Haplotypecaller producing an empty VCF file

Healthcare & Life Sciences Parabricks
1

Hello everyone,

I’m encountering an issue with GATK’s HaplotypeCaller while working with RNA-seq data using NVIDIA Parabricks. Despite following the recommended steps, the output VCF file is empty, indicating no variants were called.

Context:

  • Tool: GATK HaplotypeCaller via NVIDIA Parabricks
  • Data Type: RNA-seq
  • Reference Genome: Mus musculus (GRCm39)
  • BAM FILE: Was produced via NVIDIA Parabrick’s rna_fq2bam
docker run --rm --gpus all     -v /home/$USER:/workdir     nvcr.io/nvidia/clara/clara-parabricks:4.3.1-1     pbrun haplotypecaller     --ref /workdir/ref/Mus_musculus.GRCm39.dna.primary_assembly.fa     --in-bam /workdir/output/aligned_reads.bam     --out-variants /workdir/output/germline_variants.vcf --rna

I have checked the integrity of the BAM file with samtools as well as the contents and all seems to be okay.

Are there any pipeline’s available in parabricks similar to 3.8’s rna_gatk?

1 Like
2

I have the same issue. Did youo recieve any replies?

3

Hi @owais.siddiqi and @naomi.dyer,

We have removed some tools from Parabricks 3.8. However for RNA data we still have rna_fq2bam and starfusion.

Does that suffice or is there something more that you’re looking for?

Thank you

4

Hi, I’m not sure which tools you have removed? The tools I was attempting to usde were rna-fq2bam and haplotypecaller. I am working with Anopheles gambiae. rna-fq2bam appears to have run correctly producing bam files which look OK when examined using samtools. However, when haplotypecaller is run the vcf files only contain the header and no variants. No error messages are produced. Would this be caused by the changes to Parabricks? The STAR genome index, genome.fasta and vcf files all have the same chromosome names (2L, 2R, 3L, 3R, X). I can provide all commands and sample outputs and logs if that is any help in diagnosing the issue?
thanks

5

Hi @naomi.dyer,

It would be helpful if you can share your run command and log files that you have. I have done some digging and notice there is a --rna flag in haplotypecaller. Are you running with that on?

Thanks

6

Hi, yes that is correct, I am trying to effectively run the RNAseq short variant discovery (SNPs + Indels) pipeline. So I am first using rna-fq2bam to STAR align RNAseq reads. I’m then using haplotype caller with the --rna flag to try and call the variants from the bam files.
Is there a problem with using the --rna flag? I understood that was the flag to use if working with RNAseq data.

I should mention I couldn’t fugure out how to create the star index using nvidia fq2bam so I generated that using cpu equivalent (STAR v2.7.2a) before starting.

Here is an example command used to generate the bam
docker run --user 1003:1003 --rm --gpus all --volume /mnt/hp_scratch/Users/dyern/Clara_Parabricks/rna_seq_pop_pexa2:/workdir --workdir /workdir nvcr.io/nvidia/clara/clara-parabricks:4.3.0-1
pbrun rna_fq2bam
–in-fq /workdir/resources/reads/Unknown_BY068-001T0022_1.fq.gz /workdir/resources/reads/Unknown_BY068-001T0022_2.fq.gz
–genome-lib-dir /workdir/resources/reference/star_index
–output-dir /workdir
–ref /workdir/resources/reference/VectorBase-68_AgambiaePEST_Genome.fasta
–out-bam /workdir/results/alignments/Kisumu10.star.bam
–read-files-command zcat 2> logs/parabricks/fq2bam-Kisumu10.log

