# Tagged with #combinegvcfs 1 documentation article | 1 announcement | 24 forum discussions

### Overview

GVCF stands for Genomic VCF. A GVCF is a kind of VCF, so the basic format specification is the same as for a regular VCF (see the spec documentation here), but a Genomic VCF contains extra information.

This document explains what that extra information is and how you can use it to empower your variants analyses.

### Important caveat

What we're covering here is strictly limited to GVCFs produced by HaplotypeCaller in GATK versions 3.0 and above. The term GVCF is sometimes used simply to describe VCFs that contain a record for every position in the genome (or interval of interest) regardless of whether a variant was detected at that site or not (such as VCFs produced by UnifiedGenotyper with --output_mode EMIT_ALL_SITES). GVCFs produced by HaplotypeCaller 3.x contain additional information that is formatted in a very specific way. Read on to find out more.

### General comparison of VCF vs. gVCF

The key difference between a regular VCF and a gVCF is that the gVCF has records for all sites, whether there is a variant call there or not. The goal is to have every site represented in the file in order to do joint analysis of a cohort in subsequent steps. The records in a gVCF include an accurate estimation of how confident we are in the determination that the sites are homozygous-reference or not. This estimation is generated by the HaplotypeCaller's built-in reference model.

Note that some other tools (including the GATK's own UnifiedGenotyper) may output an all-sites VCF that looks superficially like the BP_RESOLUTION gVCFs produced by HaplotypeCaller, but they do not provide an accurate estimate of reference confidence, and therefore cannot be used in joint genotyping analyses.

### The two types of gVCFs

As you can see in the figure above, there are two options you can use with -ERC: GVCF and BP_RESOLUTION. With BP_RESOLUTION, you get a gVCF with an individual record at every site: either a variant record, or a non-variant record. With GVCF, you get a gVCF with individual variant records for variant sites, but the non-variant sites are grouped together into non-variant block records that represent intervals of sites for which the genotype quality (GQ) is within a certain range or band. The GQ ranges are defined in the ##GVCFBlock line of the gVCF header. The purpose of the blocks (also called banding) is to keep file size down, and there is no downside for the downstream analysis, so we do recommend using the -GVCF option.

### Example gVCF file

This is a banded gVCF produced by HaplotypeCaller with the -GVCF option.

As you can see in the first line, the basic file format is a valid version 4.1 VCF:

##fileformat=VCFv4.1
##ALT=<ID=NON_REF,Description="Represents any possible alternative allele at this location">
##FILTER=<ID=LowQual,Description="Low quality">
##FORMAT=<ID=AD,Number=.,Type=Integer,Description="Allelic depths for the ref and alt alleles in the order listed">
##FORMAT=<ID=GQ,Number=1,Type=Integer,Description="Genotype Quality">
##FORMAT=<ID=GT,Number=1,Type=String,Description="Genotype">
##FORMAT=<ID=MIN_DP,Number=1,Type=Integer,Description="Minimum DP observed within the GVCF block">
##FORMAT=<ID=PL,Number=G,Type=Integer,Description="Normalized, Phred-scaled likelihoods for genotypes as defined in the VCF specification">
##FORMAT=<ID=SB,Number=4,Type=Integer,Description="Per-sample component statistics which comprise the Fisher's Exact Test to detect strand bias.">
##GVCFBlock=minGQ=0(inclusive),maxGQ=5(exclusive)
##GVCFBlock=minGQ=20(inclusive),maxGQ=60(exclusive)
##GVCFBlock=minGQ=5(inclusive),maxGQ=20(exclusive)
##GVCFBlock=minGQ=60(inclusive),maxGQ=2147483647(exclusive)
##INFO=<ID=BaseQRankSum,Number=1,Type=Float,Description="Z-score from Wilcoxon rank sum test of Alt Vs. Ref base qualities">
##INFO=<ID=ClippingRankSum,Number=1,Type=Float,Description="Z-score From Wilcoxon rank sum test of Alt vs. Ref number of hard clipped bases">
##INFO=<ID=DS,Number=0,Type=Flag,Description="Were any of the samples downsampled?">
##INFO=<ID=END,Number=1,Type=Integer,Description="Stop position of the interval">
##INFO=<ID=HaplotypeScore,Number=1,Type=Float,Description="Consistency of the site with at most two segregating haplotypes">
##INFO=<ID=InbreedingCoeff,Number=1,Type=Float,Description="Inbreeding coefficient as estimated from the genotype likelihoods per-sample when compared against the Hardy-Weinberg expectation">
##INFO=<ID=MLEAC,Number=A,Type=Integer,Description="Maximum likelihood expectation (MLE) for the allele counts (not necessarily the same as the AC), for each ALT allele, in the same order as listed">
##INFO=<ID=MLEAF,Number=A,Type=Float,Description="Maximum likelihood expectation (MLE) for the allele frequency (not necessarily the same as the AF), for each ALT allele, in the same order as listed">
##INFO=<ID=MQ,Number=1,Type=Float,Description="RMS Mapping Quality">
##INFO=<ID=MQRankSum,Number=1,Type=Float,Description="Z-score From Wilcoxon rank sum test of Alt vs. Ref read mapping qualities">
##INFO=<ID=ReadPosRankSum,Number=1,Type=Float,Description="Z-score from Wilcoxon rank sum test of Alt vs. Ref read position bias">
##contig=<ID=20,length=63025520,assembly=b37>
##reference=file:///humgen/1kg/reference/human_g1k_v37.fasta


Toward the middle you see the ##GVCFBlock lines (after the ##FORMAT lines) (repeated here for clarity):

##GVCFBlock=minGQ=0(inclusive),maxGQ=5(exclusive)
##GVCFBlock=minGQ=20(inclusive),maxGQ=60(exclusive)
##GVCFBlock=minGQ=5(inclusive),maxGQ=20(exclusive)


which indicate the GQ ranges used for banding (corresponding to the boundaries [5, 20, 60]).

You can also see the definition of the MIN_DP annotation in the ##FORMAT lines.

#### Records

The first thing you'll notice, hopefully, is the <NON_REF> symbolic allele listed in every record's ALT field. This provides us with a way to represent the possibility of having a non-reference allele at this site, and to indicate our confidence either way.

The second thing to look for is the END tag in the INFO field of non-variant block records. This tells you at what position the block ends. For example, the first line is a non-variant block that starts at position 20:10000000 and ends at 20:10000116.

