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Why are there <NON_REF>s in the VCF output of genotypeGVCFs?

bifengdiebifengdie Cambridge, MA, USAMember
I have 13 samples of re-sequenced whole genomes of several insect species, they were aligned to a reference genome of a closely related species using BWA -mem. When using the pipeline of "HaplotypeCaller => genomicsDBImport => genotypeGVCFs" in GATK (Invariant site included), I ended up with VCFs that didn't pass ValidateVariants (with error messages shown in WARN), however, when invariant sites were not chosen when running genotypeGVCFs, ValidateVariants gave no error message.

The error message part reads like:

21:42:18.766 WARN ValidateVariants - ***** Input 03_vcfs_sg/Joint.final.InvariantIncluded.HiC_scaffold_9.sorted.vcf fails strict validation: one or more of the ALT allele(s) for the record at position HiC_scaffold_9:9755 are not observed at all in the sample genotypes of type: *****
21:42:18.767 WARN ValidateVariants - ***** Input 03_vcfs_sg/Joint.final.InvariantIncluded.HiC_scaffold_9.sorted.vcf fails strict validation: one or more of the ALT allele(s) for the record at position HiC_scaffold_9:9775 are not observed at all in the sample genotypes of type: *****
21:42:21.138 WARN ValidateVariants - ***** Input 03_vcfs_sg/Joint.final.InvariantIncluded.HiC_scaffold_9.sorted.vcf fails strict validation: one or more of the ALT allele(s) for the record at position HiC_scaffold_9:67171 are not observed at all in the sample genotypes of type: *****

When I went back to check the VCF file, these lines looked like:

HiC_scaffold_9 9755 . T A,<NON_REF> . . . GT:AD:DP:RGQ ./.:0,0,0:
0:0 ./.:0,0,0:0:0 ./.:0,0,0:0:0 ./.:0,0,0:0:0 ./.:0,0,0:0:0 ./.:0,0,0:0:0 ./.:0,0,0:0:0 ./.:
0,0,0:0:0 ./.:0,0,0:0:0 ./.:0,0,0:0:0 ./.:0,0,0:0:0 ./.:0,0,0:0:0 ./.:0,0,0

HiC_scaffold_9 9775 . A G,<NON_REF> . . . GT:AD:DP:RGQ ./.:0,0,0:0:0 ./.:0,0,0:0:0 ./.:0,0,0:0:0 ./.:0,0,0:0:0 ./.:0,0,0:0:0 ./.:0,0,0:0:0 ./.:0,0,0:0:0 ./.:0,0,0:0:0 ./.:0,0,0:0:0 ./.:0,0,0:0:0 ./.:0,0,0:0:0 ./.:0,0,0:0:0 ./.:0,0,0

HiC_scaffold_9 67171 . A T,<NON_REF> . . ExcessHet=3.01 GT:AD:DP:PGT:PID:RGQ ./.:0,0,0:0:.:.:0 ./.:0,0,0:0:.:.:0 ./.:0,0,0:0:.:.:0 ./.:0,0,0:0:.:.:0 ./.:0,0,0:0:.:.:0 ./.:0,0,0:0:.:.:0 ./.:0,0,0:0:.:.:0 ./.:0,0,0:0:.:.:0 ./.:0,0,0:0:.:.:0 ./.:0,0,0:0:.:.:0 ./.:0,0,0:0:.:.:0 ./.:0,0,0:0:.:.:0 ./.:0,0,0:0:0|1:67160_C_G:3

And a lot more similar lines gave out tons of similar error messages. These positions are not found in the VCF files when invariant sites are not included during joint-genotyping.

To trace the origin of these variant calls, I went back to the original GVCF files. For examples, for the variant "HiC_scaffold_9 9755", all the first 12 samples have no explicit information on this site (which means it is considered the same as the reference?) The only difference is from the last sample, which has:

HiC_scaffold_9 9755 . T A,<NON_REF> 0.01 . MLEAC=0,0;MLEAF=NaN,NaN GT ./.

and

HiC_scaffold_9 9775 . A G,<NON_REF> 0.01 . MLEAC=0,0;MLEAF=NaN,NaN GT ./.

etc.

It seems these individual genotypes determined by haplotypecaller are badly supported. Why does this information still got passed to the final VCF output after joint-genotyping?