The log file is rather long (1835 lines) so I’ve just put tha tail of it here to aid readability:
[PB Info 2024-Oct-01 21:39:59] ReadQueue:10, Stage0:0, Stage1:11, Stage1InnerVec:2000, Stage1InnerChunk:10, copyQ:0, poolQ:6, Stage2:0, Stage3 input:0
[PB Info 2024-Oct-01 21:39:59] Thread 0 windowPool:600, transPool:400, Thread 1 windowPool:200, transPool:400, Thread 2 windowPool:600, transPool:400, Thread 3 windowPool:400, transPool:400,
[PB Info 2024-Oct-01 21:39:59] PoolQ:18
[PB Info 2024-Oct-01 21:39:59] reader done
[PB Info 2024-Oct-01 21:40:00] Stage 1 gpu thread done
[PB Info 2024-Oct-01 21:40:00] Stage 1 cpu threads done
[PB Info 2024-Oct-01 21:40:00] Stage 0 gpu thread done
[PB Info 2024-Oct-01 21:40:00] Stage 0 cpu threads done
[PB Info 2024-Oct-01 21:40:00] Stage 0 clearAllQueue done
[PB Info 2024-Oct-01 21:40:00] Stage 1 clearAllQueue done
[PB Info 2024-Oct-01 21:40:00] all workers joined
[PB Info 2024-Oct-01 21:40:00] reader done
[PB Info 2024-Oct-01 21:40:00] … finished mapping
[PB Info 2024-Oct-01 21:40:01] All processing done, time 1322.708
[PB Info 2024-Oct-01 21:40:05] … finished successfully
[PB Info 2024-Oct-01 21:40:05] All done, total running time is 1328.960
[PB Info 2024-Oct-01 21:40:05] Memory free
[PB Info 2024-Oct-01 21:40:05] ------------------------------------------------------------------------------
[PB Info 2024-Oct-01 21:40:05] || Program: star ||
[PB Info 2024-Oct-01 21:40:05] || Version: 4.3.0-1 ||
[PB Info 2024-Oct-01 21:40:05] || Start Time: Tue Oct 1 21:17:56 2024 ||
[PB Info 2024-Oct-01 21:40:05] || End Time: Tue Oct 1 21:40:05 2024 ||
[PB Info 2024-Oct-01 21:40:05] || Total Time: 22 minutes 9 seconds ||
[PB Info 2024-Oct-01 21:40:05] ------------------------------------------------------------------------------
[PB Info 2024-Oct-01 21:40:08] ------------------------------------------------------------------------------
[PB Info 2024-Oct-01 21:40:08] || Parabricks accelerated Genomics Pipeline ||
[PB Info 2024-Oct-01 21:40:08] || Version 4.3.0-1 ||
[PB Info 2024-Oct-01 21:40:08] || Sorting Phase-II ||
[PB Info 2024-Oct-01 21:40:08] ------------------------------------------------------------------------------
[PB Info 2024-Oct-01 21:40:08] progressMeter - Percentage
[PB Info 2024-Oct-01 21:40:08] 0.0
[PB Info 2024-Oct-01 21:40:13] 24.9
[PB Info 2024-Oct-01 21:40:18] 62.2
[PB Info 2024-Oct-01 21:40:23] 99.9
[PB Info 2024-Oct-01 21:40:28] 99.9
[PB Info 2024-Oct-01 21:40:33] 99.9
[PB Info 2024-Oct-01 21:40:38] 99.9
[PB Info 2024-Oct-01 21:40:43] 99.9
[PB Info 2024-Oct-01 21:40:48] Sorting and Marking: 40.003 seconds
[PB Info 2024-Oct-01 21:40:48] ------------------------------------------------------------------------------
[PB Info 2024-Oct-01 21:40:48] || Program: Sorting Phase-II ||
[PB Info 2024-Oct-01 21:40:48] || Version: 4.3.0-1 ||
[PB Info 2024-Oct-01 21:40:48] || Start Time: Tue Oct 1 21:40:08 2024 ||
[PB Info 2024-Oct-01 21:40:48] || End Time: Tue Oct 1 21:40:48 2024 ||
[PB Info 2024-Oct-01 21:40:48] || Total Time: 40 seconds ||
[PB Info 2024-Oct-01 21:40:48] ------------------------------------------------------------------------------
[PB Info 2024-Oct-01 21:40:48] ------------------------------------------------------------------------------
[PB Info 2024-Oct-01 21:40:48] || Parabricks accelerated Genomics Pipeline ||
[PB Info 2024-Oct-01 21:40:48] || Version 4.3.0-1 ||
[PB Info 2024-Oct-01 21:40:48] || Marking Duplicates, BQSR ||
[PB Info 2024-Oct-01 21:40:48] ------------------------------------------------------------------------------
[PB Info 2024-Oct-01 21:40:48] Using PBBinBamFile for BAM writing
[PB Info 2024-Oct-01 21:40:48] progressMeter - Percentage
[PB Info 2024-Oct-01 21:40:58] 24.8
[PB Info 2024-Oct-01 21:41:08] 56.3
[PB Info 2024-Oct-01 21:41:18] 100.0
[PB Info 2024-Oct-01 21:41:18] BQSR and writing final BAM: 30.026 seconds
[PB Info 2024-Oct-01 21:41:18] ------------------------------------------------------------------------------
[PB Info 2024-Oct-01 21:41:18] || Program: Marking Duplicates, BQSR ||
[PB Info 2024-Oct-01 21:41:18] || Version: 4.3.0-1 ||
[PB Info 2024-Oct-01 21:41:18] || Start Time: Tue Oct 1 21:40:48 2024 ||
[PB Info 2024-Oct-01 21:41:18] || End Time: Tue Oct 1 21:41:18 2024 ||
[PB Info 2024-Oct-01 21:41:18] || Total Time: 30 seconds ||
[PB Info 2024-Oct-01 21:41:18] ------------------------------------------------------------------------------

