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suggestion for multi-samples RNAseq experiment
I've recently started working with GATK and after reading documents, tutorial and forum discussions, I set a pipeline for my experiment. I'm dealing with multi-sample RNAseq for which GATK tools are less improved and verified than DNAseq, thus I 'd like to have your suggestions.
Briefly, this is the experiment: 2 phenotypes of Sparus aurata, 8 libary per phenotype, each library consists on a pool (not barcoded) of 3 animals. Thus I have a total of** 16 samples**. My goal is to find the total number of variant sites and compare the allele frequencies between the two phenotypes. **I lack genome and SNPs database. **
Step by step:
1) I used STAR (not 2-pass) in order to map reads against my de novo assembly.
STAR --runThreadN 16 --genomeDir ./GenomeIndex --readFilesIn XXX.fastq --alignIntronMax 19 --outSAMtype BAM SortedByCoordinate --outSAMmapqUnique 60 --outFilterMultimapNmax 5 --outFilterMismatchNmax 4
2) I used the picard-tools to Mark duplicates
3) I used the picard-tools to AddOrReplaceReadGroups
4) I used the picard-tools to BuildBamIndex
5) I called the haplotype for the 16 samples with the following command:
GenomeAnalysisTK.jar -T HaplotypeCaller -R reference.fasta -I sample.bam -dontUseSoftClippedBases -ploidy 6 -ERC GVCF -o output.g.vcf
6) I used the GenotypeGVCFs to merge the samples from the same population in an unique vcf file as follow
GenomeAnalysisTK.jar -T GenotypeGVCFs -R reference.fasta -stand_call_conf 20 -stand_emit_conf 20 -ploidy 6 --variant sample1.g.vcf --variant sample2.g.vcf --variant sample3.g.vcf (8 samples) -o output_HC.vcf
Finally I'm going to filter the results with the Variant Filtration: GenomeAnalysisTK.jar -T VariantFiltration -R reference.fasta -V output.vcf -window 35 -cluster 3 -filterName FS -filter "FS > 30.0" -filterName QD -filter "QD < 2.0" -o utputFiltered.vcf
What do you think?
Now I'd like to compare the two populations, but how??? manually in excel files? vcf tools seem not to handle ploidy higher than 2.
Does anyone deal with these issues and can kindly give some tips?