Dear all,

I have been looking for an answer to my problem in all the Galaxy Support resources but with no success. I am sorry if this topic has been already discussed!

So, I am analyzing MiSeq data on the main Galaxy.
I have Fastq files from 4 paired-end samples. After having checked the quality with FastQC and groomed them, I have performed a BWA mapping, filtered the results and converted the SAM to BAM files (for each sample separately). I have then called SNPs with Freebayes and SAMtools, encountering problems in both cases.

1) SAMtools: if I run the Generate pileup tool, then the Filter pileup doesn't recognize any valid format in the files I have in my History and I cannot go on with the analysis. Why is that? What can I do?

2) I have performed variant calling with Freebayes on single BAM files and on one merged BAM files from all my four BWA mapping files. In all cases, the last column is "unknown", while it should be the name of my sample. This is not a big deal for the single vcf files, but from the merged BAM file, I cannot discriminate from which sample the SNPs were detected. I think there is a problem in the BAM files which are not properly indexed. Also Freebayes needs an RG tag.
Is there a tool in Galaxy I can use to index BAM files, adding the RG tag?

I hope someone can help me!

Thank you very much!

Debora Garzetti, PhD Student
AG Rakin
Max von Pettenkofer-Institute, LMU
Pettenkoferstra├če 9A
80336 Munich

Phone: +49 (0)89 2180 72915

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