Hi, im a newbie here. I want to know how to retain reads with zero mapping quality to be carried into the next step… Im using Map with BWA-MEM followed by samtools mpileup and varscan. I want my final output to have all variants, including the ones with multiple hits, as I am studying pseudogenes.
Most variant calling protocols will discard multi-mapping reads.
Try these two tools instead and see if they produce the results you are looking for. How to use each is on the tool form.
- Variant calling: Naive Variant Caller (NVC) - tabulate variable sites from BAM datasets (Galaxy Version 0.0.4)
- Parse those results into a summary report: Variant Annotator process variant counts (Galaxy Version 1.2)
This much older tutorial includes both tools and will offer a bit more usage help, in the context of a different experiment. Technical gotcha… the tools included are older. Use the most current version of the tools instead and you’ll be able to skip some of the steps. Example: Read groups can be directly assigned on mapping tool forms now (if you use/need those) and do not require distinct steps to add them in.
Current Galaxy Variant Analysis tutorials can be found here. None of these include the specialized variant calling tools above, but the remainder of the methods may be informative.