This generates the bam file as expected and when checked with samtools it looks good:
samtools flagstat Kisumu10.star.bam give this output
147705766 + 0 in total (QC-passed reads + QC-failed reads)
10165766 + 0 secondary
0 + 0 supplementary
64833336 + 0 duplicates
147705766 + 0 mapped (100.00% : N/A)
137540000 + 0 paired in sequencing
68770000 + 0 read1
68770000 + 0 read2
137540000 + 0 properly paired (100.00% : N/A)
137540000 + 0 with itself and mate mapped
0 + 0 singletons (0.00% : N/A)
0 + 0 with mate mapped to a different chr
0 + 0 with mate mapped to a different chr (mapQ>=5)

Then I proceeded to run haplotypecaller using the following command:
docker run --gpus all --rm --volume $(pwd):/workdir --volume $(pwd):/outputdir nvcr.io/nvidia/clara/clara-parabricks:4.3.0-1 pbrun haplotypecaller --rna --ref /workdir/resources/reference/VectorBase-68_AgambiaePEST_Genome.fasta --in-bam /workdir/results/alignments/Kisumu10.star.bam --out-variants /workdir/outputdir/Kisumu10.basic_testrun.vcf --log /workdir/outputdir/Kisumu10.basic_testrun.vcf.log

Log file looks like this:
[PB Info 2024-Oct-09 10:48:23] ------------------------------------------------------------------------------
[PB Info 2024-Oct-09 10:48:23] || Parabricks accelerated Genomics Pipeline ||
[PB Info 2024-Oct-09 10:48:23] || Version 4.3.0-1 ||
[PB Info 2024-Oct-09 10:48:23] || GPU-GATK4 HaplotypeCaller ||
[PB Info 2024-Oct-09 10:48:23] ------------------------------------------------------------------------------
[PB Info 2024-Oct-09 10:48:25] 0 /workdir/results/alignments/Kisumu10.star.bam/workdir/outputdir/Kisumu10.basic_testrun.vcf
[PB Info 2024-Oct-09 10:48:25] ProgressMeter - Current-Locus Elapsed-Minutes Regions-Processed Regions/Minute
[PB Info 2024-Oct-09 10:48:35] :0 0.2 0 0
[PB Info 2024-Oct-09 10:48:45] :0 0.3 0 0
[PB Info 2024-Oct-09 10:48:55] :0 0.5 0 0
[PB Info 2024-Oct-09 10:49:05] :0 0.7 0 0
[PB Info 2024-Oct-09 10:49:15] :0 0.8 0 0
[PB Info 2024-Oct-09 10:49:25] :0 1.0 0 0
[PB Info 2024-Oct-09 10:49:35] :0 1.2 0 0
[PB Info 2024-Oct-09 10:49:45] :0 1.3 0 0
[PB Info 2024-Oct-09 10:49:55] Total time taken: 90.290000
[PB Info 2024-Oct-09 10:49:55] ------------------------------------------------------------------------------
[PB Info 2024-Oct-09 10:49:55] || Program: GPU-GATK4 HaplotypeCaller ||
[PB Info 2024-Oct-09 10:49:55] || Version: 4.3.0-1 ||
[PB Info 2024-Oct-09 10:49:55] || Start Time: Wed Oct 9 10:48:23 2024 ||
[PB Info 2024-Oct-09 10:49:55] || End Time: Wed Oct 9 10:49:55 2024 ||
[PB Info 2024-Oct-09 10:49:55] || Total Time: 1 minute 32 seconds ||
[PB Info 2024-Oct-09 10:49:55] ------------------------------------------------------------------------------

This produces a vcf file with no variants (header only) so last line of vcf looks like this:
#CHROM POS ID REF ALT QUAL FILTER INFO FORMAT sample

I think what might be happening is that the variants are all being filtered, because the nvidia rna-fq2bam is based on STAR version 2.7.2a. The default MAPQ for unquely mapping reads is 255 (see MAPQ 255 doesn't work with GATK · Issue #1069 · alexdobin/STAR · GitHub).