#CHROM  POS ID  REF ALT QUAL    FILTER  INFO    FORMAT  NA12878
20  10000000    .   T   <NON_REF>   .   .   END=10000116    GT:DP:GQ:MIN_DP:PL  0/0:44:99:38:0,89,1385
20  10000118    .   T   <NON_REF>   .   .   END=10000210    GT:DP:GQ:MIN_DP:PL  0/0:42:99:38:0,80,1314
20  10000212    .   A   <NON_REF>   .   .   END=10000438    GT:DP:GQ:MIN_DP:PL  0/0:52:99:42:0,99,1403
20  10000439    .   T   G,<NON_REF> 1737.77 .   DP=57;MLEAC=2,0;MLEAF=1.00,0.00;MQ=221.41;MQ0=0 GT:AD:DP:GQ:PL:SB   1/1:0,56,0:56:99:1771,168,0,1771,168,1771:0,0,0,0
20  10000440    .   T   <NON_REF>   .   .   END=10000597    GT:DP:GQ:MIN_DP:PL  0/0:56:99:49:0,120,1800
20  10000598    .   T   A,<NON_REF> 1754.77 .   DP=54;MLEAC=2,0;MLEAF=1.00,0.00;MQ=185.55;MQ0=0 GT:AD:DP:GQ:PL:SB   1/1:0,53,0:53:99:1788,158,0,1788,158,1788:0,0,0,0
20  10000599    .   T   <NON_REF>   .   .   END=10000693    GT:DP:GQ:MIN_DP:PL  0/0:51:99:47:0,120,1800
20  10000695    .   G   <NON_REF>   .   .   END=10000757    GT:DP:GQ:MIN_DP:PL  0/0:48:99:45:0,120,1800
20  10000758    .   T   A,<NON_REF> 1663.77 .   DP=51;MLEAC=2,0;MLEAF=1.00,0.00;MQ=59.32;MQ0=0  GT:AD:DP:GQ:PL:SB   1/1:0,50,0:50:99:1697,149,0,1697,149,1697:0,0,0,0
20  10000759    .   A   <NON_REF>   .   .   END=10001018    GT:DP:GQ:MIN_DP:PL  0/0:40:99:28:0,65,1080
20  10001020    .   C   <NON_REF>   .   .   END=10001020    GT:DP:GQ:MIN_DP:PL  0/0:26:72:26:0,72,1080
20  10001021    .   T   <NON_REF>   .   .   END=10001021    GT:DP:GQ:MIN_DP:PL  0/0:25:37:25:0,37,909
20  10001022    .   C   <NON_REF>   .   .   END=10001297    GT:DP:GQ:MIN_DP:PL  0/0:30:87:25:0,72,831
20  10001298    .   T   A,<NON_REF> 1404.77 .   DP=41;MLEAC=2,0;MLEAF=1.00,0.00;MQ=171.56;MQ0=0 GT:AD:DP:GQ:PL:SB   1/1:0,41,0:41:99:1438,123,0,1438,123,1438:0,0,0,0
20  10001299    .   C   <NON_REF>   .   .   END=10001386    GT:DP:GQ:MIN_DP:PL  0/0:43:99:39:0,95,1226
20  10001387    .   C   <NON_REF>   .   .   END=10001418    GT:DP:GQ:MIN_DP:PL  0/0:41:42:39:0,21,315
20  10001419    .   T   <NON_REF>   .   .   END=10001425    GT:DP:GQ:MIN_DP:PL  0/0:45:12:42:0,9,135
20  10001426    .   A   <NON_REF>   .   .   END=10001427    GT:DP:GQ:MIN_DP:PL  0/0:49:0:48:0,0,1282
20  10001428    .   T   <NON_REF>   .   .   END=10001428    GT:DP:GQ:MIN_DP:PL  0/0:49:21:49:0,21,315
20  10001429    .   G   <NON_REF>   .   .   END=10001429    GT:DP:GQ:MIN_DP:PL  0/0:47:18:47:0,18,270
20  10001430    .   G   <NON_REF>   .   .   END=10001431    GT:DP:GQ:MIN_DP:PL  0/0:45:0:44:0,0,1121
20  10001432    .   A   <NON_REF>   .   .   END=10001432    GT:DP:GQ:MIN_DP:PL  0/0:43:18:43:0,18,270
20  10001433    .   T   <NON_REF>   .   .   END=10001433    GT:DP:GQ:MIN_DP:PL  0/0:44:0:44:0,0,1201
20  10001434    .   G   <NON_REF>   .   .   END=10001434    GT:DP:GQ:MIN_DP:PL  0/0:44:18:44:0,18,270
20  10001435    .   A   <NON_REF>   .   .   END=10001435    GT:DP:GQ:MIN_DP:PL  0/0:44:0:44:0,0,1130
20  10001436    .   A   AAGGCT,<NON_REF>    1845.73 .   DP=43;MLEAC=2,0;MLEAF=1.00,0.00;MQ=220.07;MQ0=0 GT:AD:DP:GQ:PL:SB   1/1:0,42,0:42:99:1886,125,0,1888,126,1890:0,0,0,0
20  10001437    .   A   <NON_REF>   .   .   END=10001437    GT:DP:GQ:MIN_DP:PL  0/0:44:0:44:0,0,0


Note that toward the end of this snippet, you see multiple consecutive non-variant block records. These were not merged into a single record because the sites they contain belong to different ranges of GQ (which are defined in the header).

GATK 3.0 was released on March 5, 2014. Highlights are listed below. Read the detailed version history overview here: http://www.broadinstitute.org/gatk/guide/version-history

One important change for those who prefer to build from source is that we now use maven instead of ant. See the relevant documentation for building the GATK with our new build system.

• This is a new GATK tool to be used for variant calling in RNA-seq data. Its purpose is to split reads that contain N Cigar operators (due to a limitation in the GATK that we will eventually handle internally) and to trim (and generally clean up) imperfect alignments.

## Haplotype Caller

• Fixed bug where dangling tail merging in the assembly graph occasionally created a cycle.
• Added experimental code to retrieve dangling heads in the assembly graph, which is needed for calling variants in RNA-seq data.
• Generally improved gVCF output by making it more accurate. This includes many updates so that the single sample gVCFs can be accurately genotyped together by GenotypeGVCFs.
• Fixed a bug in the PairHMM class where the transition probability was miscalculated resulting in probabilities larger than 1.
• Fixed bug in the function to find the best paths from an alignment graph which was causing bad genotypes to be emitted when running with multiple samples together.