I am using GATK 4.1.2.0, and the commands are as follows:

gatk HaplotypeCaller \
--java-options "-Xmx4g -XX:ParallelGCThreads=1" \
-R $RefGenome \
-I ${sp}.dedup.sorted.bam \
-O ${sp}.raw.${Interval}.g.vcf \
--emit-ref-confidence GVCF \
-L ${Interval} \

gatk GenomicsDBImport \
--java-options "-Xmx4g -XX:ParallelGCThreads=1" \
--genomicsdb-workspace-path ${PATH_TO_GENOMICDB} \
-L ${Intervals} \
--sample-name-map CohortSample_map/${IntervalName}.list \
--reader-threads 1 \

gatk GenotypeGVCFs \
--java-options "-Xmx4g -XX:ParallelGCThreads=1" \
-R $RefGenome \
-V gendb://${PATH_TO_GENOMICDB}
-O Joint.final.${Interval}.vcf \
--tmp-dir=${DIR}/tmp \
--heterozygosity=0.022 \
--include-non-variant-sites=true

How can I resolve this error message? Or is it something that should be filtered out in the first place? Thank you

Answers

  • bhanuGandhambhanuGandham Cambridge MAMember, Administrator, Broadie, Moderator admin

    Hi @bifengdie

    Could you please post the error log.

  • bifengdiebifengdie Cambridge, MA, USAMember
    Sure. Somehow I couldn't figure out by now how to use the "code" format here. Sorry for the messy presentation:

