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Changes to RNAseq pipeline - AddOrReplaceReadGroups

Hi all,

I'm currently working on an RNAseq variant calling pipeline, and it looks like there's been a change from when I last developed a pipeline. Do we no longer have to use the step to add or replace read groups with Picard? Is there a more detailed breakdown of the current best practices somewhere I've not been able to find?

Thanks!

Answers

  • bhanuGandhambhanuGandham Cambridge MAMember, Administrator, Broadie, Moderator admin

    Hi @TomWillDo

    Add or replace read groups is an additional step used to clean up your bam files if need be. It is not part of the RNAseq variant calling best practice pipeline.

  • TomWillDoTomWillDo Member
    Thank you @bhanuGandham, I'll consider removing this in the future if it doesn't affect things downstream.

    Further questions for anyone who can answer them: From memory, I recall seeing a forum post that the current best practices are a mix between GATK3 and 4. I believe I can also use GATK3 or 4 for everything, but as I have access to both tools I should probably use both for the best results. Where could I find detils on which tool to use where if this is the case?

    Also, with regards to MergeBamAlignment: would I get the unaligned reads by extracting them from STAR BAM files, using ""--outSAMunmapped Within"" and then something like SAMtools view as the unaligned BAM input?

    Thanks in advance!
  • bhanuGandhambhanuGandham Cambridge MAMember, Administrator, Broadie, Moderator admin
    edited July 29

    Hi @TomWillDo

    The workflow allows users to opt to use GATK4 for all tools instead of the default combination between GATK3 and GATK4. We have not officially released this pipeline exclusively for GATK4, although we know users who have used only GATK4 tools in the pipeline and it worked well enough that they didn't ask us to fix anything. We recommend using the GATK4 tools in the pipeline because if any errors come up we can help fix them. We do not fix any errors with GATK3 because we do not support it anymore.

    To understand what a ubam is and how to get it it take a look at this doc: https://software.broadinstitute.org/gatk/documentation/article?id=11008

  • TomWillDoTomWillDo Member
    Thank you @bhanuGandham

    Unfortunately it looks like the link within that article goes to a whole bunch of presentations, and not the specific one needed. Do you know which of the presentations I should look at? Otherwise, if I've understood it correctly, I can my samples fastq files, use the FastToSam conversion tool to obtain the unmapped BAM file which I then use for the MergeBamAlignments tool?
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