Question: Picard - MergeBamAlignment: Reads remaining on alignment iterator
Hi, I'm new to genome analysis and I need some advice on this. I'm currently follow GATK best practice, now I'm stuck at the MergeBamAlignment step of picard. I'm using the data from https://www.internationalgenome.org/, the reference genome is also downloaded from International genome database.
My command:
MergeBamAlignment -ALIGNED aln/HG02142.mapped.ILLUMINA.bwa.KHV.low_coverage.20130415.bam -UNMAPPED /aln/HG02142.unmapped.ILLUMINA.bwa.KHV.low_coverage.20130415.bam -O test.bam -R refgen/hs37d5.fa.gz -TMP_DIR tmp
I got many warning, and didn't get the output file:
WARNING 2020-02-23 21:54:56 SamAlignmentMerger Exception merging bam alignment - attempting to sort aligned reads and try again: Underlying iterator is not queryname sorted: ERR233223.95759674 2/2 101b aligned to 1:47264561-47264660. > ERR233223.14389540 2/2 101b aligned to 1:47264588-47264671.
INFO 2020-02-23 21:54:56 SamAlignmentMerger Finished reading 28356 total records from alignment SAM/BAM.
WARNING 2020-02-23 21:54:56 SAMSequenceDictionary Found sequence entry for which tags differ: 1 and tag UR has the two values: ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz and file:/data4t/KHV1000G/refgen/hs37d5.fa.gz. Using ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz
WARNING 2020-02-23 21:54:56 SAMSequenceDictionary Found sequence entry for which tags differ: 2 and tag UR has the two values: ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz and file:/data4t/KHV1000G/refgen/hs37d5.fa.gz. Using ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz
.... (many warnings in here, similar structure)
[Sun Feb 23 21:54:56 ICT 2020] picard.sam.MergeBamAlignment done. Elapsed time: 0.01 minutes.
Runtime.totalMemory()=886571008
To get help, see http://broadinstitute.github.io/picard/index.html#GettingHelp
Exception in thread "main" java.lang.IllegalStateException: Reads remaining on alignment iterator: ERR233223.100003813!
at picard.sam.AbstractAlignmentMerger.mergeAlignment(AbstractAlignmentMerger.java:558)
at picard.sam.SamAlignmentMerger.mergeAlignment(SamAlignmentMerger.java:186)
at picard.sam.MergeBamAlignment.doWork(MergeBamAlignment.java:358)
at picard.cmdline.CommandLineProgram.instanceMain(CommandLineProgram.java:305)
at picard.cmdline.PicardCommandLine.instanceMain(PicardCommandLine.java:103)
at picard.cmdline.PicardCommandLine.main(PicardCommandLine.java:113)
What have go wrong in my case? I downloaded all of my files directly from international genomes database ftp, without any modification. Please advice me on this, thank you.
I'm using the latest version of GATK and Picard.
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It could be that the reads are out of order based on querygroup instead of the queryname and that is causing the issue.
Can you run ValidateSamFile on your bams and run CountReads to make sure the counts are the same for each bam? Can you paste the entire bam header of each bam?
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I have run CountReads on both file and they return the same counts.