If I rerun with a flag to remove the filter for MAPQ being available like this:
docker run --gpus all --rm --volume $(pwd):/workdir --volume $(pwd):/outputdir nvcr.io/nvidia/clara/clara-parabricks:4.3.0-1 pbrun haplotypecaller --rna --ref /workdir/resources/reference/VectorBase-68_AgambiaePEST_Genome.fasta --disable-read-filter MappingQualityAvailableReadFilter --in-bam /workdir/results/alignments/Kisumu10.star.bam --out-variants /workdir/outputdir/Kisumu10.noMapQ_filter_testrun.vcf --log /workdir/outputdir/Kisumu10.noMapQ_filter_testrun.vcf.log

Then this does work;
Log file:
[PB Info 2024-Oct-09 10:53:59] ------------------------------------------------------------------------------
[PB Info 2024-Oct-09 10:53:59] || Parabricks accelerated Genomics Pipeline ||
[PB Info 2024-Oct-09 10:53:59] || Version 4.3.0-1 ||
[PB Info 2024-Oct-09 10:53:59] || GPU-GATK4 HaplotypeCaller ||
[PB Info 2024-Oct-09 10:53:59] ------------------------------------------------------------------------------
[PB Info 2024-Oct-09 10:54:02] 0 /workdir/results/alignments/Kisumu10.star.bam/workdir/outputdir/Kisumu10.noMapQ_filter_testrun.vcf
[PB Info 2024-Oct-09 10:54:02] ProgressMeter - Current-Locus Elapsed-Minutes Regions-Processed Regions/Minute
[PB Info 2024-Oct-09 10:54:12] 2L:4766401 0.2 37124 222744
[PB Info 2024-Oct-09 10:54:22] 2L:9883201 0.3 55775 167325
[PB Info 2024-Oct-09 10:54:32] 2L:11400001 0.5 72253 144506
[PB Info 2024-Oct-09 10:54:42] 2L:15187201 0.7 85154 127731
[PB Info 2024-Oct-09 10:54:52] 2L:17318401 0.8 95103 114123
[PB Info 2024-Oct-09 10:55:02] 2L:20496001 1.0 109615 109615
[PB Info 2024-Oct-09 10:55:12] 2L:23937601 1.2 121388 104046
[PB Info 2024-Oct-09 10:55:22] 2L:25521601 1.3 131509 98631
[PB Info 2024-Oct-09 10:55:32] 2L:27820801 1.5 139627 93084
[PB Info 2024-Oct-09 10:55:42] 2L:28238401 1.7 149060 89436
[PB Info 2024-Oct-09 10:55:52] 2L:31785601 1.8 155522 84830
[PB Info 2024-Oct-09 10:56:02] 2L:34108801 2.0 166186 83093
[PB Info 2024-Oct-09 10:56:12] 2L:37876801 2.2 180237 83186
[PB Info 2024-Oct-09 10:56:22] 2L:39705601 2.3 189365 81156
[PB Info 2024-Oct-09 10:56:32] 2L:40300801 2.5 198722 79488
[PB Info 2024-Oct-09 10:56:42] 2L:44059201 2.7 211726 79397
[PB Info 2024-Oct-09 10:56:52] 2L:46339201 2.8 224469 79224
[PB Info 2024-Oct-09 10:57:02] 2L:47481601 3.0 232065 77355
[PB Info 2024-Oct-09 10:57:12] 2L:48604801 3.2 241122 76143
[PB Info 2024-Oct-09 10:57:22] 2R:676801 3.3 248297 74489
[PB Info 2024-Oct-09 10:57:32] 2R:2880001 3.5 257713 73632
[PB Info 2024-Oct-09 10:57:42] 2R:5419201 3.7 268601 73254
[PB Info 2024-Oct-09 10:57:52] 2R:6484801 3.8 281628 73468
[PB Info 2024-Oct-09 10:58:02] 2R:8875201 4.0 291133 72783
[PB Info 2024-Oct-09 10:58:12] 2R:12379201 4.2 301802 72432
[PB Info 2024-Oct-09 10:58:22] 2R:15158401 4.3 319449 73719
[PB Info 2024-Oct-09 10:58:32] 2R:19051201 4.5 336612 74802
[PB Info 2024-Oct-09 10:58:42] 2R:20740801 4.7 342042 73294
[PB Info 2024-Oct-09 10:58:52] 2R:22171201 4.8 353034 73041
[PB Info 2024-Oct-09 10:59:02] 2R:24561601 5.0 360767 72153
[PB Info 2024-Oct-09 10:59:12] 2R:26673601 5.2 374631 72509
[PB Info 2024-Oct-09 10:59:22] 2R:28675201 5.3 381948 71615
[PB Info 2024-Oct-09 10:59:32] 2R:31420801 5.5 395880 71978
[PB Info 2024-Oct-09 10:59:42] 2R:34320001 5.7 409179 72208
