Variant calling of GBS data

Question

[analyst]

Hi all,

I have GBS data of plant and I have to call variants. But I do not have reference variation file available.

Is it okay to call variants without performing base quality score recalibration step that requires reference variation file beforehand?

Answer 1

[Pierre Lindenbaum]

Is it okay to call variants without performing base quality score recalibration step that requires reference variation file beforehand?

see https://gatk.broadinstitute.org/hc/en-us/articles/360035890471-Hard-filtering-germline-short-variants

[analyst]

Thanks Pierre Lindenbaum! Can we perform hard filtering on FreeBayes output ? And does hard filtering work same as of other variant filtration tools like snpsift, bcftools etc ?

[Pierre Lindenbaum]

No, it requires specific fields generated by gatk hapcaller

[analyst]

I am using gatK version 4.2.1.0 for variant filtration but it generates following warnings:

14:10:40.970 WARN JexlEngine - ![0,14]: 'ReadPosRankSum < -8.0;' undefined variable ReadPosRankSum 14:10:40.970 WARN JexlEngine - ![0,9]: 'MQRankSum < 0.4;' undefined variable MQRankSum 14:10:40.970 WARN JexlEngine - ![0,14]: 'ReadPosRankSum < -8.0;' undefined variable ReadPosRankSum 14:10:40.970 WARN JexlEngine - ![0,9]: 'MQRankSum < 0.4;' undefined variable MQRankSum

[Pierre Lindenbaum]

remove spaces in expressions. 'MQRankSum<0.4'

[analyst]

Right Pierre, thanks.

[analyst]

It is still giving same warning

java -jar -Xmx30G gatk.jar VariantFiltration -R reference.fa -V A_raw_snps.vcf -O A_filtered_snps.vcf --filter-name "QD_filter" -filter "QD < 2.0" --filter-name "FS_filter" -filter "FS > 60.0" --filter-name "SOR_filter" -filter "SOR > 10.0" --filter-name "MQRankSum_filter" -filter "MQRankSum<-12.5" --filter-name "ReadPosRankSum_filter" -filter "ReadPosRankSum<-8.0" --genotype-filter-expression "DP < 10" --genotype-filter-name "DP_filter" --genotype-filter-expression "GQ < 10" --genotype-filter-name "GQ_filter"

[Pierre Lindenbaum]

again, remove the spaces " "SOR > 10." -> " "SOR>10"

[analyst]

It does not output warning when I skip flags for MQRankSum and ReadPosRankSum

java -jar -Xmx30G gatk.jar VariantFiltration -R reference.fa -V A_raw_snps.vcf -O A_filtered_snps.vcf -filter-name "QD_filter" -filter "QD < 2.0" -filter-name "FS_filter" -filter "FS > 60.0" -filter-name "SOR_filter" -filter "SOR > 10.0" -genotype-filter-expression "DP < 10" -genotype-filter-name "DP_filter" -genotype-filter-expression "GQ < 10" -genotype-filter-name "GQ_filter"

[analyst]

Command used :

java -jar -Xmx30G gatk.jar VariantFiltration -R reference.fa -V A_raw_snps.vcf -O A_filtered_snps.vcf --filter-name "QD_filter" --filter-expression "QD < 2.0" --filter-name "FS_filter" --filter-expression "FS > 60.0" --filter-name "MQ_filter" --filter-expression "MQ < 40.0" --filter-name "SOR_filter" --filter-expression "SOR > 4.0" --filter-name "MQRankSum_filter" --filter-expression "MQRankSum < -12.5" --filter-name "ReadPosRankSum_filter" --filter-expression "ReadPosRankSum < -8.0" --genotype-filter-expression "DP < 10" --genotype-filter-name "DP_filter" --genotype-filter-expression "GQ < 10" --genotype-filter-name "GQ_filter"

[analyst]

Vcf file contains this information

[analyst]

[analyst]

Does GATK Haplotypecaller take care of ploidy because few plants are polyploid?