For ValidateSamFile, I use this command for both file:
java -jar $PICARD ValidateSamFile I=HG02142.mapped.ILLUMINA.bwa.KHV.low_coverage.20130415.bam MODE=SUMMARYbut they return the Error
## HISTOGRAM java.lang.String
Error Type Count
ERROR:INVALID_INDEX_FILE_POINTER 1
ERROR:MATE_NOT_FOUND 13673I checked the the header of mapped file using samtools view -H
@HD VN:1.0 SO:coordinate
@SQ SN:1 LN:249250621 M5:1b22b98cdeb4a9304cb5d48026a85128 UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:2 LN:243199373 M5:a0d9851da00400dec1098a9255ac712e UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:3 LN:198022430 M5:fdfd811849cc2fadebc929bb925902e5 UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:4 LN:191154276 M5:23dccd106897542ad87d2765d28a19a1 UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:5 LN:180915260 M5:0740173db9ffd264d728f32784845cd7 UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:6 LN:171115067 M5:1d3a93a248d92a729ee764823acbbc6b UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:7 LN:159138663 M5:618366e953d6aaad97dbe4777c29375e UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:8 LN:146364022 M5:96f514a9929e410c6651697bded59aec UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:9 LN:141213431 M5:3e273117f15e0a400f01055d9f393768 UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:10 LN:135534747 M5:988c28e000e84c26d552359af1ea2e1d UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:11 LN:135006516 M5:98c59049a2df285c76ffb1c6db8f8b96 UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:12 LN:133851895 M5:51851ac0e1a115847ad36449b0015864 UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:13 LN:115169878 M5:283f8d7892baa81b510a015719ca7b0b UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:14 LN:107349540 M5:98f3cae32b2a2e9524bc19813927542e UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:15 LN:102531392 M5:e5645a794a8238215b2cd77acb95a078 UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:16 LN:90354753 M5:fc9b1a7b42b97a864f56b348b06095e6 UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:17 LN:81195210 M5:351f64d4f4f9ddd45b35336ad97aa6de UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:18 LN:78077248 M5:b15d4b2d29dde9d3e4f93d1d0f2cbc9c UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:19 LN:59128983 M5:1aacd71f30db8e561810913e0b72636d UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:20 LN:63025520 M5:0dec9660ec1efaaf33281c0d5ea2560f UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:21 LN:48129895 M5:2979a6085bfe28e3ad6f552f361ed74d UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:22 LN:51304566 M5:a718acaa6135fdca8357d5bfe94211dd UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:X LN:155270560 M5:7e0e2e580297b7764e31dbc80c2540dd UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:Y LN:59373566 M5:1fa3474750af0948bdf97d5a0ee52e51 UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:MT LN:16569 M5:c68f52674c9fb33aef52dcf399755519 UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:GL000207.1 LN:4262 M5:f3814841f1939d3ca19072d9e89f3fd7 UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:GL000226.1 LN:15008 M5:1c1b2cd1fccbc0a99b6a447fa24d1504 UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:GL000229.1 LN:19913 M5:d0f40ec87de311d8e715b52e4c7062e1 UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:GL000231.1 LN:27386 M5:ba8882ce3a1efa2080e5d29b956568a4 UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:GL000210.1 LN:27682 M5:851106a74238044126131ce2a8e5847c UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:GL000239.1 LN:33824 M5:99795f15702caec4fa1c4e15f8a29c07 UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:GL000235.1 LN:34474 M5:118a25ca210cfbcdfb6c2ebb249f9680 UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:GL000201.1 LN:36148 M5:dfb7e7ec60ffdcb85cb359ea28454ee9 UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:GL000247.1 LN:36422 M5:7de00226bb7df1c57276ca6baabafd15 UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:GL000245.1 LN:36651 M5:89bc61960f37d94abf0df2d481ada0ec UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:GL000197.1 LN:37175 M5:6f5efdd36643a9b8c8ccad6f2f1edc7b UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:GL000203.1 LN:37498 M5:96358c325fe0e70bee73436e8bb14dbd UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:GL000246.1 LN:38154 M5:e4afcd31912af9d9c2546acf1cb23af2 UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:GL000249.1 LN:38502 M5:1d78abec37c15fe29a275eb08d5af236 UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:GL000196.1 LN:38914 M5:d92206d1bb4c3b4019c43c0875c06dc0 UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:GL000248.1 LN:39786 M5:5a8e43bec9be36c7b49c84d585107776 UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:GL000244.1 LN:39929 M5:0996b4475f353ca98bacb756ac479140 UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:GL000238.1 LN:39939 M5:131b1efc3270cc838686b54e7c34b17b UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:GL000202.1 LN:40103 M5:06cbf126247d89664a4faebad130fe9c UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:GL000234.1 LN:40531 M5:93f998536b61a56fd0ff47322a911d4b UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:GL000232.1 LN:40652 M5:3e06b6741061ad93a8587531307057d8 UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:GL000206.1 LN:41001 M5:43f69e423533e948bfae5ce1d45bd3f1 UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:GL000240.1 LN:41933 M5:445a86173da9f237d7bcf41c6cb8cc62 UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:GL000236.1 LN:41934 M5:fdcd739913efa1fdc64b6c0cd7016779 UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:GL000241.1 LN:42152 M5:ef4258cdc5a45c206cea8fc3e1d858cf UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:GL000243.1 LN:43341 M5:cc34279a7e353136741c9fce79bc4396 UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:GL000242.1 LN:43523 M5:2f8694fc47576bc81b5fe9e7de0ba49e UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:GL000230.1 LN:43691 M5:b4eb71ee878d3706246b7c1dbef69299 UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:GL000237.1 LN:45867 M5:e0c82e7751df73f4f6d0ed30cdc853c0 UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:GL000233.1 LN:45941 M5:7fed60298a8d62ff808b74b6ce820001 UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:GL000204.1 LN:81310 M5:efc49c871536fa8d79cb0a06fa739722 UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:GL000198.1 LN:90085 M5:868e7784040da90d900d2d1b667a1383 UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:GL000208.1 LN:92689 M5:aa81be49bf3fe63a79bdc6a6f279abf6 UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:GL000191.1 LN:106433 M5:d75b436f50a8214ee9c2a51d30b2c2cc UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:GL000227.1 LN:128374 M5:a4aead23f8053f2655e468bcc6ecdceb UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:GL000228.1 LN:129120 M5:c5a17c97e2c1a0b6a9cc5a6b064b714f UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:GL000214.1 LN:137718 M5:46c2032c37f2ed899eb41c0473319a69 UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:GL000221.1 LN:155397 M5:3238fb74ea87ae857f9c7508d315babb UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:GL000209.1 LN:159169 M5:f40598e2a5a6b26e84a3775e0d1e2c81 UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:GL000218.1 LN:161147 M5:1d708b54644c26c7e01c2dad5426d38c UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:GL000220.1 LN:161802 M5:fc35de963c57bf7648429e6454f1c9db UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:GL000213.1 LN:164239 M5:9d424fdcc98866650b58f004080a992a UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:GL000211.1 LN:166566 M5:7daaa45c66b288847b9b32b964e623d3 UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:GL000199.1 LN:169874 M5:569af3b73522fab4b40995ae4944e78e UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:GL000217.1 LN:172149 M5:6d243e18dea1945fb7f2517615b8f52e UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:GL000216.1 LN:172294 M5:642a232d91c486ac339263820aef7fe0 UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:GL000215.1 LN:172545 M5:5eb3b418480ae67a997957c909375a73 UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:GL000205.1 LN:174588 M5:d22441398d99caf673e9afb9a1908ec5 UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:GL000219.1 LN:179198 M5:f977edd13bac459cb2ed4a5457dba1b3 UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:GL000224.1 LN:179693 M5:d5b2fc04f6b41b212a4198a07f450e20 UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:GL000223.1 LN:180455 