## CombineGVCFs

• This is a new GATK tool to be used in the Haplotype Caller pipeline with large cohorts. Its purpose is to combine any number of gVCF files into a single merged gVCF. One would use this tool for hierarchical merges of the data when there are too many samples in the project to throw at all at once to GenotypeGVCFs.

## GenotypeGVCFs

• This is a new GATK tool to be used in the Haplotype Caller pipeline. Its purpose is to take any number of gVCF files and to genotype them in order to produce a VCF with raw SNP and indel calls.

• This is a new GATK tool that might be useful to some. Given a VCF file, this tool will generate simulated reads that support the variants present in the file.

## Unified Genotyper

• Fixed bug when clipping long reads in the HMM; some reads were incorrectly getting clipped.

## Variant Recalibrator

• Added the capability to pass in a single file containing a list of VCFs (must end in ".list") instead of having to enumerate all of the files on the command-line. Duplicate entries are not allowed in the list (but the same file can be present in separate lists).

• Removed from the GATK. It was a valiant attempt, but ultimately we found a better way to process large cohorts. Reduced BAMs are no longer supported in the GATK.

## Variant Annotator

• Improved the FisherStrand (FS) calculation when used in large cohorts. When the table gets too large, we normalize it down to values that are more reasonable. Also, we don't include a particular sample's contribution unless we observe both ref and alt counts for it. We expect to improve on this even further in a future release.
• Improved the QualByDepth (QD) calculation when used in large cohorts. Now, when the AD annotation is present for a given genotype then we only use its depth for QD if the variant depth > 1. Note that this only works in the gVCF pipeline for now.
• In addition, fixed the normalization for indels in QD (which was over-penalizing larger events).

## Combine Variants

• Added the capability to pass in a single file containing a list of VCFs (must end in ".list") instead of having to enumerate all of the files on the command-line. Duplicate entries are not allowed in the list (but the same file can be present in separate lists).

## Select Variants

• Fixed a huge bug where selecting out a subset of samples while using multi-threading (-nt) caused genotype-level fields (e.g. AD) to get swapped among samples. This was a bad one.
• Fixed a bug where selecting out a subset of samples at multi-allelic sites occasionally caused the alternate alleles to be re-ordered but the AD values were not updated accordingly.

## CalculateGenotypePosteriors

• Fixed bug where it wasn't checking for underflow and occasionally produced bad likelihoods.
• It no longer strips out the AD annotation from genotypes.
• AC/AF/AN counts are updated after fixing genotypes.
• Updated to handle cases where the AC (and MLEAC) annotations are not good (e.g. they are greater than AN somehow).

## Indel Realigner

• Fixed bug where a realigned read can sometimes get partially aligned off the end of the contig.

• Updated the tool to use the VCF 4.1 framework for phasing; it now uses HP tags instead of '|' to convey phase information.

## Miscellaneous

• Thanks to Phillip Dexheimer for several Queue related fixes and patches.
• Thanks to Nicholas Clarke for patches to the timer which occasionally had negative elapsed times.
• Providing an empty BAM list no results in a user error.
• Fixed a bug in the gVCF writer where it was dropping the first few reference blocks at the beginnings of all but the first chromosome. Also, several unnecessary INFO field annotations were dropped from the output.
• Logger output now goes to STDERR instead of STDOUT.
• Picard, Tribble, and Variant jars updated to version 1.107.1683.

I have been trying to use CombinegVCFs on gVCF file produce by HaplotypeCaller in GVCF mode. The output VCF file doesn't seem to have any data in the genotype field: (just a dot)

chr1 95849 . T . . END=95850 GT:DP:GQ:MIN_DP:PL .:48:89:47:0,90 .:100:99:3:0,106 chr1 95851 . T . . END=95856 GT:DP:GQ:MIN_DP:PL .:48:44:47:0,45 .:100:99:3:0,106 chr1 95857 . GAA G,GA, . . DP=50;MQ=60.23;MQ0=0 GT:AD:DP:MIN_DP:PL:SB .:0,6,41,0:47:.:1150,935,0,1090:0,0,18,23 .:.:100:3:0,106,106,106 chr1 95858 . A . . END=95859 GT:DP:GQ:MIN_DP:PL . .:100:99:3:0,106 chr1 95860 . A . . END=96535 GT:DP:GQ:MIN_DP:PL .:44:99:27:0,1038 .:100:99:3:0,106 chr1 96536 . A G, . . DP=95;MQ=57.24;MQ0=0 GT:AD:DP:MIN_DP:PL:SB .:0,25,0:25:.:792,0,792:0,0,13,12 .:.:70:70:0,0,0 chr1 96537 . G . . END=96823 GT:DP:GQ:MIN_DP:PL .:37:99:23:0,380 .:68:99:51:0,374 chr1 96824 . C . . . GT:DP:GQ:MIN_DP:PL .:31:25:31:0,26 .:51:57:51:0,58

This is the command I used:

java -Xmx45g -Djava.io.tmpdir=/home/LANPARK/mboursnell/javatempdir -jar /opt/gatk/GenomeAnalysisTK.jar -R /home/genetics/strep_equi/strep_equi.fasta -T CombineGVCFs -V 17-1-2-5_NL_S12_L001_R1_001.gVCF -V 17-1-2-6_NL_S9_L001_R1_001.gVCF -o combined_1a.vcf -S STRICT

I used the following command to combine 3 VCF files which are outputs of HaplotypeCaller:

java -jar data/GenomeAnalysisTK-3.2-2/GenomeAnalysisTK.jar \ -R data/ucsc.hg19.fasta \ -T CombineGVCFs \ --variant data/47V_post.ERC.vcf \ --variant data/48V_post.ERC.vcf \ --variant data/49V_post.ERC.vcf \ --out data/Combined_3files.vcf

However, after combined all 3 files, in the output final VCF, I can only see ./. genotypes. What is the problem? how I can to fix this? Thanks

GATK team,

I currently have many WES gVCFs called with GATK 3.x HaplotypeCaller, and I'm now looking to combine them and run GenotypeGVCFs. Unfortunately, I forgot to add the "-L" argument to HC to reduce the size of the resulting gVCFs, and CombineGVCFs looks like it's taking much longer than I expect it to.

Is there any potential problem with using the "-L" argument to SelectVariants to reduce the size of my gVCFs and then use those smaller gVCFs in the CombineGVCFs stage (and beyond), or do I have to re-call HaplotypeCaller again? Would it be better to extend the boundaries of the target file by a certain amount to avoid recalling HaplotypeCaller?

