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Variant Discovery in High-Throughput Sequencing Data

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ApplyBQSR

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Wondessen Ayalew

REQUIRED for all errors and issues:
a) GATK version used: GATK4.3  
b) Exact command used: gatk ApplyBQSR -R ../reference/Bos_taurus.ARS-UCD1.2.dna.toplevel.fa -I ../data/mapped/ABigar/Abi1_dedup.bam --bqsr-recal-file ../data/mapped/ABigar/AB_haplo/Abi1_recal.table1 -O ../data/mapped/ABigar/AB_haplo/Abi1_recal.bam
c) Entire program log:
Dear All, I hope everyone is doing well. When I run ApplyBQSR some of my samples generate all the chromosomes with MT as indicated below. The other samples came from a similar pipeline that missed MT after lists of chromosomes. In addition, "IntelInflater - Zero Bytes Written : 0" warning message and unmapped regions are generated at the end of the output. Therefore, I sincerely request your suggestions about the output to proceed to the next step. Thank you in advance for your valuable support!

The last portion of the result is posted below ....

22:40:14.206 INFO  ProgressMeter -          X:126181720            117.3             211182000        1800853.9
22:40:24.231 INFO  ProgressMeter -          X:128930737            117.4             211409000        1800224.7
22:40:34.253 INFO  ProgressMeter -          X:132515206            117.6             211701000        1800150.8
22:40:44.264 INFO  ProgressMeter -          X:136218098            117.8             211997000        1800113.8
22:40:54.265 INFO  ProgressMeter -              MT:2801            117.9             212241000        1799638.8
22:41:04.266 INFO  ProgressMeter - NKLS02002208.1:4545561            118.1             212535000        1799588.0
22:41:14.268 INFO  ProgressMeter - NKLS02000910.1:966969            118.3             212839000        1799621.9
22:41:24.278 INFO  ProgressMeter - NKLS02002210.1:706467            118.4             213084000        1799155.7
22:41:34.300 INFO  ProgressMeter - NKLS02002206.1:164260            118.6             213393000        1799227.0
22:41:44.321 INFO  ProgressMeter - NKLS02002204.1:314438            118.8             213704000        1799315.4
22:41:54.346 INFO  ProgressMeter - NKLS02001547.1:74827            118.9             214029000        1799520.2
22:42:04.378 INFO  ProgressMeter - NKLS02000119.1:55243            119.1             214358000        1799756.3
22:42:14.401 INFO  ProgressMeter - NKLS02001545.1:63062            119.3             214681000        1799943.7
22:42:24.409 INFO  ProgressMeter - NKLS02000350.1:59646            119.4             214919000        1799422.7
22:42:34.437 INFO  ProgressMeter - NKLS02001786.1:89090            119.6             215221000        1799433.2
22:42:44.441 INFO  ProgressMeter - NKLS02001967.1:51106            119.8             215536000        1799558.2
22:42:54.468 INFO  ProgressMeter - NKLS02000966.1:82063            119.9             215839000        1799577.1
22:43:04.489 INFO  ProgressMeter - NKLS02001026.1:119477            120.1             216154000        1799697.4
22:43:14.505 INFO  ProgressMeter - NKLS02001289.1:78180            120.3             216492000        1800009.7
22:43:24.517 INFO  ProgressMeter - NKLS02002005.1:90153            120.4             216722000        1799425.8
22:43:34.516 INFO  ProgressMeter - NKLS02000975.1:73447            120.6             217043000        1799600.7
22:43:44.540 INFO  ProgressMeter -  NKLS02001054.1:9675            120.8             217358000        1799719.7
22:43:54.566 INFO  ProgressMeter - NKLS02001478.1:11884            120.9             217599000        1799225.6
22:44:04.587 INFO  ProgressMeter - NKLS02000183.1:36852            121.1             217932000        1799493.9
22:44:14.589 INFO  ProgressMeter - NKLS02000790.1:17570            121.3             218243000        1799585.1
22:44:24.600 INFO  ProgressMeter - NKLS02000111.1:34997            121.4             218469000        1798973.3
22:44:34.630 INFO  ProgressMeter - NKLS02001241.1:28790            121.6             218775000        1799016.7
