I am new to NGS data analysis and I'm working in a multiple-sample variant calling workflow. I have Illumina-Miseq fastq files (paired end, raw reads) for a father, mother and child trio, one pair for each individual, totalling 6 files. I could trim, align, do the pre-processing and variant calling for each individual pair separately (I'm skipping indel-realignment and quality recalibration, for the sake of simplicity, as this workflow is intended for learning only), but I wish to merge the samples into a single file. I wish that the alignment step (with BWA-MEN), the pre-processing steps (with Picard) and the variant calling step (with FreeBayes), are done at once for all samples, if possible and correct, while taking in consideration the correct paired end mates and the respective read groups (when applicable).

My final goal is to obtain a single vcf file from which I'll compute the total number of different kinds of variants.

At which step, in which file format and with which Galaxy tools can I merge the samples in a manner that I can get correct, meaninful results at the variant calling step?

avoid to merge the VCFS : call them at the same time:

    samtools mpileup [options...] father.bam mother.bam child.bam | bcftools call  [options...] > result.vcf

or something like:

    java -jar GATK.jar -T HaplotypCaller [options...] -o result.vcf -i father.bam -i mother.bam child.bam 

or you can use GATK + gVCF calling : http://gatkforums.broadinstitute.org/gatk/discussion/4017/what-is-a-gvcf-and-how-is-it-different-from-a-regular-vcf


or you can merge the 3 VCF later with https://software.broadinstitute.org/gatk/documentation/tooldocs/3.5-0/org_broadinstitute_gatk_tools_walkers_variantutils_CombineVariants.php , but you'll get some missing genotypes (./.) where you can't tell if the sample was HOM_REF or if there was not enough information to do the calling.


