCCGCCCCGAAGCCGCGGGACCCCGAGACCCTTGCCCCGGGAGAGGCCCAGGCGCCTTTACCCGGTTTCATTTTCAGTTTAGGCCAAAAATCCCCCCGGGTTGGTCGGGGCGGGGCGGGGCTCGGGGGACTGGGCTGACCGCGGGGTCGGGGCCAGGTTCTCACACCATCCAGATAATGTATGGCTGCGACGTGGGGCCGGACGGGCGCTTCCTCCGCGGGTACCGGCAGGACGCCTACGACGGCAAGGATTACATCGCCCTGAACGAGGACCTGCGCTCTTGGACCGCGGCGGACATGGCAGCTCAGATCACCAAGCGCAAGTGGGAGGCGGTCCATGCGGCGGAGCAGCGGAGAGTCTACCTGGAGGGCCGGTGCGTGGACGGGCTCCGCAGATACCTGGAGAACGGGAAGGAGACGCTGCAGCGCACGGGTACCAGGGGCCACGGGGCGCCTCCCTGATCGCCTATAGATCTCCCGGGCTGGCCTCCCACAAGGAGGGGAGACAATTGGGACCAACACTAGAATATCACCCTCCCTCTGGTCCTGAGGGAGAGGAATCCTCCTGGGTTTCCAGATCCTGTACCAGAGAGTGACTCTGAGGTTCCGCCCTGCTCTCTGACACAATTAAGGNAGGTCAGGACCAGAAGTCGCTGTTCCCTTCTCAGGGAATAGAAGATTATCCCAGGTGCCTGTGTCCAGGCTGGTGTCTGGGTTCTGTGCTCTCTTCCCCATCCCGGGTGTCCTGTCCATTCTCAAGATGGCCACATGCGTGCTGGTGGAGTGTCCCATGACAGATGCAAAATGCCTGAATTTTCTGACTCTTCCCGTCAGACCCCCCCAAGACACATATGACCCACCACCCCATCTCTGACCATGAGGCCACCCTGAGGTGCTGGGCCCTGGGCTTCTACCCTGCGGAGATCACACTGACCTGGCAGCGGGATGGGGAGGACCAGACCCAGGACACGGAGCTCGTGGAGACCAGGCCTGCAGGGGATGGAACCTTCCAGAAGTGGGCGGCTGTGGTGGTGCCTTCTGGAGAGGAGCAGAGATACACCTGCCATGTGCAGCATGAGGGTCTGCCCAAGCCCCTCACCCTGAGATGGGGTAAGGAGGGAGATGGGGGTGTCATGTCTCTTAGGGAAAGCAGGAGCCTCTCTGGAGACCTTTAGCAGGGTCAGGGCCCCTCACCTTCCCCTCTTTTCCCAGAGCTGTCTTCCCAGCCCACCATCCCCATCGTGGGCATCATTGCTGGCCTGGTTCTCCTTGGAGCTGTGATCACTGGAGCTGTGGTCGCTGCCGTGATGTGGAGGAGGAAGAGCTCAGGTGGAGAAGGGGTGAAGGGTGGGGTCTGAGATTTCTTGTCTCACTGAGGGTTCCAAGCCCCAGCTAGAAATGTGCCCTGTCTCATTACTGGGAAGCACCTTCCACAATCATGGGCCGACCCAGCCTGGGCCCTGTGTGCCAGCACTTACTCTTTTGTAAAGCACCTGTTAAAATGAAGGACAGATTTATCACCTTGATTANGGAAGGTCCCTGAGGACAGACCTCAGGAGGGCTATTGGTCCAGGACCCACACCTGCTTTCTTCATGTTTCCTGATCCCGCCCTGGGTCTGCAGTCACACATTTCTGGAAACTTCTCTGGGGTCCAAGACTAGGAGGTTCCTCTAGGACCTTAAGGCCCTGGCTCCTTTCTGGTATCTCACAGGACATTTTCTTCCCACAGATAGAAAAGGAGGGAGTTACACTCAGGCTGCAAGTAAGTATGAAGGAGGCTGATGCCTGAGGTCCTTGGGATATTGTGTTTGGGAGCCCATGGGGGAGCTCACCCACCCCACAATTCCTCCTCTAGCCACATCTTCTGTGGGATCTGACCAGGTTCTGTTTTTGTTCTACCCCAGGCAGTGACAGTGCCCAGGGCTCTGATGTGTCTCTCACAGCTTGTAAAGGTGAGAGCTTGGAGGGCCTGATGTGTGTTGGGTGTTGGGTGGAACAGTGGACACAGCTGTGCTATGGGGTTTCTTTGCGTTGGATGTATTGAGCATGCGATGGGCTGTTTAAGGTGTGACCCCTCACTGTGATGGATATGAATTTGTTCATGAATATTTTTTTCTATAGTGTGAGACAGCTGCCTTGTGTGGGACTGAGAGGCAAGAGTTGTTCCTGCCCTTCC