22:44:44.653 INFO  ProgressMeter - NKLS02000483.1:22422            121.8             219094000        1799168.4
22:44:54.656 INFO  ProgressMeter - NKLS02001553.1:22892            121.9             219401000        1799226.2
22:45:04.671 INFO  ProgressMeter - NKLS02001553.1:58333            122.1             219715000        1799338.2
22:45:14.697 INFO  ProgressMeter - NKLS02000941.1:45041            122.3             220030000        1799455.4
22:45:24.725 INFO  ProgressMeter - NKLS02000194.1:41972            122.4             220332000        1799465.6
22:45:34.732 INFO  ProgressMeter -  NKLS02001530.1:3825            122.6             220650000        1799611.5
22:45:44.734 INFO  ProgressMeter - NKLS02001530.1:17993            122.8             220979000        1799847.7
22:45:54.750 INFO  ProgressMeter - NKLS02001461.1:42262            122.9             221198000        1799185.2
22:46:04.757 INFO  ProgressMeter - NKLS02000540.1:20640            123.1             221512000        1799298.3
22:46:14.763 INFO  ProgressMeter - NKLS02001746.1:13640            123.3             221825000        1799403.2
22:46:24.790 INFO  ProgressMeter - NKLS02001746.1:28894            123.4             222120000        1799357.0
22:46:34.811 INFO  ProgressMeter - NKLS02001746.1:37298            123.6             222437000        1799490.3
22:46:44.838 INFO  ProgressMeter - NKLS02001746.1:44394            123.8             222758000        1799654.1
22:46:54.858 INFO  ProgressMeter - NKLS02001746.1:50459            123.9             222989000        1799093.0
22:47:04.886 INFO  ProgressMeter - NKLS02001746.1:57347            124.1             223311000        1799264.7
22:47:14.893 INFO  ProgressMeter - NKLS02000995.1:21702            124.3             223635000        1799457.1
22:47:24.921 INFO  ProgressMeter -  NKLS02001584.1:1774            124.4             223943000        1799515.4
22:47:34.942 INFO  ProgressMeter - NKLS02001917.1:29982            124.6             224265000        1799687.5
22:47:44.946 INFO  ProgressMeter - NKLS02000752.1:40352            124.8             224596000        1799935.4
22:47:54.946 INFO  ProgressMeter - NKLS02002110.1:31043            124.9             224812000        1799263.2
22:48:04.954 INFO  ProgressMeter - NKLS02002110.1:49248            125.1             225132000        1799422.1
22:48:14.961 INFO  ProgressMeter -  NKLS02000393.1:7626            125.3             225458000        1799628.8
22:48:24.970 INFO  ProgressMeter - NKLS02000393.1:50242            125.4             225696000        1799133.1
22:48:34.973 INFO  ProgressMeter - NKLS02001242.1:25710            125.6             226025000        1799364.2
22:48:44.983 INFO  ProgressMeter - NKLS02001242.1:32515            125.8             226349000        1799553.4
22:48:54.995 INFO  ProgressMeter - NKLS02001242.1:34145            125.9             226594000        1799114.5
22:49:05.012 INFO  ProgressMeter - NKLS02001242.1:42572            126.1             226905000        1799198.8
22:49:15.021 INFO  ProgressMeter -  NKLS02000055.1:8303            126.3             227227000        1799372.0
22:49:25.051 INFO  ProgressMeter - NKLS02000055.1:16285            126.4             227479000        1798986.1
22:49:35.076 INFO  ProgressMeter - NKLS02000055.1:25862            126.6             227789000        1799060.7
22:49:45.085 INFO  ProgressMeter - NKLS02000055.1:34025            126.8             228103000        1799170.0
22:49:55.090 INFO  ProgressMeter - NKLS02000055.1:39572            126.9             228349000        1798744.8
22:50:05.106 INFO  ProgressMeter - NKLS02000055.1:47190            127.1             228666000        1798876.1
22:50:16.733 INFO  ProgressMeter - NKLS02001580.1:38781            127.3             228989000        1798675.1
22:50:26.733 INFO  ProgressMeter -  NKLS02001057.1:2087            127.5             229312000        1798857.3
22:50:36.734 INFO  ProgressMeter - NKLS02000261.1:20528            127.6             229631000        1799007.4
