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  • QC using Samtools

    Hi all,

    I am trying to learn how to do QC with RNA-Seq data and I've been referring to this pdf file that was the RNA-Seq Blog. In one of the steps it recommends that you filter out:
    1. Reads that don't align
    2. Alignments that are not primary
    3. Alignments Fails QC
    4. Alignments that are PCR or optical duplicates


    It then has the following commands

    Code:
    samtools view -F 1796 -S -bt mm9.fa.fai GSM521256_exp_filtered.sam > GSM521256_exp.ba
    samtools rmdup GSM521256_exp.bam GSM521256_exp.nodup.bam
    In the -F parameter we are already filtering out for PCR and Optical duplicates are we not? Then what does the rmdup step add to the filtering since it's already filtered out (or supposedly filtered out) PCR and Optical duplicates in the last step.

    Also, what determines if an alignment fails QC? is it the mapping quality?

    Thanks,

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