Thanks,

John Wallace

Hi,

In the documentation for CombineGVCFs it says:

CombineGVCFs is meant to be used for hierarchical merging of gVCFs that will eventually be input into GenotypeGVCFs. One would use this tool when needing to genotype too large a number of individual gVCFs; instead of passing them all in to GenotypeGVCFs, one would first use CombineGVCFs on smaller batches of samples and then pass these combined gVCFs to GenotypeGVCFs.

Do you have any guidelines for this? I am trying to use genotypeGVCFS on 12 gVCF files and it doesn't work, so can you advise how I should I "pre-merge" them? Two batches of 6? Three batches of 3?

Thanks,

Mike

Currently I am following GATK best practice for using HC 3.0+, however I'm splitting my calls to chromosomal regions (-L). Next are the following step I perform working up to GenotypeGVCF and my question.

1 - I use CatVariants (following HC) to merge all 25 chromosome gvcf files into a single gvcf file per individual.
2 - I use CombineGVCF to merge 2 .. n number of individuals together. This is done because some analysis have 300+ individuals. 3- I then use CombineGVCF again to merge all the file from step 2 into one large gvcf file for one large joint GenotypeGVCF step. 4 - GenotypeGVCF is done again based on chromosomal regions (-L), which is followed by a additional CatVariants before VQSR.

The question I have this this: Given the size of the analysis I have noticed that my CombineGVCF done in step 3 can take anywhere from 4-8 hours. I was wondering if I could change this step to use CombineVariants and have the result be the same (unlost data). The main reason for this would be because GATK currently allow CombineVariants to use the -nt option.

Thanks for you time and work.

--Shawn

Hi GATK-ers,

I have been given ~2000 gVCFs generated by Illumina (one sample per gVCF). Though they are in standard gVCF format, they were generated by an Illumina pipeline (https://support.basespace.illumina.com/knowledgebase/articles/147078-gvcf-file if you're really curious) and not the Haplotype Caller. As a result (I think ... ), the GATK doesn't want to process them (I have tried CombineGVCFs and GenotypeGVCFs to no avail). Is there a GATK walker or some other tool that will make my gVCFs GATK-friendly? I need to be able to merge this data together to make it analyze-able because in single-sample VCF format it's pretty useless at the moment.

My only other thought has been to expand all the ref blocks of data and then merge everything together, but this seems like it will result in the creation of a massive amount of data.

Any suggestions you may have are greatly appreciated!!!

Sara

Hi,

I am using GATK v3.2.2 following the recommended practices (...HC -> CombineGVCFs -> GenotypeGVCFs ...) and while looking through suspicious variants I came across a few hetz with AD=X,0. Tracing them back I found two inconsistencies (bugs?);

1) Reordering of genotypes when combining gvcfs while the AD values are kept intact, which leads to an erronous AD for a heterozygous call. Also, I find it hard to understand why the 1bp insertion is emitted in the gvcf - there is no reads supporting it:

• single sample gvcf 1 26707944 . A AG,G,<NON_REF> 903.73 . [INFO] GT:AD:DP:GQ:PL:SB 0/2:66,0,36,0:102:99:1057,1039,4115,0,2052,1856,941,3051,1925,2847:51,15,27,9

• combined gvcf 1 26707944 . A G,AG,<NON_REF> . . [INFO] GT:AD:DP:MIN_DP:PL:SB [other_samples] ./.:66,0,36,0:102:.:1057,0,1856,1039,2052,4115,941,1925,3051,2847:51,15,27,9 [other_samples]

• vcf
1 26707944 . A G 3169.63 . [INFO] [other_samples] 0/1:66,0:102:99:1057,0,1856 [other_samples]

2) Incorrect AD is taken while genotyping gvcf files:

• single sample gvcf: 1 1247185 rs142783360 AG A,<NON_REF> 577.73 . [INFO] GT:AD:DP:GQ:PL:SB 0/1:13,20,0:33:99:615,0,361,654,421,1075:7,6,17,3
• combined gvcf 1 1247185 rs142783360 AG A,<NON_REF> . . [INFO] [other_samples] ./.:13,20,0:33:.:615,0,361,654,421,1075:7,6,17,3 [other_samples]

• vcf
1 1247185 . AG A 569.95 . [INFO] [other_samples] 0/1:13,0:33:99:615,0,361 [other_samples]

I have found multiple such cases here, and no errors nor warnings in the logs. I checked also with calls that I had done before on these samples, but in a smaller batch. There the AD values were correct, but there were plenty of other hetz with AD=X,0... I haven't looked closer into those.

Are these bugs that have been fixed in 3.3? Or maybe my brain is not working properly today and I miss sth obvious?

Best regards, Paweł

Hi,

I am combining gcvf files into single gvcf files by chromosome, using CombineGVCFs, in order to run GenotypeGVCFs. When I checked the first gvcf file generated by CombineGVCFs, I noticed that at each position, all the alleles were missing.

For example, at position 16050036, this is what comes up in the final gvcf file: 22 16050036 . A C,<NON_REF> . . BaseQRankSum=-7.360e-01;ClippingRankSum=-7.360e-01;DP=4;MQ=27.00;MQ0=0;MQRankSum=-7.360e-01;ReadPosRankSum=0.736 GT:AD:DP:MIN_DP:PL:SB ./.:1,2,0:3:.:55,0,23,58,29,86:1,0,2,0 ./.:.:1:1:0,0,0,0,0,0 ./.:.:0:0:0,0,0,0,0,0

But if we just take one of the precursor gvcf files (one individual), we clearly see the genotype at that site: 22 16050036 . A C,<NON_REF> 26.80 . BaseQRankSum=-0.736;ClippingRankSum=-0.736;DP=3;MLEAC=1,0;MLEAF=0.500,0.00;MQ=27.00;MQ0=0;MQRankSum=-0.736;ReadPosRankSum=0.736 GT:AD:DP:GQ:PL:SB 0/1:1,2,0:3:23:55,0,23,58,29,86:1,0,2,0