[PB Info 2024-Oct-09 10:59:52] 2R:38803201 5.8 431566 73982
[PB Info 2024-Oct-09 11:00:02] 2R:43128001 6.0 447785 74630
[PB Info 2024-Oct-09 11:00:12] 2R:48264001 6.2 469453 76127
[PB Info 2024-Oct-09 11:00:22] 2R:50966401 6.3 484400 76484
[PB Info 2024-Oct-09 11:00:32] 2R:54931201 6.5 499599 76861
[PB Info 2024-Oct-09 11:00:42] 2R:57451201 6.7 512872 76930
[PB Info 2024-Oct-09 11:00:52] 3L:3192001 6.8 558400 81717
[PB Info 2024-Oct-09 11:01:02] 3L:8323201 7.0 570813 81544
[PB Info 2024-Oct-09 11:01:12] 3L:12681601 7.2 588374 82098
[PB Info 2024-Oct-09 11:01:22] 3L:15787201 7.3 603631 82313
[PB Info 2024-Oct-09 11:01:32] 3L:18230401 7.5 614768 81969
[PB Info 2024-Oct-09 11:01:42] 3L:20961601 7.7 624765 81491
[PB Info 2024-Oct-09 11:01:52] 3L:26913601 7.8 648714 82814
[PB Info 2024-Oct-09 11:02:02] 3L:31392001 8.0 663978 82997
[PB Info 2024-Oct-09 11:02:12] 3L:34632001 8.2 675457 82709
[PB Info 2024-Oct-09 11:02:22] 3L:37449601 8.3 690167 82820
[PB Info 2024-Oct-09 11:02:32] 3L:41760001 8.5 708425 83344
[PB Info 2024-Oct-09 11:02:42] 3R:1123201 8.7 716255 82644
[PB Info 2024-Oct-09 11:02:52] 3R:3216001 8.8 723273 81879
[PB Info 2024-Oct-09 11:03:02] 3R:3835201 9.0 731280 81253
[PB Info 2024-Oct-09 11:03:12] 3R:5092801 9.2 744724 81242
[PB Info 2024-Oct-09 11:03:22] 3R:8625601 9.3 757654 81177
[PB Info 2024-Oct-09 11:03:32] 3R:13262401 9.5 777460 81837
[PB Info 2024-Oct-09 11:03:42] 3R:18494401 9.7 794747 82215
[PB Info 2024-Oct-09 11:03:52] 3R:20760001 9.8 811421 82517
[PB Info 2024-Oct-09 11:04:02] 3R:24465601 10.0 825074 82507
[PB Info 2024-Oct-09 11:04:12] 3R:28142401 10.2 838170 82442
[PB Info 2024-Oct-09 11:04:22] 3R:30672001 10.3 848943 82155
[PB Info 2024-Oct-09 11:04:32] 3R:34713601 10.5 858959 81805
[PB Info 2024-Oct-09 11:04:42] 3R:37152001 10.7 874923 82024
[PB Info 2024-Oct-09 11:04:52] 3R:44323201 10.8 890589 82208
[PB Info 2024-Oct-09 11:05:02] 3R:46982401 11.0 909907 82718
[PB Info 2024-Oct-09 11:05:12] 3R:49483201 11.2 918663 82268
[PB Info 2024-Oct-09 11:05:22] UNKN:26428801 11.3 1027564 90667
[PB Info 2024-Oct-09 11:05:32] X:878401 11.5 1075921 93558
[PB Info 2024-Oct-09 11:05:42] X:1780801 11.7 1087566 93219
[PB Info 2024-Oct-09 11:05:52] X:5750401 11.8 1107240 93569
[PB Info 2024-Oct-09 11:06:02] X:8016001 12.0 1122733 93561
[PB Info 2024-Oct-09 11:06:12] X:11654401 12.2 1139458 93654
[PB Info 2024-Oct-09 11:06:22] X:14438401 12.3 1160493 94094
[PB Info 2024-Oct-09 11:06:32] X:19651201 12.5 1199452 95956
[PB Info 2024-Oct-09 11:06:42] AAAB01008965:48001 12.7 1209199 95463
[PB Info 2024-Oct-09 11:06:52] Mt:4801 12.8 1209199 94223
[PB Info 2024-Oct-09 11:07:03] Total time taken: 781.434274
[PB Info 2024-Oct-09 11:07:03] ------------------------------------------------------------------------------
[PB Info 2024-Oct-09 11:07:03] || Program: GPU-GATK4 HaplotypeCaller ||
[PB Info 2024-Oct-09 11:07:03] || Version: 4.3.0-1 ||
[PB Info 2024-Oct-09 11:07:03] || Start Time: Wed Oct 9 10:53:59 2024 ||
[PB Info 2024-Oct-09 11:07:03] || End Time: Wed Oct 9 11:07:03 2024 ||
[PB Info 2024-Oct-09 11:07:03] || Total Time: 13 minutes 4 seconds ||
[PB Info 2024-Oct-09 11:07:03] ------------------------------------------------------------------------------