[mourisl]

Great. With the correct coordinate information, I think T1K should work on your BAM files. Please let me know how it works.

[Pharmacogenetecist]

Great. With the correct coordinate information, I think T1K should work on your BAM files. Please let me know how it works.

As above, I ran it and got the following error:

[Mon May 1 10:57:41 2023] run-t1k begins. [Mon May 1 10:57:41 2023] SYSTEM CALL: /home/smaggo/T1K/T1Kmaster_05012023/bam-extractor -b Clinical3.0.bam -t 1 -f /home/smaggo/T1K/T1Kmaster_05012023/REFFILES/_dna_coord.fa -o /home/smaggo/T1K/T1Kmaster_05012023/OUTPUT/_candidate [Mon May 1 10:57:42 2023] Start to extract candidate reads from bam file. Unknown genome name: chrHSCHR6_MHC_QBL system /home/smaggo/T1K/T1Kmaster_05012023/bam-extractor -b Clinical3.0.bam -t 1 -f /home/smaggo/T1K/T1Kmaster_05012023/REFFILES/_dna_coord.fa -o /home/smaggo/T1K/T1Kmaster_05012023/OUTPUT/_candidate failed: 256 at /home/smaggo/T1K/T1Kmaster_05012023/run-t1k line 59.

[mourisl]

Sorry for the delayed reply. I think you still used "/home/smaggo/T1K/T1Kmaster_05012023/REFFILES/_dna_coord.fa" as the input for T1K, which should be "REFFILES_dna_coord.fa". Please let me know whether this would work.

I would suggest to use --od to specify the output directory and -o to specify the output prefix for better file name readability.

[Pharmacogenetecist]

Sorry for the delayed reply. I think you still used "/home/smaggo/T1K/T1Kmaster_05012023/REFFILES/_dna_coord.fa" as the input for T1K, which should be "REFFILES_dna_coord.fa". Please let me know whether this would work.

I would suggest to use --od to specify the output directory and -o to specify the output prefix for better file name readability.

I still get an error unfortunately

I re-created the reference files with a may2023 prefix. t1k-build.pl -o may2023 -d "/home/smaggo/T1K/T1Kmaster_05012023/hlaidx/hla.dat" -g "/home/smaggo/T1K/Homo_sapiens.GRCh37.75.gtf"

Then I used the run t1k run-t1k -b "/mmfs1/research/smaggo/Clinical3.0.bam" -f "/home/smaggo/T1K/May2023_REF/may2023/may2023_dna_seq.fa" -c "/home/smaggo/T1K/May2023_REF/may2023/may2023_dna_coord.fa" --preset hla -o "/home/smaggo/T1K/OUTPUT_test/"

ERROR: [Tue May 9 11:31:00 2023] Start to extract candidate reads from bam file. Unknown genome name: chrHSCHR6_MHC_QBL system /home/smaggo/T1K/T1Kmaster_05012023/bam-extractor -b /mmfs1/research/smaggo/Clinical3.0.bam -t 1 -f /home/smaggo/T1K/May2023_REF/may2023/may2023_dna_coord.fa -o /home/smaggo/T1K/OUTPUT_test/_candidate failed: 256 at /home/smaggo/T1K/T1Kmaster_05012023/run-t1k line 59.