The command I'm using to generate these files is:

java -Xmx1g -jar GenomeAnalysisTK.jar -T CombineGVCFs -R hs37d5.fa -V vcfs.chr${numchr}.new.list -o mergeGvcf_${numchr}.vcf -L {numchr} where numchr is a variable previously defined (indicating the chromosome number). It seems that all the information is being taken into account except the actual genotypes. How do I solve this problem? Thanks, Alva Hi GATK team, i would like to seek opinion from your team to find the best workflow that best fit my data. Previously i've been exploring both variant calling algorithms UnifiedGenotyper and HaplotypeCaller, and i would love to go for UnifiedGenotyper considering of the sensitivity and the analysis runtime. Due to my experimental cohort samples grows from time to time, so i've opt for single sample calling follow by joint-analysis using combineVariants instead of doing multiple-samples variant calling. However by doing so, i've experience few drawbacks from it (this issue was discussed at few forums). For a particular SNP loci, we wouldn't know whether the "./." reported for some of the samples are due to no reads covering that particular loci, or it doesn't pass certain criteria during variant calling performed previously, or it is a homo-reference base (which i concern this most and can't cope to lost this information). Then, i found this "gvcf", and it is potentially to solve my problem (Thanks GATK team for always understand our researcher's need)!! Again, i'm insist of opt for unifiedGenotyper instead of haplotypeCaller to generate the gvcf, and reading from the forum at https://www.broadinstitute.org/gatk/guide/tagged?tag=gvcf, i would assume that as VCFs produced by "UnifiedGenotyper with --output_mode EMIT_ALL_SITES" to be something alike with the gvcf file produced by HaplotyperCaller. However i couldn't joint them up using either "CombineVariants" or "CombineGVCFs", most probably i think "UnifiedGenotyper with --output_mode EMIT_ALL_SITES" doesn't generate gvcf format. Can you please give me some advice to BEST fit my need and with minimum runtime (UnifiedGenotyper would be my best choice), is there any method to joint the ALL_SITES vcf file produced by UnifiedGenotyper which i might probably missed out from the GATK page? Hi, CombineGVCFs is failing to combine these three files if we provide an interval file. a.vcf: #CHROM POS ID REF ALT QUAL FILTER INFO FORMAT A 1 2337033 . G <NON_REF> . . END=2337368 GT:DP:GQ:MIN_DP:PL 0/0:216:99:42:0,99,1485 b.vcf: #CHROM POS ID REF ALT QUAL FILTER INFO FORMAT B 1 2337033 . G <NON_REF> . . END=2337297 GT:DP:GQ:MIN_DP:PL 0/0:114:99:48:0,120,1800 c.vcf: #CHROM POS ID REF ALT QUAL FILTER INFO FORMAT C 1 2337033 . G <NON_REF> . . END=2337336 GT:DP:GQ:MIN_DP:PL 0/0:134:99:35:0,105,950 intervals.bed #Chrom Start End Gene 0 Strand 1 2337194 2337283 PEX10 0 - If I run CombineGVCFs without -L I get what you would expect: java -Xmx1G -jar GenomeAnalysisTK-3.3-0/GenomeAnalysisTK.jar -T CombineGVCFs -R human_g1k_v37.fasta -V a.vcf -V b.vcf -V c.vcf -o combined.vcf #CHROM POS ID REF ALT QUAL FILTER INFO FORMAT A B C 1 2337033 . G <NON_REF> . . END=2337297 GT:DP:GQ:MIN_DP:PL ./.:216:99:42:0,99,1485 ./.:114:99:48:0,120,1800 ./.:134:99:35:0,105,950 1 2337298 . C <NON_REF> . . END=2337336 GT:DP:GQ:MIN_DP:PL ./.:216:99:42:0,99,1485 ./. ./.:134:99:35:0,105,950 1 2337337 . C <NON_REF> . . END=2337368 GT:DP:GQ:MIN_DP:PL ./.:216:99:42:0,99,1485 ./. ./. However if I pass -L, I get no combined data at all: java -Xmx1G -jar GenomeAnalysisTK-3.3-0/GenomeAnalysisTK.jar -T CombineGVCFs -R human_g1k_v37.fasta -V a.vcf -V b.vcf -V c.vcf -o combined.vcf -L intervals.bed #CHROM POS ID REF ALT QUAL FILTER INFO FORMAT A B C Notice that this interval file specifies an interval right in the middle of the GVCF band all three files have. However, CombineGVCFs is completely ignoring the data in those bands. Best regards, Carlos Hi all, I am attempting to use the HaplotyperCaller / CombineGVCFs / GenotypeGVCFs to call variants on chrom X and Y of 769 samples (356 males, 413 females) sequenced at 12x coverage (WG sequening, but right not only calling X and Y). I have called the samples according to the best practises using the HaplotypeCaller, using ploidy = 1 for males on X and Y and ploidy =2 for females on X, e.g.: INFO 16:28:45,750 HelpFormatter - Program Args: -R /gcc/resources/b37/indices/human_g1k_v37.fa -T HaplotypeCaller -L X -ploidy 1 -minPruning 3 --emitRefConfidence GVCF --variant_index_type LINEAR --variant_index_parameter 128000 -I /target/gpfs2/gcc/groups/gonl/projects/trio-analysis/rawdata_release2/A102.human_g1k_v37.trio_realigned.bam --sample_name A102a -o /gcc/groups/gonl/tmp01/lfrancioli/chromX/hc/results/A102a.chrX.hc.g.vcf Then I have used CombineGVCFs to combine my samples in batches of 100 samples. Now I am attempting to genotype them and I face the same issue on both X (males + females) and Y (males only): It starts running fine and then just hang on a certain position. At first it crashed asking for additional memory but now with 24Gb or memory it simply stays at a single position for 24hrs until my job eventually stops due to walltime. Here is the chrom X output: INFO 15:00:39,501 HelpFormatter - Program Args: -R /gcc/resources/b37/indices/human_g1k_v37.fa -T GenotypeGVCFs -ploidy 1 --dbsnp /gcc/resources/b37/snp/dbSNP/dbsnp_138.b37.vcf -stand_call_conf 10 -stand_emit_conf 10 --max_alternate_alleles 4 -o /gcc/groups/gonl/tmp01/lfrancioli/chromX/hc/results/gonl.chrX.hc.vcf -L X -V /gcc/groups/gonl/tmp01/lfrancioli/chromX/hc/results/gonl.chrX.hc.1.g.vcf -V /gcc/groups/gonl/tmp01/lfrancioli/chromX/hc/results/gonl.chrX.hc.2.g.vcf -V /gcc/groups/gonl/tmp01/lfrancioli/chromX/hc/results/gonl.chrX.hc.3.g.vcf -V /gcc/groups/gonl/tmp01/lfrancioli/chromX/hc/results/gonl.chrX.hc.4.g.vcf -V /gcc/groups/gonl/tmp01/lfrancioli/chromX/hc/results/gonl.chrX.hc.5.g.vcf -V /gcc/groups/gonl/tmp01/lfrancioli/chromX/hc/results/gonl.chrX.hc.6.g.vcf -V /gcc/groups/gonl/tmp01/lfrancioli/chromX/hc/results/gonl.chrX.hc.7.g.vcf -V /gcc/groups/gonl/tmp01/lfrancioli/chromX/hc/results/gonl.chrX.hc.8.g.vcf INFO 15:00:39,507 HelpFormatter - Executing as lfrancioli@targetgcc15-mgmt on Linux 3.0.80-0.5-default amd64; Java HotSpot(TM) 64-Bit Server VM 1.7.0_51-b13. INFO 15:00:39,507 HelpFormatter - Date/Time: 2014/11/12 15:00:39 INFO 15:00:39,508 HelpFormatter - -------------------------------------------------------------------------------- INFO 15:00:39,508 HelpFormatter - -------------------------------------------------------------------------------- INFO 15:00:40,951 GenomeAnalysisEngine - Strictness is SILENT INFO 15:00:41,153 GenomeAnalysisEngine - Downsampling Settings: Method: BY_SAMPLE, Target Coverage: 1000 INFO 15:57:53,416 RMDTrackBuilder - Writing Tribble index to disk for file /gcc/groups/gonl/tmp01/lfrancioli/chromX/hc/results/gonl.chrX.hc.4.g.vcf.idx INFO 17:09:39,597 RMDTrackBuilder - Writing Tribble index to disk for file /gcc/groups/gonl/tmp01/lfrancioli/chromX/hc/results/gonl.chrX.hc.5.g.vcf.idx INFO 18:21:00,656 RMDTrackBuilder - Writing Tribble index to disk for file /gcc/groups/gonl/tmp01/lfrancioli/chromX/hc/results/gonl.chrX.hc.6.g.vcf.idx INFO 19:30:46,624 RMDTrackBuilder - Writing Tribble index to disk for file /gcc/groups/gonl/tmp01/lfrancioli/chromX/hc/results/gonl.chrX.hc.7.g.vcf.idx INFO 20:22:38,368 RMDTrackBuilder - Writing Tribble index to disk for file /gcc/groups/gonl/tmp01/lfrancioli/chromX/hc/results/gonl.chrX.hc.8.g.vcf.idx WARN 20:26:45,716 FSLockWithSharedLockAcquisitionTask - WARNING: Unable to lock file /gcc/resources/b37/snp/dbSNP/dbsnp_138.b37.vcf.idx because an IOException occurred with message: No locks available. INFO 20:26:45,718 RMDTrackBuilder - Could not acquire a shared lock on index file /gcc/resources/b37/snp/dbSNP/dbsnp_138.b37.vcf.idx, falling back to using an in-memory index for this GATK run. INFO 20:33:29,491 IntervalUtils - Processing 155270560 bp from intervals INFO 20:33:29,628 GenomeAnalysisEngine - Preparing for traversal INFO 20:33:29,635 GenomeAnalysisEngine - Done preparing for traversal INFO 20:33:29,636 ProgressMeter - [INITIALIZATION COMPLETE; STARTING PROCESSING] INFO 20:33:29,637 ProgressMeter - | processed | time | per 1M | | total | remaining INFO 20:33:29,638 ProgressMeter - Location | sites | elapsed | sites | completed | runtime | runtime INFO 20:33:29,948 GenotypeGVCFs - Notice that the -ploidy parameter is ignored in GenotypeGVCFs tool as this is automatically determined by the input variant files INFO 20:33:59,642 ProgressMeter - X:65301 0.0 30.0 s 49.6 w 0.0% 19.8 h 19.8 h INFO 20:34:59,820 ProgressMeter - X:65301 0.0 90.0 s 149.1 w 0.0% 59.4 h 59.4 h ... INFO 20:52:01,064 ProgressMeter - X:177301 0.0 18.5 m 1837.7 w 0.1% 11.3 d 11.2 d INFO 20:53:01,066 ProgressMeter - X:177301 0.0 19.5 m 1936.9 w 0.1% 11.9 d 11.9 d ... INFO 14:58:25,243 ProgressMeter - X:177301 0.0 18.4 h 15250.3 w 0.1% 96.0 w 95.9 w INFO 14:59:38,112 ProgressMeter - X:177301 0.0 18.4 h 15250.3 w 0.1% 96.1 w 96.0 w INFO 15:00:47,482 ProgressMeter - X:177301 0.0 18.5 h 15250.3 w 0.1% 96.2 w 96.1 w =>> PBS: job killed: walltime 86440 exceeded limit 86400