    ```
    Using GATK jar /n/holylfs/LABS/mallet_lab/txiong/Software/gatk-4.1.2.0/gatk-package-4.1.2.0-local.jar
    Running:
    java -Dsamjdk.use_async_io_read_samtools=false -Dsamjdk.use_async_io_write_samtools=true -Dsamjdk.use_async_io_write_tribble=false -Dsamjdk.compression_level=2 -jar /n/holylfs/LABS/mallet_lab/txiong/Software/gatk-4.1.2.0/gatk-package-4.1.2.0-local.jar ValidateVariants -R 00_genome_Pbianor/Pb.fa -V 03_vcfs_sg/Joint.final.InvariantIncluded.HiC_scaffold_9.sorted.vcf --validation-type-to-exclude IDS --warn-on-errors true
    21:42:08.099 INFO NativeLibraryLoader - Loading libgkl_compression.so from jar:file:/n/holylfs/LABS/mallet_lab/txiong/Software/gatk-4.1.2.0/gatk-package-4.1.2.0-local.jar!/com/intel/gkl/native/libgkl_compression.so
    Aug 01, 2019 9:42:10 PM shaded.cloud_nio.com.google.auth.oauth2.ComputeEngineCredentials runningOnComputeEngine
    INFO: Failed to detect whether we are running on Google Compute Engine.
    21:42:10.846 INFO ValidateVariants - ------------------------------------------------------------
    21:42:10.847 INFO ValidateVariants - The Genome Analysis Toolkit (GATK) v4.1.2.0
    21:42:10.848 INFO ValidateVariants - For support and documentation go to [LINK REMOVED BY MYSELF IN THE FORUM]
    21:42:10.859 INFO ValidateVariants - Executing as [email protected] on Linux v3.10.0-957.12.1.el7.x86_64 amd64
    21:42:10.860 INFO ValidateVariants - Java runtime: Java HotSpot(TM) 64-Bit Server VM v1.8.0_45-b14
    21:42:10.860 INFO ValidateVariants - Start Date/Time: August 1, 2019 9:42:08 PM GMT-05:00
    21:42:10.860 INFO ValidateVariants - ------------------------------------------------------------
    21:42:10.860 INFO ValidateVariants - ------------------------------------------------------------
    21:42:10.861 INFO ValidateVariants - HTSJDK Version: 2.19.0
    21:42:10.861 INFO ValidateVariants - Picard Version: 2.19.0
    21:42:10.861 INFO ValidateVariants - HTSJDK Defaults.COMPRESSION_LEVEL : 2
    21:42:10.862 INFO ValidateVariants - HTSJDK Defaults.USE_ASYNC_IO_READ_FOR_SAMTOOLS : false
    21:42:10.862 INFO ValidateVariants - HTSJDK Defaults.USE_ASYNC_IO_WRITE_FOR_SAMTOOLS : true
    21:42:10.862 INFO ValidateVariants - HTSJDK Defaults.USE_ASYNC_IO_WRITE_FOR_TRIBBLE : false
    21:42:10.862 INFO ValidateVariants - Deflater: IntelDeflater
    21:42:10.862 INFO ValidateVariants - Inflater: IntelInflater
    21:42:10.863 INFO ValidateVariants - GCS max retries/reopens: 20
    21:42:10.863 INFO ValidateVariants - Requester pays: disabled
    21:42:10.863 INFO ValidateVariants - Initializing engine
    21:42:12.010 INFO FeatureManager - Using codec VCFCodec to read file file:///n/scratchlfs/mallet_lab/txiong/Research/2019_Achillides_Sequencing/03_vcfs_sg/Joint.final.InvariantIncluded.HiC_scaffold_9.sorted.vcf
    21:42:13.840 INFO ValidateVariants - Done initializing engine
    21:42:13.841 INFO ProgressMeter - Starting traversal
    21:42:13.841 INFO ProgressMeter - Current Locus Elapsed Minutes Variants Processed Variants/Minute
    21:42:18.766 WARN ValidateVariants - ***** Input 03_vcfs_sg/Joint.final.InvariantIncluded.HiC_scaffold_9.sorted.vcf fails strict validation: one or more of the ALT allele(s) for the record at position HiC_scaffold_9:9755 are not observed at all in the sample genotypes of type: *****
    21:42:18.767 WARN ValidateVariants - ***** Input 03_vcfs_sg/Joint.final.InvariantIncluded.HiC_scaffold_9.sorted.vcf fails strict validation: one or more of the ALT allele(s) for the record at position HiC_scaffold_9:9775 are not observed at all in the sample genotypes of type: *****
    21:42:21.138 WARN ValidateVariants - ***** Input 03_vcfs_sg/Joint.final.InvariantIncluded.HiC_scaffold_9.sorted.vcf fails strict validation: one or more of the ALT allele(s) for the record at position HiC_scaffold_9:67171 are not observed at all in the sample genotypes of type: *****
    21:42:21.139 WARN ValidateVariants - ***** Input 03_vcfs_sg/Joint.final.InvariantIncluded.HiC_scaffold_9.sorted.vcf fails strict validation: one or more of the ALT allele(s) for the record at position HiC_scaffold_9:67183 are not observed at all in the sample genotypes of type: *****
    21:42:21.146 WARN ValidateVariants - ***** Input 03_vcfs_sg/Joint.final.InvariantIncluded.HiC_scaffold_9.sorted.vcf fails strict validation: one or more of the ALT allele(s) for the record at position HiC_scaffold_9:67196 are not observed at all in the sample genotypes of type: *****
    21:42:21.147 WARN ValidateVariants - ***** Input 03_vcfs_sg/Joint.final.InvariantIncluded.HiC_scaffold_9.sorted.vcf fails strict validation: one or more of the ALT allele(s) for the record at position HiC_scaffold_9:67204 are not observed at all in the sample genotypes of type: *****
    21:42:21.147 WARN ValidateVariants - ***** Input 03_vcfs_sg/Joint.final.InvariantIncluded.HiC_scaffold_9.sorted.vcf fails strict validation: one or more of the ALT allele(s) for the record at position HiC_scaffold_9:67205 are not observed at all in the sample genotypes of type: *****