M5:399dfa03bf32022ab52a846f7ca35b30 UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:GL000195.1 LN:182896 M5:5d9ec007868d517e73543b005ba48535 UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:GL000212.1 LN:186858 M5:563531689f3dbd691331fd6c5730a88b UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:GL000222.1 LN:186861 M5:6fe9abac455169f50470f5a6b01d0f59 UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:GL000200.1 LN:187035 M5:75e4c8d17cd4addf3917d1703cacaf25 UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:GL000193.1 LN:189789 M5:dbb6e8ece0b5de29da56601613007c2a UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:GL000194.1 LN:191469 M5:6ac8f815bf8e845bb3031b73f812c012 UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:GL000225.1 LN:211173 M5:63945c3e6962f28ffd469719a747e73c UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:GL000192.1 LN:547496 M5:325ba9e808f669dfeee210fdd7b470ac UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:NC_007605 LN:171823 M5:6743bd63b3ff2b5b8985d8933c53290a UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:hs37d5 LN:35477943 M5:5b6a4b3a81a2d3c134b7d14bf6ad39f1 UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@RG ID:ERR233223 LB:HG02142_I_bc_pelib_1057 SM:HG02142 PI:496 CN:MPIMG PL:ILLUMINA DS:SRP001516
@PG ID:bwa_index PN:bwa VN:0.5.9-r16 CL:bwa index -a bwtsw $reference_fasta
@PG ID:bwa_aln_fastq PN:bwa PP:bwa_index VN:0.5.9-r16 CL:bwa aln -q 15 -f $sai_file $reference_fasta $fastq_file
@PG ID:bwa_sam PN:bwa PP:bwa_aln_fastq VN:0.5.9-r16 CL:bwa sampe -a 1488 -r $rg_line -f $sam_file $reference_fasta $sai_file(s) $fastq_file(s)
@PG ID:sam_to_fixed_bam PN:samtools PP:bwa_sam VN:0.1.17 (r973:277) CL:samtools view -bSu $sam_file | samtools sort -n -o - samtools_nsort_tmp | samtools fixmate /dev/stdin /dev/stdout | samtools sort -o - samtools_csort_tmp | samtools fillmd -u - $reference_fasta > $fixed_bam_file
@PG ID:gatk_target_interval_creator PN:GenomeAnalysisTK PP:sam_to_fixed_bam VN:1.2-29-g0acaf2d CL:java $jvm_args -jar GenomeAnalysisTK.jar -T RealignerTargetCreator -R $reference_fasta -o $intervals_file -known $known_indels_file(s)
@PG ID:bam_realignment_around_known_indels PN:GenomeAnalysisTK PP:gatk_target_interval_creator VN:1.2-29-g0acaf2d CL:java $jvm_args -jar GenomeAnalysisTK.jar -T IndelRealigner -R $reference_fasta -I $bam_file -o $realigned_bam_file -targetIntervals $intervals_file -known $known_indels_file(s) -LOD 0.4 -model KNOWNS_ONLY -compress 0 --disable_bam_indexing
@PG ID:bam_count_covariates PN:GenomeAnalysisTK PP:bam_realignment_around_known_indels VN:1.2-29-g0acaf2d CL:java $jvm_args -jar GenomeAnalysisTK.jar -T CountCovariates -R $reference_fasta -I $bam_file -recalFile $bam_file.recal_data.csv -knownSites $known_sites_file(s) -l INFO -L '1;2;3;4;5;6;7;8;9;10;11;12;13;14;15;16;17;18;19;20;21;22;X;Y;MT' -cov ReadGroupCovariate -cov QualityScoreCovariate -cov CycleCovariate -cov DinucCovariate
@PG ID:bam_recalibrate_quality_scores PN:GenomeAnalysisTK PP:bam_count_covariates VN:1.2-29-g0acaf2d CL:java $jvm_args -jar GenomeAnalysisTK.jar -T TableRecalibration -R $reference_fasta -recalFile $bam_file.recal_data.csv -I $bam_file -o $recalibrated_bam_file -l INFO -compress 0 --disable_bam_indexing
@PG ID:bam_calculate_bq PN:samtools PP:bam_recalibrate_quality_scores VN:0.1.17 (r973:277) CL:samtools calmd -Erb $bam_file $reference_fasta > $bq_bam_file
@PG ID:bam_merge PN:picard PP:bam_calculate_bq VN:1.53 CL:java $jvm_args -jar MergeSamFiles.jar INPUT=$bam_file(s) OUTPUT=$merged_bam VALIDATION_STRINGENCY=SILENT
@PG ID:bam_mark_duplicates PN:picard PP:bam_merge VN:1.53 CL:java $jvm_args -jar MarkDuplicates.jar INPUT=$bam_file OUTPUT=$markdup_bam_file ASSUME_SORTED=TRUE METRICS_FILE=/dev/null VALIDATION_STRINGENCY=SILENT
@PG ID:bam_merge.1 PN:picard PP:bam_mark_duplicates VN:1.53 CL:java $jvm_args -jar MergeSamFiles.jar INPUT=$bam_file(s) OUTPUT=$merged_bam VALIDATION_STRINGENCY=SILENT
@CO $known_indels_file(s) = ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_mapping_resources/ALL.wgs.indels_mills_devine_hg19_leftAligned_collapsed_double_hit.indels.sites.vcf.gz
@CO $known_indels_file(s) .= ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_mapping_resources/ALL.wgs.low_coverage_vqsr.20101123.indels.sites.vcf.gz