Also - I tried using the .fa file in the hlaidx directory and also got the same error.

[mourisl]

I see. It seems there are some genes that are only represented on the alternative chroms in your gtf file. I think you can directly substitute all the terms like "chrHSCHR6_MHC_QBL" to "chr6" in the fa file

For the hladix directory, do you mean the hla reference file from https://github.com/mourisl/T1K_manuscript_evaluation? They do not have the HSCHR6 sequences, so I don't think it will have the same error. Could you please check whether the .fa file has been overwritten? Thank you.

[Pharmacogenetecist]

The file is fine, not overwritten.

My question was - when running t1k.build - it generates a dna_coord.fa and a dna_seq.fa When I run t1k on a bam file, should I use the Newly generated dna_seq.fa as -f or should I use the hlaidx_dna_seq.fa file in the hlaidx directory generated from (perl t1k-build.pl -o hlaidx --download IPD-IMGT/HLA)

[mourisl]

These two files non-coord fa files should be the same, because the "dat" file used for the dna_coord.fa file was downloaded through the "--download" command before. The issue in your case is that the dna_coord.fa contains chromosomes not in the BAM file, so you need to substitute the alternative chrom names to chr6.

[Pharmacogenetecist]

Perfect, so I had to replace two alternative terms in the coord.fa file

sed 's/chrHSCHR6_MHC_QBL/chr6/g' and sed 's/chrHSCHR6_MHC_SSTO/chr6/g'

This resolved the issue, thank you.

[mourisl]

On a second thought, directly changing the chrom name will force T1K to extract reads aligned to the beginning of the chr6 (because the coordinate range is quite small I guess). So probably changing them to "chr6 -1 -1" or "chr19 -1 -1" (the default) might be safer.

[Pharmacogenetecist]

On a second thought, directly changing the chrom name will force T1K to extract reads aligned to the beginning of the chr6 (because the coordinate range is quite small I guess). So probably changing them to "chr6 -1 -1" or "chr19 -1 -1" (the default) might be safer.

I can do that and see if it changes the results for a few bam files and report back. To confirm for HLA you are suggesting for the coord.fa I do sed -i 's/chrHSCHR6_MHC_QBL/chr6 -1 -1/g; sed 's/chrHSCHR6_MHC_SSTO/chr6 -1 -1/g'

[mourisl]

There should be two numbers following chrHSCHR6_MHC_QBL in the original coord fa file as the coordinate range. Those two numbers should be replaced by -1 -1.

[Pharmacogenetecist]

Okay so I will replace the numbers below with -1 and -1

grep 'chrHSCHR6_MHC_QBL' "/home/smaggo/T1K/May2023_REF/may2023/may2023_dna_coord.fa"