22:50:46.747 INFO  ProgressMeter - NKLS02002178.1:12357            127.8             229952000        1799169.9
22:50:56.768 INFO  ProgressMeter - NKLS02002178.1:25083            128.0             230202000        1798775.4
22:51:06.777 INFO  ProgressMeter - NKLS02001083.1:23995            128.1             230521000        1798923.2
22:51:16.781 INFO  ProgressMeter - NKLS02000731.1:10387            128.3             230783000        1798627.5
22:51:26.808 INFO  ProgressMeter - NKLS02000731.1:15552            128.5             231113000        1798856.5
22:51:36.833 INFO  ProgressMeter - NKLS02000731.1:22076            128.6             231429000        1798976.7
22:51:46.865 INFO  ProgressMeter - NKLS02000731.1:35983            128.8             231671000        1798520.1
22:51:56.873 INFO  ProgressMeter - NKLS02001828.1:19323            129.0             231997000        1798721.7
22:52:06.899 INFO  ProgressMeter - NKLS02000470.1:30046            129.1             232331000        1798980.6
22:52:16.910 INFO  ProgressMeter - NKLS02000255.1:31608            129.3             232609000        1798809.5
22:52:26.921 INFO  ProgressMeter - NKLS02000203.1:15850            129.5             232919000        1798885.5
22:52:36.938 INFO  ProgressMeter -  NKLS02001257.1:5004            129.6             233250000        1799122.1
22:52:46.957 INFO  ProgressMeter - NKLS02001042.1:31393            129.8             233530000        1798964.7
22:52:56.986 INFO  ProgressMeter - NKLS02000134.1:13968            130.0             233854000        1799144.0
22:53:07.008 INFO  ProgressMeter - NKLS02001180.1:15927            130.1             234181000        1799347.5
22:53:17.021 INFO  ProgressMeter - NKLS02000368.1:17027            130.3             234510000        1799567.9
22:53:27.049 INFO  ProgressMeter -  NKLS02000402.1:8181            130.5             234844000        1799822.6
22:53:37.056 INFO  ProgressMeter - NKLS02001029.1:18438            130.6             235174000        1800050.8
22:53:47.083 INFO  ProgressMeter - NKLS02001231.1:22035            130.8             235463000        1799960.5
22:53:57.104 INFO  ProgressMeter - NKLS02001523.1:18900            131.0             235785000        1800123.9
22:54:07.114 INFO  ProgressMeter - NKLS02000075.1:10836            131.1             236105000        1800273.7
22:54:17.142 INFO  ProgressMeter - NKLS02000075.1:25339            131.3             236352000        1799863.4
22:54:27.156 INFO  ProgressMeter -  NKLS02000798.1:8737            131.5             236681000        1800081.2
22:54:37.165 INFO  ProgressMeter -  NKLS02001040.1:9184            131.7             236995000        1800185.1
22:54:47.177 INFO  ProgressMeter -  NKLS02001245.1:3942            131.8             237278000        1800053.2
22:54:57.178 INFO  ProgressMeter - NKLS02001811.1:20320            132.0             237593000        1800166.5
22:55:07.191 INFO  ProgressMeter - NKLS02001807.1:15348            132.2             237904000        1800246.6
22:55:17.221 INFO  ProgressMeter -  NKLS02002189.1:9259            132.3             238194000        1800163.9
22:55:27.245 INFO  ProgressMeter -  NKLS02001293.1:8790            132.5             238509000        1800271.5
22:55:37.257 INFO  ProgressMeter - NKLS02000275.1:14151            132.7             238841000        1800509.7
22:55:47.286 INFO  ProgressMeter - NKLS02000275.1:22099            132.8             239110000        1800269.1
22:55:57.287 INFO  ProgressMeter - NKLS02000275.1:22136            133.0             239412000        1800283.6
22:56:07.292 INFO  ProgressMeter - NKLS02002056.1:25059            133.2             239742000        1800507.4
22:56:17.309 INFO  ProgressMeter - NKLS02001176.1:13224            133.3             240060000        1800637.9
22:56:27.311 INFO  ProgressMeter -  NKLS02000331.1:2882            133.5             240384000        1800816.5
22:56:37.312 INFO  ProgressMeter -  NKLS02001725.1:7194            133.7             240708000        1800994.8
22:56:47.334 INFO  ProgressMeter - NKLS02002125.1:10320            133.8             241035000        1801190.4