This produces a vcf file with 872132 variants.

7

Continued from last post.
Alternatively I have also tried rerunning the rna-fq2bam with a flag to label uniquely mapped reads as having MAPQ 60 as suggested by Alex Dobin in the STAR github (MAPQ 255 doesn't work with GATK · Issue #1069 · alexdobin/STAR · GitHub)

Command:
docker run --user 1003:1003 --rm --gpus all --volume $(pwd):/workdir --volume $(pwd)outputdir --workdir /workdir nvcr.io/nvidia/clara/clara-parabricks:4.3.0-1 pbrun rna_fq2bam --in-fq /workdir/resources/reads/Unknown_BY068-001T0022_1.fq.gz /workdir/resources/reads/Unknown_BY068-001T0022_2.fq.gz --read-files-command zcat --out-sam-mapq-unique 60 --genome-lib-dir /workdir/resources/reference/star_index --output-dir /workdir --ref /workdir/resources/reference/VectorBase-68_AgambiaePEST_Genome.fasta --out-bam /workdir/outputdir/Kisumu10_mqpq60.star.bam --logfile /workdir/outputdir/Kisumu10_mqpq60.star.bam.log

Log file (again, long, so last few lines)

Then rerun haplotypecaller with all filters in place:
try haplotype caller on this with filters in place:
docker run --gpus all --rm --volume $(pwd):/workdir --volume $(pwd):/outputdir nvcr.io/nvidia/clara/clara-parabricks:4.3.0-1 pbrun haplotypecaller --rna --ref /workdir/resources/reference/VectorBase-68_AgambiaePEST_Genome.fasta --in-bam /workdir/outputdir/Kisumu10_mqpq60.star.bam --out-variants /workdir/outputdir/Kisumu10.from_MapQ60bam.vcf --log /workdir/outputdir/Kisumu10.from_MapQ60bam.vcf