I would really appreciate if you could give me some pointer as how to handle this situation.

Thanks! Laurent

Dear all,

I am dealing with something that looks like a bug in the combineGVCF routine, or at least something I do not understand. I am looking at a variant that I know exists (Sanger sequencing validated). The individual gVCF looks clean:

6 30888174 . G C, 897.77 . GT:AD:DP:GQ:PL:SB 0/1:39,33,0:72:99:926,0,1131,1044,1230,2274:13,26,13,20

which I think means 39 reads for a G and 33 reads for C. So a convincing heterozygous call, as evidenced by the probabilities that follow. After combining with other gVCF files, I get 6 30888174 . G C 870.96 0/1:39,0:72:99:926,0,1131

So my 33 reads supporting a "C" have disappeared. While the call remains 0/1, this creates various QC issues and that call ends up being filtered out. I was wondering if there was any logic behind this, or if this could be a bug of some sort?

Any comments would be very welcome. This is based on a recent version of GATK (nightly, as of 1-2 weeks ago I think).

I try to combine 56 low coverage exomes gvcf produced by Haplotypecaller GVCF mode. It take 5+ days if I perform the combination of 56gvcf all in one go. I try CombineGVCFs on per chr basis using -L with command as

java $JAVAmem -jar$GATKPATH/GenomeAnalysisTK.jar -T CombineGVCFs \ -R "$REFfasta" \ -L 1 \ --variant$IN/001.AllChr.raw.snps.indels.gvcf \ to --variant $IN/056.AllChr.raw.snps.indels.gvcf \ -o$OUT/mergeChr1.raw.snps.indels.gvcf

After getting the combined files per chromosome, am I right that I should use CatVariants to combine them to a mega file rather than using CombineGVCFs again ?