HLA-DRB301:01:02:01 chrHSCHR6_MHC_QBL 32417557 32430644 - HLA-DRB301:01:02:03 chrHSCHR6_MHC_QBL 32417557 32430644 - HLA-DRB301:01:02:04 chrHSCHR6_MHC_QBL 32417557 32430644 - HLA-DRB301:01:02:05 chrHSCHR6_MHC_QBL 32417557 32430644 - HLA-DRB301:01:02:06 chrHSCHR6_MHC_QBL 32417557 32430644 - HLA-DRB301:62:01:01 chrHSCHR6_MHC_QBL 32417557 32430644 - HLA-DRB301:62:01:02 chrHSCHR6_MHC_QBL 32417557 32430644 - HLA-DRB302:02:01:01 chrHSCHR6_MHC_QBL 32417557 32430644 - HLA-DRB302:02:01:02 chrHSCHR6_MHC_QBL 32417557 32430644 - HLA-DRB302:02:01:03 chrHSCHR6_MHC_QBL 32417557 32430644 - HLA-DRB302:02:01:04 chrHSCHR6_MHC_QBL 32417557 32430644 - HLA-DRB302:02:01:05 chrHSCHR6_MHC_QBL 32417557 32430644 - HLA-DRB302:02:01:06 chrHSCHR6_MHC_QBL 32417557 32430644 - HLA-DRB302:02:01:07 chrHSCHR6_MHC_QBL 32417557 32430644 - HLA-DRB302:02:01:08 chrHSCHR6_MHC_QBL 32417557 32430644 - HLA-DRB302:02:01:09 chrHSCHR6_MHC_QBL 32417557 32430644 - HLA-DRB302:02:01:10 chrHSCHR6_MHC_QBL 32417557 32430644 - HLA-DRB302:02:01:11 chrHSCHR6_MHC_QBL 32417557 32430644 - HLA-DRB302:02:01:12 chrHSCHR6_MHC_QBL 32417557 32430644 - HLA-DRB302:02:01:13 chrHSCHR6_MHC_QBL 32417557 32430644 - HLA-DRB302:02:01:14 chrHSCHR6_MHC_QBL 32417557 32430644 - HLA-DRB302:02:01:15 chrHSCHR6_MHC_QBL 32417557 32430644 - HLA-DRB302:02:01:16 chrHSCHR6_MHC_QBL 32417557 32430644 - HLA-DRB302:02:01:17 chrHSCHR6_MHC_QBL 32417557 32430644 - HLA-DRB302:22:01 chrHSCHR6_MHC_QBL 32417557 32430644 - HLA-DRB302:24 chrHSCHR6_MHC_QBL 32417557 32430644 - HLA-DRB302:32 chrHSCHR6_MHC_QBL 32417557 32430644 - HLA-DRB303:01:01:01 chrHSCHR6_MHC_QBL 32417557 32430644 - HLA-DRB303:01:01:02 chrHSCHR6_MHC_QBL 32417557 32430644 - HLA-DRB303:01:01:03 chrHSCHR6_MHC_QBL 32417557 32430644 - HLA-DRB303:01:01:04 chrHSCHR6_MHC_QBL 32417557 32430644 - HLA-DRB303:01:01:05 chrHSCHR6_MHC_QBL 32417557 32430644 - HLA-DRB303:01:03:01 chrHSCHR6_MHC_QBL 32417557 32430644 - HLA-DRB303:01:03:02 chrHSCHR6_MHC_QBL 32417557 32430644 -

[mourisl]

I think the "chrHSCHR6_MHC_QB" should be removed after the command. I just noticed that the coordinate 32417557-32430644 seems to be the real coordinate for HLA genes on chr6. So your previous sed 's/chrHSCHR6_MHC_QBL/chr6/g' and sed 's/chrHSCHR6_MHC_SSTO/chr6/g' command should be fine.

[Pharmacogenetecist]

I think the "chrHSCHR6_MHC_QB" should be removed after the command. I just noticed that the coordinate 32417557-32430644 seems to be the real coordinate for HLA genes on chr6. So your previous sed 's/chrHSCHR6_MHC_QBL/chr6/g' and sed 's/chrHSCHR6_MHC_SSTO/chr6/g' command should be fine.