This ran

log file:
[PB Info 2024-Oct-09 11:52:22] ------------------------------------------------------------------------------
[PB Info 2024-Oct-09 11:52:22] || Parabricks accelerated Genomics Pipeline ||
[PB Info 2024-Oct-09 11:52:22] || Version 4.3.0-1 ||
[PB Info 2024-Oct-09 11:52:22] || GPU-GATK4 HaplotypeCaller ||
[PB Info 2024-Oct-09 11:52:22] ------------------------------------------------------------------------------
[PB Info 2024-Oct-09 11:52:24] 0 /workdir/outputdir/Kisumu10_mqpq60.star.bam/workdir/outputdir/Kisumu10.from_MapQ60bam.vcf
[PB Info 2024-Oct-09 11:52:24] ProgressMeter - Current-Locus Elapsed-Minutes Regions-Processed Regions/Minute
[PB Info 2024-Oct-09 11:52:34] 2L:4862401 0.2 33626 201756
[PB Info 2024-Oct-09 11:52:44] 2L:7804801 0.3 50966 152898
[PB Info 2024-Oct-09 11:52:54] 2L:10339201 0.5 58116 116232
[PB Info 2024-Oct-09 11:53:04] 2L:12998401 0.7 72760 109140
[PB Info 2024-Oct-09 11:53:14] 2L:15081601 0.8 82855 99426
[PB Info 2024-Oct-09 11:53:24] 2L:17030401 1.0 91969 91969
[PB Info 2024-Oct-09 11:53:34] 2L:18000001 1.2 99575 85350
[PB Info 2024-Oct-09 11:53:44] 2L:21196801 1.3 114236 85677
[PB Info 2024-Oct-09 11:53:54] 2L:24052801 1.5 123869 82579
[PB Info 2024-Oct-09 11:54:04] 2L:26649601 1.7 135617 81370
[PB Info 2024-Oct-09 11:54:14] 2L:28238401 1.8 141516 77190
[PB Info 2024-Oct-09 11:54:24] 2L:30926401 2.0 153882 76941
[PB Info 2024-Oct-09 11:54:34] 2L:32371201 2.2 163660 75535
[PB Info 2024-Oct-09 11:54:44] 2L:35649601 2.3 178729 76598
[PB Info 2024-Oct-09 11:54:54] 2L:39556801 2.5 188901 75560
[PB Info 2024-Oct-09 11:55:04] 2L:40156801 2.7 199810 74928
[PB Info 2024-Oct-09 11:55:14] 2L:44611201 2.8 217919 76912
[PB Info 2024-Oct-09 11:55:24] 2L:46627201 3.0 228543 76181
[PB Info 2024-Oct-09 11:55:34] 2L:48508801 3.2 237891 75123
[PB Info 2024-Oct-09 11:55:44] 2R:619201 3.3 250440 75132
[PB Info 2024-Oct-09 11:55:54] 2R:3580801 3.5 261587 74739
[PB Info 2024-Oct-09 11:56:04] 2R:5812801 3.7 274837 74955
[PB Info 2024-Oct-09 11:56:14] 2R:8798401 3.8 287200 74921
[PB Info 2024-Oct-09 11:56:24] 2R:10617601 4.0 300612 75153
[PB Info 2024-Oct-09 11:56:34] 2R:14827201 4.2 319198 76607
[PB Info 2024-Oct-09 11:56:44] 2R:18345601 4.3 337494 77883
[PB Info 2024-Oct-09 11:56:54] 2R:20942401 4.5 344525 76561
[PB Info 2024-Oct-09 11:57:04] 2R:22891201 4.7 357726 76655
[PB Info 2024-Oct-09 11:57:14] 2R:27182401 4.8 373504 77276
[PB Info 2024-Oct-09 11:57:24] 2R:28392001 5.0 386246 77249
[PB Info 2024-Oct-09 11:57:34] 2R:32894401 5.2 402758 77953
[PB Info 2024-Oct-09 11:57:44] 2R:36148801 5.3 431319 80872
[PB Info 2024-Oct-09 11:57:54] 2R:42369601 5.5 457247 83135
[PB Info 2024-Oct-09 11:58:04] 2R:48715201 5.7 477956 84345
[PB Info 2024-Oct-09 11:58:14] 2R:54374401 5.8 497823 85341
[PB Info 2024-Oct-09 11:58:24] 2R:57595201 6.0 520407 86731
[PB Info 2024-Oct-09 11:58:34] 3L:7166401 6.2 567808 92076
[PB Info 2024-Oct-09 11:58:44] 3L:8620801 6.3 589062 93009
[PB Info 2024-Oct-09 11:58:54] 3L:16468801 6.5 606880 93366
[PB Info 2024-Oct-09 11:59:04] 3L:20548801 6.7 622046 93306
[PB Info 2024-Oct-09 11:59:14] 3L:24744001 6.8 648331 94875
[PB Info 2024-Oct-09 11:59:24] 3L:31713601 7.0 668271 95467
[PB Info 2024-Oct-09 11:59:34] 3L:36835201 7.2 688846 96118