    21:42:21.148 WARN ValidateVariants - ***** Input 03_vcfs_sg/Joint.final.InvariantIncluded.HiC_scaffold_9.sorted.vcf fails strict validation: one or more of the ALT allele(s) for the record at position HiC_scaffold_9:67220 are not observed at all in the sample genotypes of type: *****
    21:42:21.148 WARN ValidateVariants - ***** Input 03_vcfs_sg/Joint.final.InvariantIncluded.HiC_scaffold_9.sorted.vcf fails strict validation: one or more of the ALT allele(s) for the record at position HiC_scaffold_9:67241 are not observed at all in the sample genotypes of type: *****
    21:42:21.148 WARN ValidateVariants - ***** Input 03_vcfs_sg/Joint.final.InvariantIncluded.HiC_scaffold_9.sorted.vcf fails strict validation: one or more of the ALT allele(s) for the record at position HiC_scaffold_9:67249 are not observed at all in the sample genotypes of type: *****
    21:42:21.149 WARN ValidateVariants - ***** Input 03_vcfs_sg/Joint.final.InvariantIncluded.HiC_scaffold_9.sorted.vcf fails strict validation: one or more of the ALT allele(s) for the record at position HiC_scaffold_9:67250 are not observed at all in the sample genotypes of type: *****
    21:42:21.149 WARN ValidateVariants - ***** Input 03_vcfs_sg/Joint.final.InvariantIncluded.HiC_scaffold_9.sorted.vcf fails strict validation: one or more of the ALT allele(s) for the record at position HiC_scaffold_9:67260 are not observed at all in the sample genotypes of type: *****
    21:42:21.149 WARN ValidateVariants - ***** Input 03_vcfs_sg/Joint.final.InvariantIncluded.HiC_scaffold_9.sorted.vcf fails strict validation: one or more of the ALT allele(s) for the record at position HiC_scaffold_9:67267 are not observed at all in the sample genotypes of type: *****
    21:42:21.151 WARN ValidateVariants - ***** Input 03_vcfs_sg/Joint.final.InvariantIncluded.HiC_scaffold_9.sorted.vcf fails strict validation: one or more of the ALT allele(s) for the record at position HiC_scaffold_9:67268 are not observed at all in the sample genotypes of type: *****
    21:42:21.151 WARN ValidateVariants - ***** Input 03_vcfs_sg/Joint.final.InvariantIncluded.HiC_scaffold_9.sorted.vcf fails strict validation: one or more of the ALT allele(s) for the record at position HiC_scaffold_9:67269 are not observed at all in the sample genotypes of type: *****
    21:42:21.151 WARN ValidateVariants - ***** Input 03_vcfs_sg/Joint.final.InvariantIncluded.HiC_scaffold_9.sorted.vcf fails strict validation: one or more of the ALT allele(s) for the record at position HiC_scaffold_9:67280 are not observed at all in the sample genotypes of type: *****
    21:42:21.155 WARN ValidateVariants - ***** Input 03_vcfs_sg/Joint.final.InvariantIncluded.HiC_scaffold_9.sorted.vcf fails strict validation: one or more of the ALT allele(s) for the record at position HiC_scaffold_9:67629 are not observed at all in the sample genotypes of type: *****
    21:42:21.155 WARN ValidateVariants - ***** Input 03_vcfs_sg/Joint.final.InvariantIncluded.HiC_scaffold_9.sorted.vcf fails strict validation: one or more of the ALT allele(s) for the record at position HiC_scaffold_9:67630 are not observed at all in the sample genotypes of type: *****
    21:42:24.242 INFO ProgressMeter - HiC_scaffold_9:128354 0.2 124000 716624.9
    21:42:24.308 WARN ValidateVariants - ***** Input 03_vcfs_sg/Joint.final.InvariantIncluded.HiC_scaffold_9.sorted.vcf fails strict validation: one or more of the ALT allele(s) for the record at position HiC_scaffold_9:132080 are not observed at all in the sample genotypes of type: *****
    21:42:24.310 WARN ValidateVariants - ***** Input 03_vcfs_sg/Joint.final.InvariantIncluded.HiC_scaffold_9.sorted.vcf fails strict validation: one or more of the ALT allele(s) for the record at position HiC_scaffold_9:132089 are not observed at all in the sample genotypes of type: *****
    21:42:32.421 WARN ValidateVariants - ***** Input 03_vcfs_sg/Joint.final.InvariantIncluded.HiC_scaffold_9.sorted.vcf fails strict validation: one or more of the ALT allele(s) for the record at position HiC_scaffold_9:481713 are not observed at all in the sample genotypes of type: *****
    21:42:32.421 WARN ValidateVariants - ***** Input 03_vcfs_sg/Joint.final.InvariantIncluded.HiC_scaffold_9.sorted.vcf fails strict validation: one or more of the ALT allele(s) for the record at position HiC_scaffold_9:481714 are not observed at all in the sample genotypes of type: *****
    21:42:32.422 WARN ValidateVariants - ***** Input 03_vcfs_sg/Joint.final.InvariantIncluded.HiC_scaffold_9.sorted.vcf fails strict validation: one or more of the ALT allele(s) for the record at position HiC_scaffold_9:481715 are not observed at all in the sample genotypes of type: *****
    21:42:32.424 WARN ValidateVariants - ***** Input 03_vcfs_sg/Joint.final.InvariantIncluded.HiC_scaffold_9.sorted.vcf fails strict validation: one or more of the ALT allele(s) for the record at position HiC_scaffold_9:481716 are not observed at all in the sample genotypes of type: *****
    ```