Thanks

Kin

## Hi this is pretty much a feature request for something I think would be useful, I mentioned it briefly at the Brussels workshop and it seems like it might be possible.

In a couple of projects I'm involved in we have done low coverage (2-10x whole genome) exploratory sequencing for a large number of individuals (similar to 1K genomes, around 1,200 individuals between the two projects) and have recently processed these individuals using the new N+1 pipeline, generating gVCFs.

Now going forward we are adding additional sequence for a decent number of these individuals (from the same PCR free library) to improve genome coverage and the accuracy of genotypes (target 30x) in individuals and Trios of interest. We thus want to combine the new sequence (20x) with the older sequence (6-10x) to get as much coverage as possible. To do this I understand that currently I would need to rerun the GATK HaplotypeCaller on both the old and new BAMs at once, generating a new gVCF then track down the individuals in our previous combined gVCFs and remove them so I can Genotype the old gVCF minus the low coverage samples + the new gVCFs. Following that process I have to reprocess the old data multiple times and subset old combined gVCF files if new data comes in which is rather painful and computationally wasteful.

Ideally it would instead be possible to run GATK HaplotypeCaller just on the new sequence generating a second new gVCF that only has data for the new 20x coverage, then combine it somehow with the old gVCF merging the data from both the old and new gVCFs and resulting in a single final VCF record for this sample which has utilised the data from both the old and new gVCFs. I guess this could either be run as a separate tool to merge/combine old and new gVCFs or be done automatically by the GenotypeGVCFs tool.

This would also be useful from a work flow point of view, as we have limited computational resources and storage it's preferable that we process data as soon as it comes off the sequencer through to the gVCF stage to save space and allow us to archive the BAM files while keeping the gVCFs for when we run GenotypeGVCF on all the current data. At the moment I have to keep the BAMs for an individual in working space until I'm sure I've got all the sequence for that individual (and as mentioned above that can change in the future) then generate the gVCFs. Being able to flow sequence data through the cluster to gVCF stage as soon as it becomes available and then later merge the gVCFs when additional lanes are completed would make things a lot simpler from a resource management and pipeline design.

If this is possible it would be greatly appreciated if it could be implemented. Thanks!

Hi,
when using scatter with CombineGVCFs walkers I get the following error: "You have asked for an interval that cuts in the middle of one or more gVCF blocks. Please note that this will cause you to lose records that don't end within your interval"
CombineGVCFs need to see all the data at once? Or at least all the chromosome at once? If it need it, I think the automatic scatter-gather for this class in scala script is working by 'LOCI' instead of 'CONTIG'.
Thanks,
Ester

Hello all,

I have a quick question about the results of an CombineGVCFs file while creating large background files. Prior to combining the files a region of the .gvcf file from HC looks like this.

#CHROM POS ID REF ALT QUAL FILTER INFO FORMAT SRR070473 13 19408518 . A <NON_REF> . . END=19409266 GT:DP:GQ:MIN_DP:PL 0/0:0:0:0:0,0,0 13 19409267 . T <NON_REF> . . END=19409323 GT:DP:GQ:MIN_DP:PL 0/0:4:12:2:0,6,60 13 19409324 . C <NON_REF> . . END=19409400 GT:DP:GQ:MIN_DP:PL 0/0:15:42:8:0,24,299

If you noticed the first individual looks like this SRR070473, with a 0/0:0:0:0:0,0,0 recorded, but after combining the file in batches of 200, the same information will be recorded as no call.

#CHROM POS ID REF ALT QUAL FILTER INFO FORMAT SRR070473 SRR070477 SRR070505 SRR070516 SRR070517 SRR070772 SRR070779 SRR070796 SRR0 13 19408518 . A <NON_REF> . . END=19408519 GT:DP:GQ:MIN_DP:PL ./. ./. ./. ./. ./. ./. ./. ./. ./. ./. ./. ./.

The issue I'm seeing is when you attempt to use GenotypeGVCFs I get the following an error similar to this if using the file containing no call notation.

##### ERROR MESSAGE: cannot merge genotypes from samples without PLs; sample ERR031932 does not have likelihoods at position 1:10929

I have run HaplotypeCaller (Version=3.1-1-g07a4bf8) on 99 WGS samples and created my GVCFs. I did this in 60+ intervals to enable faster turn around time. I then merged the GVCFs with CombineGVCFs (Version=3.1-1-g07a4bf8) However, when I try to call variants with java -jar $GATK -T GenotypeGVCFs -V WGS.gvcf.gz -o VCF/WGS.vcf.gz -R$GENOME -nt 8 it errors out complaining about PL values. The stack trace is here:

##### ERROR stack trace

java.lang.IllegalStateException: The original PLs do not have enough values; accessing index 10011 but size is 10000 at org.broadinstitute.sting.utils.variant.GATKVariantContextUtils.generatePLs(GATKVariantContextUtils.java:1593) at org.broadinstitute.sting.utils.variant.GATKVariantContextUtils.mergeRefConfidenceGenotypes(GATKVariantContextUtils.java:1530) at org.broadinstitute.sting.utils.variant.GATKVariantContextUtils.referenceConfidenceMerge(GATKVariantContextUtils.java:1095) at org.broadinstitute.sting.gatk.walkers.variantutils.GenotypeGVCFs.map(GenotypeGVCFs.java:183) at org.broadinstitute.sting.gatk.walkers.variantutils.GenotypeGVCFs.map(GenotypeGVCFs.java:110) at org.broadinstitute.sting.gatk.traversals.TraverseLociNano$TraverseLociMap.apply(TraverseLociNano.java:267) at org.broadinstitute.sting.gatk.traversals.TraverseLociNano$TraverseLociMap.apply(TraverseLociNano.java:255) at org.broadinstitute.sting.utils.nanoScheduler.NanoScheduler.executeSingleThreaded(NanoScheduler.java:274) at org.broadinstitute.sting.utils.nanoScheduler.NanoScheduler.execute(NanoScheduler.java:245) at org.broadinstitute.sting.gatk.traversals.TraverseLociNano.traverse(TraverseLociNano.java:144) at org.broadinstitute.sting.gatk.traversals.TraverseLociNano.traverse(TraverseLociNano.java:92) at org.broadinstitute.sting.gatk.traversals.TraverseLociNano.traverse(TraverseLociNano.java:48) at org.broadinstitute.sting.gatk.executive.ShardTraverser.call(ShardTraverser.java:98) at java.util.concurrent.FutureTask$Sync.innerRun(FutureTask.java:334) at java.util.concurrent.FutureTask.run(FutureTask.java:166) at java.util.concurrent.ThreadPoolExecutor.runWorker(ThreadPoolExecutor.java:1110) at java.util.concurrent.ThreadPoolExecutor$Worker.run(ThreadPoolExecutor.java:603) at java.lang.Thread.run(Thread.java:722)

##### ERROR ------------------------------------------------------------------------------------------

In the short term, is there a hidden setting I can use to skip over these, rather than completely throwing up?