22:56:57.356 INFO  ProgressMeter -  NKLS02000632.1:5292            134.0             241369000        1801437.8
22:57:07.379 INFO  ProgressMeter - NKLS02000727.1:20651            134.2             241692000        1801602.3
22:57:17.395 INFO  ProgressMeter -  NKLS02000450.1:2484            134.3             241982000        1801522.3
22:57:27.411 INFO  ProgressMeter - NKLS02001535.1:15976            134.5             242284000        1801531.7
22:57:37.415 INFO  ProgressMeter - NKLS02000048.1:13717            134.7             242610000        1801722.0
22:57:47.418 INFO  ProgressMeter -  NKLS02001954.1:8837            134.8             242894000        1801600.5
22:57:57.426 INFO  ProgressMeter -  NKLS02001121.1:8008            135.0             243202000        1801656.0
22:58:07.446 INFO  ProgressMeter -  NKLS02000327.1:7430            135.2             243534000        1801886.3
22:58:17.475 INFO  ProgressMeter -  NKLS02001925.1:7030            135.3             243812000        1801714.9
22:58:27.494 INFO  ProgressMeter -  NKLS02000520.1:7211            135.5             244121000        1801775.0
22:58:37.502 INFO  ProgressMeter - NKLS02000468.1:10266            135.7             244442000        1801925.9
22:58:48.941 INFO  ProgressMeter - NKLS02000468.1:14963            135.8             244721000        1801450.8
22:58:58.943 INFO  ProgressMeter -  NKLS02000587.1:3254            136.0             245026000        1801485.4
22:59:08.959 INFO  ProgressMeter - NKLS02000587.1:12684            136.2             245346000        1801626.9
22:59:18.963 INFO  ProgressMeter - NKLS02000599.1:12408            136.3             245667000        1801778.0
22:59:28.971 INFO  ProgressMeter - NKLS02002070.1:10346            136.5             245983000        1801891.3
22:59:38.980 INFO  ProgressMeter -  NKLS02002142.1:9005            136.7             246306000        1802055.3
22:59:48.983 INFO  ProgressMeter -  NKLS02001633.1:4158            136.8             246592000        1801949.8
22:59:58.993 INFO  ProgressMeter -  NKLS02000858.1:1958            137.0             246917000        1802128.0
23:00:08.996 INFO  ProgressMeter -  NKLS02001556.1:3012            137.2             247236000        1802263.0
23:00:19.013 INFO  ProgressMeter - NKLS02000784.1:10866            137.3             247484000        1801877.9
23:00:29.047 INFO  ProgressMeter -  NKLS02000047.1:6048            137.5             247814000        1802086.4
23:00:39.077 INFO  ProgressMeter -   NKLS02001379.1:365            137.7             248136000        1802237.1
23:00:49.077 INFO  ProgressMeter -  NKLS02001119.1:7252            137.8             248403000        1801995.0
23:00:59.085 INFO  ProgressMeter -  NKLS02000395.1:2351            138.0             248730000        1802186.5
23:01:09.091 INFO  ProgressMeter -  NKLS02001549.1:7797            138.2             249064000        1802428.6
23:01:19.093 INFO  ProgressMeter -  NKLS02001549.1:8728            138.3             249383000        1802562.6
23:01:29.093 INFO  ProgressMeter -  NKLS02001249.1:4972            138.5             249702000        1802696.7
23:01:39.103 INFO  ProgressMeter -  NKLS02001671.1:4679            138.7             250021000        1802828.3
23:01:49.105 INFO  ProgressMeter -  NKLS02001453.1:1923            138.8             250290000        1802601.2
23:01:59.121 INFO  ProgressMeter -             unmapped            139.0             250614000        1802767.2
23:02:09.153 INFO  ProgressMeter -             unmapped            139.2             250946000        1802986.9
23:02:19.179 INFO  ProgressMeter -             unmapped            139.4             251233000        1802884.5
23:02:29.200 INFO  ProgressMeter -             unmapped            139.5             251575000        1803177.5
23:02:39.225 INFO  ProgressMeter -             unmapped            139.7             251914000        1803447.6
23:02:49.245 INFO  ProgressMeter -             unmapped            139.9             252181000        1803203.2
23:02:49.837 WARN  IntelInflater - Zero Bytes Written : 0
23:02:49.840 INFO  ApplyBQSR - 0 read(s) filtered by: WellformedReadFilter