@CO $known_sites_file(s) = ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_mapping_resources/ALL.wgs.dbsnp.build135.snps.sites.vcf.gzThe header of unmapped file
@HD VN:1.0 SO:coordinate
@SQ SN:1 LN:249250621 M5:1b22b98cdeb4a9304cb5d48026a85128 UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:2 LN:243199373 M5:a0d9851da00400dec1098a9255ac712e UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:3 LN:198022430 M5:fdfd811849cc2fadebc929bb925902e5 UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:4 LN:191154276 M5:23dccd106897542ad87d2765d28a19a1 UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:5 LN:180915260 M5:0740173db9ffd264d728f32784845cd7 UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:6 LN:171115067 M5:1d3a93a248d92a729ee764823acbbc6b UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:7 LN:159138663 M5:618366e953d6aaad97dbe4777c29375e UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:8 LN:146364022 M5:96f514a9929e410c6651697bded59aec UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:9 LN:141213431 M5:3e273117f15e0a400f01055d9f393768 UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:10 LN:135534747 M5:988c28e000e84c26d552359af1ea2e1d UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:11 LN:135006516 M5:98c59049a2df285c76ffb1c6db8f8b96 UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:12 LN:133851895 M5:51851ac0e1a115847ad36449b0015864 UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:13 LN:115169878 M5:283f8d7892baa81b510a015719ca7b0b UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:14 LN:107349540 M5:98f3cae32b2a2e9524bc19813927542e UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:15 LN:102531392 M5:e5645a794a8238215b2cd77acb95a078 UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:16 LN:90354753 M5:fc9b1a7b42b97a864f56b348b06095e6 UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:17 LN:81195210 M5:351f64d4f4f9ddd45b35336ad97aa6de UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:18 LN:78077248 M5:b15d4b2d29dde9d3e4f93d1d0f2cbc9c UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:19 LN:59128983 M5:1aacd71f30db8e561810913e0b72636d UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:20 LN:63025520 M5:0dec9660ec1efaaf33281c0d5ea2560f UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:21 LN:48129895 M5:2979a6085bfe28e3ad6f552f361ed74d UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:22 LN:51304566 M5:a718acaa6135fdca8357d5bfe94211dd UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:X LN:155270560 M5:7e0e2e580297b7764e31dbc80c2540dd UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:Y LN:59373566 M5:1fa3474750af0948bdf97d5a0ee52e51 UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:MT LN:16569 M5:c68f52674c9fb33aef52dcf399755519 UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:GL000207.1 LN:4262 M5:f3814841f1939d3ca19072d9e89f3fd7 UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:GL000226.1 LN:15008 M5:1c1b2cd1fccbc0a99b6a447fa24d1504 UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:GL000229.1 LN:19913 M5:d0f40ec87de311d8e715b52e4c7062e1 UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:GL000231.1 LN:27386 M5:ba8882ce3a1efa2080e5d29b956568a4 UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:GL000210.1 LN:27682 M5:851106a74238044126131ce2a8e5847c UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:GL000239.1 LN:33824 M5:99795f15702caec4fa1c4e15f8a29c07 UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:GL000235.1 LN:34474 M5:118a25ca210cfbcdfb6c2ebb249f9680 UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:GL000201.1 LN:36148 M5:dfb7e7ec60ffdcb85cb359ea28454ee9 UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:GL000247.1 LN:36422 M5:7de00226bb7df1c57276ca6baabafd15 UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:GL000245.1 LN:36651 M5:89bc61960f37d94abf0df2d481ada0ec UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:GL000197.1 LN:37175 M5:6f5efdd36643a9b8c8ccad6f2f1edc7b UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:GL000203.1 LN:37498 M5:96358c325fe0e70bee73436e8bb14dbd UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:GL000246.1 LN:38154 M5:e4afcd31912af9d9c2546acf1cb23af2 UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:GL000249.1 LN:38502 M5:1d78abec37c15fe29a275eb08d5af236 UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:GL000196.1 LN:38914 M5:d92206d1bb4c3b4019c43c0875c06dc0 UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:GL000248.1 LN:39786 M5:5a8e43bec9be36c7b49c84d585107776 UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:GL000244.1 LN:39929 M5:0996b4475f353ca98bacb756ac479140 UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:GL000238.1 LN:39939 M5:131b1efc3270cc838686b54e7c34b17b UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:GL000202.1 LN:40103 M5:06cbf126247d89664a4faebad130fe9c UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:GL000234.1 LN:40531 M5:93f998536b61a56fd0ff47322a911d4b UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:GL000232.1 LN:40652 M5:3e06b6741061ad93a8587531307057d8 UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:GL000206.1 LN:41001 M5:43f69e423533e948bfae5ce1d45bd3f1 UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:GL000240.1 LN:41933 M5:445a86173da9f237d7bcf41c6cb8cc62 UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:GL000236.1 LN:41934 M5:fdcd739913efa1fdc64b6c0cd7016779 UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:GL000241.1 LN:42152 M5:ef4258cdc5a45c206cea8fc3e1d858cf UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:GL000243.1 LN:43341 M5:cc34279a7e353136741c9fce79bc4396 UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:GL000242.1 LN:43523 M5:2f8694fc47576bc81b5fe9e7de0ba49e UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:GL000230.1 LN:43691 M5:b4eb71ee878d3706246b7c1dbef69299 UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:GL000237.1 LN:45867 M5:e0c82e7751df73f4f6d0ed30cdc853c0 UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:GL000233.1 LN:45941 M5:7fed60298a8d62ff808b74b6ce820001 UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:GL000204.1 LN:81310 M5:efc49c871536fa8d79cb0a06fa739722 UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:GL000198.1 LN:90085 M5:868e7784040da90d900d2d1b667a1383 UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:GL000208.1 LN:92689 M5:aa81be49bf3fe63a79bdc6a6f279abf6 UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:GL000191.1 LN:106433 M5:d75b436f50a8214ee9c2a51d30b2c2cc UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:GL000227.1 LN:128374 M5:a4aead23f8053f2655e468bcc6ecdceb UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:GL000228.1 LN:129120 M5:c5a17c97e2c1a0b6a9cc5a6b064b714f UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:GL000214.1 LN:137718 M5:46c2032c37f2ed899eb41c0473319a69 UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:GL000221.1 LN:155397 M5:3238fb74ea87ae857f9c7508d315babb UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:GL000209.1 LN:159169 M5:f40598e2a5a6b26e84a3775e0d1e2c81 UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:GL000218.1 LN:161147 M5:1d708b54644c26c7e01c2dad5426d38c UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:GL000220.1 LN:161802 M5:fc35de963c57bf7648429e6454f1c9db UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:GL000213.1 LN:164239 M5:9d424fdcc98866650b58f004080a992a UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:GL000211.1 LN:166566 M5:7daaa45c66b288847b9b32b964e623d3 UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:GL000199.1 LN:169874 M5:569af3b73522fab4b40995ae4944e78e UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:GL000217.1 LN:172149 M5:6d243e18dea1945fb7f2517615b8f52e UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:GL000216.1 LN:172294 M5:642a232d91c486ac339263820aef7fe0 UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:GL000215.1 LN:172545 M5:5eb3b418480ae67a997957c909375a73 UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:GL000205.1 LN:174588 M5:d22441398d99caf673e9afb9a1908ec5 UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:GL000219.1 LN:179198 M5:f977edd13bac459cb2ed4a5457dba1b3 UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:GL000224.1 LN:179693 M5:d5b2fc04f6b41b212a4198a07f450e20 UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:GL000223.1 LN:180455 M5:399dfa03bf32022ab52a846f7ca35b30 UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:GL000195.1 LN:182896 M5:5d9ec007868d517e73543b005ba48535 UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:GL000212.1 