    The warning goes on similarly
  • bhanuGandhambhanuGandham Cambridge MAMember, Administrator, Broadie, Moderator admin

    Hi @bifengdie

    This looks like they are all only warnings and not an error so it should be fine. We have not tested joint genotyping on insect samples so I am not entirely sure how joint calling would affect the variant callset. Are you getting any variants calls that have a confident genotype call?

  • bifengdiebifengdie Cambridge, MA, USAMember
    Hi @bhanuGandham

    I am actually getting ERROR messages but I manually suppress them to become WARNINGs such that everything can be written in the log files.

    The strange behaviors also include that I consistently got very low quality scores across invariant sites in the final VCFs, but the variant sites have very good qualities. Even if that variant site is embedded inside a block of bad-quality invariants. For instance (trimmed to show only the first three samples):

    ```
    HiC_scaffold_9 70504 . C . 24.87 LowQual DP=22 GT:AD:DP:RGQ ./.:0,0:0 ./.:0,0:0 ./.:0,0:0
    HiC_scaffold_9 70505 . T . 25.59 LowQual DP=23 GT:AD:DP:RGQ ./.:0,0:0 ./.:0,0:0 ./.:0,0:0
    HiC_scaffold_9 70506 . A . 25.04 LowQual DP=23 GT:AD:DP:RGQ ./.:0,0:0 ./.:0,0:0 ./.:0,0:0
    HiC_scaffold_9 70507 . A . 25.59 LowQual DP=23 GT:AD:DP:RGQ ./.:0,0:0 ./.:0,0:0 ./.:0,0:0
    HiC_scaffold_9 70508 . T . 25.59 LowQual DP=23 GT:AD:DP:RGQ ./.:0,0:0 ./.:0,0:0 ./.:0,0:0
    HiC_scaffold_9 70509 . T . 25.04 LowQual DP=22 GT:AD:DP:RGQ ./.:0,0:0 ./.:0,0:0 ./.:0,0:0
    HiC_scaffold_9 70510 . A . 25.04 LowQual DP=23 GT:AD:DP:RGQ ./.:0,0:0 ./.:0,0:0 ./.:0,0:0
    HiC_scaffold_9 70511 . A . 25.59 LowQual DP=22 GT:AD:DP:RGQ ./.:0,0:0 ./.:0,0:0 ./.:0,0:0
    HiC_scaffold_9 70512 . A . 25.59 LowQual DP=22 GT:AD:DP:RGQ ./.:0,0:0 ./.:0,0:0 ./.:0,0:0
    HiC_scaffold_9 70513 . A G 144.78 . AC=6;AF=1.00;AN=6;DP=16;ExcessHet=3.0103;FS=0.000;MLEAC=3;MLEAF=0.500;MQ
    HiC_scaffold_9 70514 . T . 25.41 LowQual DP=16 GT:AD:DP:RGQ ./.:0,0:0 ./.:0,0:0 ./.:0,0:0
    HiC_scaffold_9 70515 . C . 25.41 LowQual DP=16 GT:AD:DP:RGQ ./.:0,0:0 ./.:0,0:0 ./.:0,0:0
    HiC_scaffold_9 70516 . C . 25.41 LowQual DP=16 GT:AD:DP:RGQ ./.:0,0:0 ./.:0,0:0 ./.:0,0:0
    HiC_scaffold_9 70517 . G . 25.41 LowQual DP=16 GT:AD:DP:RGQ ./.:0,0:0 ./.:0,0:0 ./.:0,0:0
    HiC_scaffold_9 70518 . T . 25.41 LowQual DP=16 GT:AD:DP:RGQ ./.:0,0:0 ./.:0,0:0 ./.:0,0:0
    HiC_scaffold_9 70519 . G . 25.41 LowQual DP=17 GT:AD:DP:RGQ ./.:0,0:0 ./.:0,0:0 ./.:0,0:0
    HiC_scaffold_9 70520 . A . 25.41 LowQual DP=17 GT:AD:DP:RGQ ./.:0,0:0 ./.:0,0:0 ./.:0,0:0
    HiC_scaffold_9 70521 . C T 93.19 . AC=4;AF=0.500;AN=8;DP=17;ExcessHet=0.1902;FS=0.000;MLEAC=2;MLEAF=0.250;M
    HiC_scaffold_9 70522 . G . 25.55 LowQual DP=19 GT:AD:DP:RGQ ./.:0,0:0 ./.:0,0:0 ./.:0,0:0
    HiC_scaffold_9 70523 . A . 25.55 LowQual DP=19 GT:AD:DP:RGQ ./.:0,0:0 ./.:0,0:0 ./.:0,0:0
    HiC_scaffold_9 70524 . C . 24.81 LowQual DP=19 GT:AD:DP:RGQ ./.:0,0:0 ./.:0,0:0 ./.:0,0:0
    HiC_scaffold_9 70525 . A . 24.81 LowQual DP=19 GT:AD:DP:RGQ ./.:0,0:0 ./.:0,0:0 ./.:0,0:0
    HiC_scaffold_9 70526 . G . 0.03 LowQual DP=19;ExcessHet=3.01;MQ=31.00 GT:DP:RGQ 0/0:0:0 0/0:0:0 0/0:0:0