[PB Info 2024-Oct-09 11:59:44] 3L:38529601 7.3 710773 96923
[PB Info 2024-Oct-09 11:59:54] 3R:768001 7.5 719242 95898
[PB Info 2024-Oct-09 12:00:04] 3R:3216001 7.7 727104 94839
[PB Info 2024-Oct-09 12:00:14] 3R:4876801 7.8 735590 93905
[PB Info 2024-Oct-09 12:00:24] 3R:8601601 8.0 751921 93990
[PB Info 2024-Oct-09 12:00:34] 3R:11352001 8.2 769125 94178
[PB Info 2024-Oct-09 12:00:44] 3R:18100801 8.3 791641 94996
[PB Info 2024-Oct-09 12:00:54] 3R:20808001 8.5 809196 95199
[PB Info 2024-Oct-09 12:01:04] 3R:26486401 8.7 826281 95340
[PB Info 2024-Oct-09 12:01:14] 3R:28147201 8.8 838434 94917
[PB Info 2024-Oct-09 12:01:24] 3R:33494401 9.0 852887 94765
[PB Info 2024-Oct-09 12:01:34] 3R:35620801 9.2 863836 94236
[PB Info 2024-Oct-09 12:01:44] 3R:41788801 9.3 888436 95189
[PB Info 2024-Oct-09 12:01:54] 3R:45307201 9.5 908089 95588
[PB Info 2024-Oct-09 12:02:04] 3R:49272001 9.7 921783 95356
[PB Info 2024-Oct-09 12:02:14] UNKN:29361601 9.8 1043300 106098
[PB Info 2024-Oct-09 12:02:24] X:1252801 10.0 1079509 107950
[PB Info 2024-Oct-09 12:02:34] X:3019201 10.2 1095519 107755
[PB Info 2024-Oct-09 12:02:44] X:7603201 10.3 1118509 108242
[PB Info 2024-Oct-09 12:02:54] X:9456001 10.5 1131815 107791
[PB Info 2024-Oct-09 12:03:04] X:15691201 10.7 1156637 108434
[PB Info 2024-Oct-09 12:03:14] X:21753601 10.8 1199713 110742
[PB Info 2024-Oct-09 12:03:24] Mt:1 11.0 1209199 109927
[PB Info 2024-Oct-09 12:03:34] Mt:14401 11.2 1209199 108286
[PB Info 2024-Oct-09 12:03:45] Total time taken: 681.142182
[PB Info 2024-Oct-09 12:03:45] ------------------------------------------------------------------------------
[PB Info 2024-Oct-09 12:03:45] || Program: GPU-GATK4 HaplotypeCaller ||
[PB Info 2024-Oct-09 12:03:45] || Version: 4.3.0-1 ||
[PB Info 2024-Oct-09 12:03:45] || Start Time: Wed Oct 9 11:52:22 2024 ||
[PB Info 2024-Oct-09 12:03:45] || End Time: Wed Oct 9 12:03:45 2024 ||
[PB Info 2024-Oct-09 12:03:45] || Total Time: 11 minutes 23 seconds ||
[PB Info 2024-Oct-09 12:03:45] ------------------------------------------------------------------------------

last line of vcf:
Mt 13081 . T C 305592.06 . AC=2;AF=1.00;AN=2;BaseQRankSum=0.363;DP=7446;ExcessHet=0.0000;FS=3.032;MLEAC=2;MLEAF=1.00;MQ=60.00;MQRankSum=-0.202;QD=35.89;ReadPosRankSum=7.731;SOR=0.050 GT:AD:DP:GQ:PL 1/1:41,7248:7289:99:305606,20413,0

number of variants called: 872132 (same as with the --disable-read-filter MappingQualityAvailebleReadFilter option with the bam file without changing the label from 255 to 60)

8

Summary of reply: I think I have solved my issue of the no variants. This can be solved be either
a. adding --disable-read-filter MappingQualityAvailableReadFilter to the haplotypecaller command
b. adding --out-sam-mapq-unique 60 to the rna-fq2bam command
The number of snps called on my one sample test was the same.
While haplotypecaller is impressively fast, fq2bam is only about 6 times faster than running CPU equivalent on my hardware.
It would be extremely helpful to add a warning about this to the docs - it is not specific to my data as I repeated the same progress on the sample human file from Getting The Sample Data - NVIDIA Docs (pretending it was RNAseq data) with the same outcome.