LN:186858 M5:563531689f3dbd691331fd6c5730a88b UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:GL000222.1 LN:186861 M5:6fe9abac455169f50470f5a6b01d0f59 UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:GL000200.1 LN:187035 M5:75e4c8d17cd4addf3917d1703cacaf25 UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:GL000193.1 LN:189789 M5:dbb6e8ece0b5de29da56601613007c2a UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:GL000194.1 LN:191469 M5:6ac8f815bf8e845bb3031b73f812c012 UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:GL000225.1 LN:211173 M5:63945c3e6962f28ffd469719a747e73c UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:GL000192.1 LN:547496 M5:325ba9e808f669dfeee210fdd7b470ac UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:NC_007605 LN:171823 M5:6743bd63b3ff2b5b8985d8933c53290a UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@SQ SN:hs37d5 LN:35477943 M5:5b6a4b3a81a2d3c134b7d14bf6ad39f1 UR:ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human
@RG ID:ERR233223 LB:HG02142_I_bc_pelib_1057 SM:HG02142 PI:496 CN:MPIMG PL:ILLUMINA DS:SRP001516
@PG ID:bwa_index PN:bwa VN:0.5.9-r16 CL:bwa index -a bwtsw $reference_fasta
@PG ID:bwa_aln_fastq PN:bwa PP:bwa_index VN:0.5.9-r16 CL:bwa aln -q 15 -f $sai_file $reference_fasta $fastq_file
@PG ID:bwa_sam PN:bwa PP:bwa_aln_fastq VN:0.5.9-r16 CL:bwa sampe -a 1488 -r $rg_line -f $sam_file $reference_fasta $sai_file(s) $fastq_file(s)
@PG ID:sam_to_fixed_bam PN:samtools PP:bwa_sam VN:0.1.17 (r973:277) CL:samtools view -bSu $sam_file | samtools sort -n -o - samtools_nsort_tmp | samtools fixmate /dev/stdin /dev/stdout | samtools sort -o - samtools_csort_tmp | samtools fillmd -u - $reference_fasta > $fixed_bam_file
@PG ID:gatk_target_interval_creator PN:GenomeAnalysisTK PP:sam_to_fixed_bam VN:1.2-29-g0acaf2d CL:java $jvm_args -jar GenomeAnalysisTK.jar -T RealignerTargetCreator -R $reference_fasta -o $intervals_file -known $known_indels_file(s)
@PG ID:bam_realignment_around_known_indels PN:GenomeAnalysisTK PP:gatk_target_interval_creator VN:1.2-29-g0acaf2d CL:java $jvm_args -jar GenomeAnalysisTK.jar -T IndelRealigner -R $reference_fasta -I $bam_file -o $realigned_bam_file -targetIntervals $intervals_file -known $known_indels_file(s) -LOD 0.4 -model KNOWNS_ONLY -compress 0 --disable_bam_indexing
@PG ID:bam_count_covariates PN:GenomeAnalysisTK PP:bam_realignment_around_known_indels VN:1.2-29-g0acaf2d CL:java $jvm_args -jar GenomeAnalysisTK.jar -T CountCovariates -R $reference_fasta -I $bam_file -recalFile $bam_file.recal_data.csv -knownSites $known_sites_file(s) -l INFO -L '1;2;3;4;5;6;7;8;9;10;11;12;13;14;15;16;17;18;19;20;21;22;X;Y;MT' -cov ReadGroupCovariate -cov QualityScoreCovariate -cov CycleCovariate -cov DinucCovariate
@PG ID:bam_recalibrate_quality_scores PN:GenomeAnalysisTK PP:bam_count_covariates VN:1.2-29-g0acaf2d CL:java $jvm_args -jar GenomeAnalysisTK.jar -T TableRecalibration -R $reference_fasta -recalFile $bam_file.recal_data.csv -I $bam_file -o $recalibrated_bam_file -l INFO -compress 0 --disable_bam_indexing
@PG ID:bam_calculate_bq PN:samtools PP:bam_recalibrate_quality_scores VN:0.1.17 (r973:277) CL:samtools calmd -Erb $bam_file $reference_fasta > $bq_bam_file
@PG ID:bam_merge PN:picard PP:bam_calculate_bq VN:1.53 CL:java $jvm_args -jar MergeSamFiles.jar INPUT=$bam_file(s) OUTPUT=$merged_bam VALIDATION_STRINGENCY=SILENT
@PG ID:bam_mark_duplicates PN:picard PP:bam_merge VN:1.53 CL:java $jvm_args -jar MarkDuplicates.jar INPUT=$bam_file OUTPUT=$markdup_bam_file ASSUME_SORTED=TRUE METRICS_FILE=/dev/null VALIDATION_STRINGENCY=SILENT
@PG ID:bam_merge.1 PN:picard PP:bam_mark_duplicates VN:1.53 CL:java $jvm_args -jar MergeSamFiles.jar INPUT=$bam_file(s) OUTPUT=$merged_bam VALIDATION_STRINGENCY=SILENT
@CO $known_indels_file(s) = ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_mapping_resources/ALL.wgs.indels_mills_devine_hg19_leftAligned_collapsed_double_hit.indels.sites.vcf.gz
@CO $known_indels_file(s) .= ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_mapping_resources/ALL.wgs.low_coverage_vqsr.20101123.indels.sites.vcf.gz
@CO $known_sites_file(s) = ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_mapping_resources/ALL.wgs.dbsnp.build135.snps.sites.vcf.gzI'm new to this so I don't know to check which file is sorted in which order.