23:02:49.840 INFO  ProgressMeter -             unmapped            139.9             252199945        1803210.8
23:02:49.840 INFO  ProgressMeter - Traversal complete. Processed 252199945 total reads in 139.9 minutes.
23:02:50.929 INFO  ApplyBQSR - Shutting down engine
[22 February 2023 at 23:02:50 CET] org.broadinstitute.hellbender.tools.walkers.bqsr.ApplyBQSR done. Elapsed time: 139.90 minutes.
Runtime.totalMemory()=2579496960

 

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    David Roazen

    Hi,

    Could you please clarify whether your recalibrated output file (Abi1_recal.bam) is missing any reads that were present in the input file (Abi1_dedup.bam)? Are you saying that there are MT-aligned reads present in the input bam that are not showing up in the recalibrated output bam?

    You might also try running with the additional options --use-jdk-inflater and --use-jdk-deflater and see if that makes a difference.

    Regards,

    David

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    Wondessen Ayalew

    Dear David,
    Thank you for your kind replay. When I check the recalibrated bam file MT is avilable.

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    Wondessen Ayalew
    • Edited

    While i am running haplotypecaller, it took more than 6 days for one sample to generate g.vcf file. Menwhile, I am convinced to call only the MT variants and continue the nuclear variants later.
    Would you please provide me support in this regard.
    Thank you once again for your remarkable support.

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    Atal Saha

    Hi Wondessen Ayalew,

    Did you get any support on this? I am having the same problem - it took a week to finish generating g.vcf for 1 sample. The command I used:

    /gpfs/gpfs0/software/rhel7/eucleia/gatk-4.2.6.1/gatk --java-options "-Xmx260G" HaplotypeCaller -R /gpfs/gpfs0/scratch/Ecogenome/monkfish/reference_genome_monkfish/chr_level_assembly/LOPHIUS_GENOME_and_ANNOTATION/bf2_chromosomelevel.masked.fasta  -I  /gpfs/gpfs0/scratch/Ecogenome/reSultS/bam/Sample_15-LOP-095.dedup.fixed.bam -O /gpfs/gpfs0/scratch/Ecogenome/reSultS/hapcaller/monkfish/Sample_15-LOP-095.g.vcf > Sample_15-LOP-095.log

    so, surprised that even with 260 GB memory its so slow! Would be grateful if anyone has suggestion that can help speeding up the process.

     

    thanks,

    Atal

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    David Roazen

    Hi Atal Saha / Wondessen Ayalew,

    The most common way to speed up HaplotypeCaller is to parallelize by genomic interval using the -L option, either in a local cluster or on the cloud, and then combine the outputs using MergeVcfs or CombineGVCFs. The basic idea is to run HaplotypeCaller many times in parallel, each with a different -L interval, and then merge the outputs at the end. Users typically will parallelize at least by chromosome, and often more finely. We publish a cloud-based workflow in Terra that can do this parallelization for you here. 

    If you don't have access to a cluster, and don't want to run on the cloud, you can try running HaplotypeCallerSpark, which is able to parallelize HaplotypeCaller using multiple threads on a local machine.

    One other thing you should check is whether you're running on an Intel/AMD CPU, or another architecture such as M1. GATK does not currently have good support for M1 chips, and tools like HaplotypeCaller will run very slowly on such machines.

    Regards,
    David

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    Atal Saha

    Hi David,

    Thanks very much for your reply on this.

    Running Spark version was much faster, but I am worried about warning about this version (as it says that we should not use spark if we care about results!). Are results from spark reliable? also spark is not generating .idx files. Does spark not generate .idx file by default as like the normal haplotypecaller?

    thanks for your help with this,

    Atal

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    David Roazen

    Hi Atal Saha,

    HaplotypeCallerSpark is a thin wrapper that just calls directly into the regular HaplotypeCaller code, so the results should be extremely close. However, because the Spark version "shards" the input data across multiple threads, there may be calling artifacts near the shard boundaries -- though a lot of work has been done to minimize this possibility. It's also possible that certain arguments that work for regular HaplotypeCaller may not work with HaplotypeCallerSpark. For these reasons, we hesitate to endorse the Spark version for clinical / production use, but for more casual purposes it should be perfectly fine to use. You may need to manually index the output VCF (eg., using GATK's IndexFeatureFile) after running the Spark version of the tool. 

    What we actually do in production here at the Broad is to use an interval list with carefully-chosen split points at areas of the reference that are filled with N's (such as at the centromeres), and then launch many HaplotypeCaller tasks at once to call variants for these intervals. This approach eliminates the possibility of calling artifacts near the interval boundaries.

    Regards,
    David

     

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    Atal Saha

    Thanks very much, David.

     

    I did manage to produce all my vcf files. Seemed using 20-30 gb memory was a good solution.

    However, I accidentally did not include -ERC GVCF option while running haplotypecalling and now struglling to run combine vcfs and to run jointgenotyping. Running haplotypecalling one more time for all my samples will again take 2 weeks, so, is there a way out here?

     

    Thanks again,

    Atal

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    David Roazen

    Hi Atal Saha,

    Unfortunately, without the reference confidence scores produced by -ERC GVCF you will be unable to run joint genotyping using GATK. I'm afraid your only option is to re-call your samples.

    Sorry!

    David

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    Atal Saha

    Thanks again, David.

    Re-calling started.

     

    cheers

    Atal

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