    ```

    Basically I have no invariant sites with a good quality score. In the above example, site 70513 and 70521 have nice quality scores, and they are both variant sites. Has anyone reported similar problems before? I have not found one on the forum. Thanks for helping!
  • bifengdiebifengdie Cambridge, MA, USAMember
    Is it mandatory to use

    --emit-ref-confidence BP_RESOLUTION

    in HaplotypeCaller if I need to analyze all sites in GenotypeGVCFs? I thought GVCF rather than BP_RESOLUTION would work too, since it include the invariant haplotype blocks, but this post (#11665) suggests it is necessary.
  • bhanuGandhambhanuGandham Cambridge MAMember, Administrator, Broadie, Moderator admin
    edited August 6

    Hi @bifengdie

    We have not tested our pipelines on insect samples so unfortunately I am not sure why you are seeing these results. Maybe someone from our zoo and garden community forum might be able to help you out with this question or www.biostars.org or www.seqanswers.com

    Sorry I wasn't of more help.

  • bifengdiebifengdie Cambridge, MA, USAMember
    Ah OK, thank you any way! @bhanuGandham
  • bifengdiebifengdie Cambridge, MA, USAMember
    Just as a follow-up, I used `-ERC BP_RESOLUTION` in HaplotypeCaller to see whether it will make any difference, and it did!

    For one sample, here is a haplotype block when using `-ERC GVCF`:

    HiC_scaffold_10 271 . T <NON_REF> . . END=284 GT:DP:GQ:MIN_DP:PL 0/0:21:0:20:0,0,0

    And it looks like this when using `-ERC BP_RESOLUTION`:

    HiC_scaffold_10 271 . T <NON_REF> . . . GT:AD:DP:GQ:PL 0/0:15,0:15:45:0,45,490
    HiC_scaffold_10 272 . A <NON_REF> . . . GT:AD:DP:GQ:PL 0/0:15,0:15:45:0,45,490
    HiC_scaffold_10 273 . C <NON_REF> . . . GT:AD:DP:GQ:PL 0/0:15,0:15:45:0,45,478
    HiC_scaffold_10 274 . T <NON_REF> . . . GT:AD:DP:GQ:PL 0/0:15,0:15:45:0,45,490
    HiC_scaffold_10 275 . A <NON_REF> . . . GT:AD:DP:GQ:PL 0/0:16,0:16:48:0,48,486
    HiC_scaffold_10 276 . T <NON_REF> . . . GT:AD:DP:GQ:PL 0/0:16,0:16:48:0,48,498
    HiC_scaffold_10 277 . G <NON_REF> . . . GT:AD:DP:GQ:PL 0/0:16,0:16:48:0,48,472
    HiC_scaffold_10 278 . T <NON_REF> . . . GT:AD:DP:GQ:PL 0/0:17,0:17:51:0,51,496
    HiC_scaffold_10 279 . A <NON_REF> . . . GT:AD:DP:GQ:PL 0/0:17,0:17:51:0,51,506
    HiC_scaffold_10 280 . G <NON_REF> . . . GT:AD:DP:GQ:PL 0/0:17,0:17:51:0,51,494
    HiC_scaffold_10 281 . T <NON_REF> . . . GT:AD:DP:GQ:PL 0/0:17,0:17:51:0,51,506
    HiC_scaffold_10 282 . G <NON_REF> . . . GT:AD:DP:GQ:PL 0/0:18,0:18:54:0,54,530
    HiC_scaffold_10 283 . A <NON_REF> . . . GT:AD:DP:GQ:PL 0/0:18,0:18:54:0,54,530
    HiC_scaffold_10 284 . T <NON_REF> . . . GT:AD:DP:GQ:PL 0/0:21,0:21:63:0,63,617

    If you look at the GQ field, they are quite different!
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