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I have tried two cases using Sort by queryname:
1, Sorting unmapped file then MergeAlignment
INFO 2020-02-25 12:13:30 SamAlignmentMerger Processing SAM file(s): [aln/HG01842.mapped.ILLUMINA.bwa.KHV.low_coverage.20120522.bam] WARNING 2020-02-25 12:13:30 SAMSequenceDictionary Found sequence entry for which tags differ: 1 and tag UR has the two values: ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human and ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz. Using ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human (...) WARNING 2020-02-25 12:13:31 SAMSequenceDictionary Found sequence entry for which tags differ: hs37d5 and tag UR has the two values: ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human and ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz. Using ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human [Tue Feb 25 12:13:31 ICT 2020] picard.sam.MergeBamAlignment done. Elapsed time: 0.01 minutes. Runtime.totalMemory()=886571008 To get help, see http://broadinstitute.github.io/picard/index.html#GettingHelp Exception in thread "main" java.lang.IllegalStateException: Reads remaining on alignment iterator: ERR042514.10000846! at picard.sam.AbstractAlignmentMerger.mergeAlignment(AbstractAlignmentMerger.java:558) at picard.sam.SamAlignmentMerger.mergeAlignment(SamAlignmentMerger.java:186) at picard.sam.MergeBamAlignment.doWork(MergeBamAlignment.java:358) at picard.cmdline.CommandLineProgram.instanceMain(CommandLineProgram.java:305) at picard.cmdline.PicardCommandLine.instanceMain(PicardCommandLine.java:103) at picard.cmdline.PicardCommandLine.main(PicardCommandLine.java:113)2, Sorting both files then MergeAlignment
INFO 2020-02-25 12:11:56 SamAlignmentMerger Processing SAM file(s): [HG 01842.mapped.sorted.bam] WARNING 2020-02-25 12:11:56 SAMSequenceDictionary Found sequence entry for which tags differ: 1 and tag UR has the two values: ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human and ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz. Using ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human (...) WARNING 2020-02-25 12:11:57 SAMSequenceDictionary Found sequence entry for which tags differ: hs37d5 and tag UR has the two values: ftp://ftp.1000genomes.ebi.ac .uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human and ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz. Using ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/hs37d5.fa.gz AS:NCBI37 SP:Human [Tue Feb 25 12:11:57 ICT 2020] picard.sam.MergeBamAlignment done. Elapsed time: 0.01 minutes. Runtime.totalMemory()=886571008 To get help, see http://broadinstitute.github.io/picard/index.html#GettingHelp Exception in thread "main" java.lang.IllegalStateException: Reads remaining on alignment iterator: ERR042514.10000846! at picard.sam.AbstractAlignmentMerger.mergeAlignment(AbstractAlignmentMerger.java:558) at picard.sam.SamAlignmentMerger.mergeAlignment(SamAlignmentMerger.java:186) at picard.sam.MergeBamAlignment.doWork(MergeBamAlignment.java:358) at picard.cmdline.CommandLineProgram.instanceMain(CommandLineProgram.java:305) at picard.cmdline.PicardCommandLine.instanceMain(PicardCommandLine.java:103) at picard.cmdline.PicardCommandLine.main(PicardCommandLine.java:113)And it still output nothing
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Hi Nguyen Nguyen Thanh, Can you try determining why the SAM files are giving you these errors by following Step 2 in this tutorial ?
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