Grch37 coord file with above replaced with chr6 and coordinates replaced with -1 -1 HLA_coord_final.zip

grch37 coord file with just chr6 replacement, coordinates NOT replaced HLACHR6_coord.zip

[Pharmacogenetecist]

FYI for KIR, I had to replace: chrHSCHR19LRC_COX2_CTG1 chrHSCHR19LRC_LRC_I_CTG1 chrHSCHR19LRC_PGF2_CTG1

[Pharmacogenetecist]

@mourisl

I tried both coord files, I notice I often get only one allele typed. From the HLACHR6_coord.fa HLA-A 2 HLA-A01:01:01 376.480559 60 HLA-A31:01:02 357.771933 60 HLA-B 1 HLA-B08:01:01 643.179775 60 . 0 -1 HLA-C 1 HLA-C07:01:01 484.807227 60 . 0 -1 HLA-E 2 HLA-E01:03:02 370.566252 60 HLA-E01:01:01 264.346833 60 HLA-F 1 HLA-F01:01:01 674.582891 60 . 0 -1 HLA-G 2 HLA-G01:03:01 419.324082 60 HLA-G01:01:02 399.898646 60 HLA-H 2 HLA-H02:09 393.706988 60 HLA-H02:01:01 334.563341 60 HLA-J 1 HLA-J01:01:01 711.575585 60 . 0 -1 HLA-K 2 HLA-K01:01:01 249.162239 60 HLA-K01:03 78.179975 30 HLA-L 2 HLA-L01:01:01 271.263463 60 HLA-L01:01:02 245.396821 60 HLA-N 1 HLA-N01:01:01 23.371648 21 . 0 -1 HLA-P 2 HLA-P01:01:01 120.031930 60 HLA-P02:01:01 30.182660 15 HLA-S 1 HLA-S01:02:01 383.227843 60 . 0 -1 HLA-T 2 HLA-T03:01 119.519752 60 HLA-T01:01:01 104.328029 60 HLA-U 1 HLA-U01:01:01 454.054033 60 . 0 -1 HLA-V 1 HLA-V01:01:01 355.351064 60 . 0 -1 HLA-W 2 HLA-W01:01:01 112.604365 60 HLA-W03:01:01 43.600433 25 HLA-Y 1 HLA-Y*02:01 267.909705 60 . 0 -1

From the HLA_coord_final.fa file HLA-A 2 HLA-A01:01:01 376.480750 60 HLA-A31:01:02 357.772026 60 HLA-B 1 HLA-B08:01:01 643.178796 60 . 0 -1 HLA-C 1 HLA-C07:01:01 484.802549 60 . 0 -1 HLA-E 2 HLA-E01:03:02 370.645486 60 HLA-E01:01:01 264.284152 60 HLA-F 1 HLA-F01:01:01 674.582891 60 . 0 -1 HLA-G 2 HLA-G01:03:01 419.324083 60 HLA-G01:01:02 399.898645 60 HLA-H 2 HLA-H02:09 393.706988 60 HLA-H02:01:01 334.563022 60 HLA-J 1 HLA-J01:01:01 711.569739 60 . 0 -1 HLA-K 2 HLA-K01:01:01 249.160390 60 HLA-K01:03 78.163022 30 HLA-L 2 HLA-L01:01:01 271.251620 60 HLA-L01:01:02 245.406286 60 HLA-N 1 HLA-N01:01:01 23.371648 21 . 0 -1 HLA-P 2 HLA-P01:01:01 120.025770 60 HLA-P02:01:01 30.188810 15 HLA-S 1 HLA-S01:02:01 383.227843 60 . 0 -1 HLA-T 2 HLA-T03:01 119.519752 60 HLA-T01:01:01 104.328029 60 HLA-U 1 HLA-U01:01:01 454.054033 60 . 0 -1 HLA-V 1 HLA-V01:01:01 355.351064 60 . 0 -1 HLA-W 2 HLA-W01:01:01 112.555746 60 HLA-W03:01:01 43.655422 25 HLA-Y 1 HLA-Y*02:01 267.909705 60 . 0 -1

[mourisl]

In the case of homozygous alleles, T1K will output one. In your case, the sample probably has homozygous HLA-B01:01 and HLA-C07:01.

[Pharmacogenetecist]

In the case of homozygous alleles, T1K will output one. In your case, the sample probably has homozygous HLA-B_01:01 and HLA-C_07:01.

Perfect, yes, I can confirm. I checked with some positive controls. Thank you.