Dear all,

I am using combinegVCF to put together some gVCF files and I am seeing a massive speed difference with or without the -L option to specify a target region. It goes in the wrong direction, i.e. using the -L option to limit the target massively INCREASES the computation time. See:

INFO 11:22:44,538 GenomeAnalysisEngine - Done preparing for traversal

INFO 11:22:44,538 ProgressMeter - [INITIALIZATION COMPLETE; STARTING PROCESSING]

INFO 11:22:44,539 ProgressMeter - Location processed.sites runtime per.1M.sites completed total.runtime remaining

INFO 11:23:14,550 ProgressMeter - 1:1217334 1.24e+05 30.0 s 4.0 m 0.1% 8.4 h 8.4 h

INFO 11:23:44,554 ProgressMeter - 1:1290472 1.60e+05 60.0 s 6.3 m 0.1% 12.8 h 12.8 h

INFO 11:24:44,564 ProgressMeter - 1:1412532 1.93e+05 120.0 s 10.4 m 0.2% 21.6 h 21.6 h

INFO 11:25:44,571 ProgressMeter - 1:1469759 2.06e+05 3.0 m 14.6 m 0.2% 30.3 h 30.2 h

and without the -L flag: INFO 11:18:32,796 ProgressMeter - [INITIALIZATION COMPLETE; STARTING PROCESSING]

INFO 11:18:32,796 ProgressMeter - Location processed.sites runtime per.1M.sites completed total.runtime remaining

INFO 11:19:02,802 ProgressMeter - 1:1823201 1.00e+06 30.0 s 30.0 s 0.1% 14.2 h 14.2 h

INFO 11:20:02,808 ProgressMeter - 1:5999901 5.00e+06 90.0 s 18.0 s 0.2% 12.9 h 12.9 h

INFO 11:21:02,817 ProgressMeter - 1:10308601 1.00e+07 2.5 m 15.0 s 0.3% 12.5 h 12.5 h

INFO 11:22:02,824 ProgressMeter - 1:14672301 1.40e+07 3.5 m 15.0 s 0.5% 12.3 h 12.3 h

I appreciate these are just the beginning of the runs but it appears to be stable and the -L option makes a real mess of things. I am using the latest nightly build right now, and this is maybe part of the pbm? But something does not see to make sense here.

My aim is to compute"genome-wide" gVCFs and then, when I combine into combinedgVCFs, restrict to exome target. So this is probably a step I cannot avoid.

Any thoughts on this?

Hi there:

I'm trying to figure out if I need to run CombineGVCFs before GenotypeGVCFs. The documentation said "One would use this tool when needing to genotype too large a number of individual gVCFs". Is there a ballpark number for "too large a number of individuals"? For example, if I have 1000 individuals, should I use CombineGVCFs first? If so, is there a recommended chuck size? It seems CombineGVCFs does not take nt/nct option and when I tried to combine 600 files it's estimated to take a week.

Thanks.

I've run into the following bug while running GenotypeGVCFs:

##### ERROR MESSAGE: cannot merge genotypes from samples without PLs; sample <ID redacted> does not have likelihoods at position 1:1115551

The input file in question is a gVCF produced by merging a large number of smaller gVCFs using CombineGVCFs (all tasks were run using version 3.1). What's happening is that the position 1115551 doesn't exist in that particular sample:

#CHROM  POS     ID      REF     ALT     QUAL    FILTER  INFO    FORMAT  <Sample_ID>
1       1115552 .       C       <NON_REF>       .       .       END=1115552     GT:DP:GQ:MIN_DP:PL      0/0:15:0:15:0,0,31


But when the sample is combined with other samples, that position gets filled in with a simple "0/0", without any PLs (or any of the other fields, including AD, DP, GQ, etc.), which causes the GenotypeGVCFs to choke.

I can imagine there might be other scenarios that will result in a "0/0" genotype field, so perhaps the easiest way to fix this would be to make sure that any "0/0" actually gets output as "./.:.:.:.:.".

Thanks,

Grace

I've got 300 gvcfs as a results of a Queue pipeline, that I want to combine. When I run CombineGVCFs (GATK v3.1-1) this however seems fairly slow:

INFO  15:24:22,100 ProgressMeter -        Location processed.sites  runtime per.1M.sites completed total.runtime remaining
INFO  15:57:52,778 ProgressMeter -      1:11456201        1.10e+07   33.5 m        3.0 m      0.4%         6.4 d     6.3 d
INFO  15:58:52,780 ProgressMeter -      1:11805001        1.10e+07   34.5 m        3.1 m      0.4%         6.4 d     6.3 d
INFO  15:59:52,781 ProgressMeter -      1:12140201        1.20e+07   35.5 m        3.0 m      0.4%         6.4 d     6.3 d


Is there a way of improving the performance of this merge? 6 days seems like a lot, but of course not unfeasible. Likewise, what kind of performance could I expect in the GenotypeGVCFs step?

ERROR MESSAGE: cannot merge genotypes from samples without PLs; sample Y257873-1B does not have likelihoods at position Y:3640996

Similar error happens in other samples at other chrs as well.

The gvcf files were produced by HC.

GATK Team,

First of all, I'm very excited about v3.x GATK, you guys and gals on the GATK Team are doing awesome work!

I have a question: How do I get CombineGVCFs to forward the sample-level annotations calculated by HC with the --emitRefConfidence GVCF option enabled? I tried passing the same annotation flags to CombineGVCFs that I passed to HC, but none of the annotations beyond the standard set were incorporated. Annotations such as StrandBiasBySample require access to the underlying reads and forwarding these annotations to the CombineGVCFs output would be less prohibitive than running VariantAnnotator on my 20 WGS datasets...

it happens with CombineGVCFs, I didn't check other functions.

Running in the current dir /site/ne/data/ngs/var, the error will be

ERROR MESSAGE: Couldn't read file /site/ne/data/ngs/var because file does not exist

It took me a while to figure out it's due to the empty line.