Library type and difference between call-up & pileup

[aayushraman]

Thank you for developing the tool. Appreciate it. I have a question regarding the Library type and strand information. I am using the Illumina TruSeq Stranded Total RNA kit and I validate using a couple of different tools, including RSeQC where I do get the fr-firststrand and also looked into the info provided in different blog post including the one here.

1) I am confused with the option of stranded library type provided by JACUSA which are RF-FIRSTSTRAND and FR-SECONDSTRAND. Can you please explain which one I need to use?

2) What is the different between the call-2 and the pileup method?

Thank you!

[piechottam]

1. Check for instance this post or even better Illumina, page 17 "Library Type".
2. call-2 does variant calling and by default only outputs sites with differences. pileup outputs everything (~ samtools mpileup).

[aayushraman]

Thanks for the prompt reply. The difference between call-2 and pileup is clear to me. So, is the difference between pileup in Jacusa vs. mpileup in the samtools the use of library type (-P argument) used by JACUSA, whereas the samtools does not use that information?

Also, I still need help understanding the stranded library type or annotation used by JACUSA. In both blog posts, I see the use of FR-FIRSTSTRAND and not RF-FIRSTSTRAND which is used by JACUSA.

[piechottam]

Yes, to your first question - The mapping qual, start, and end are missing in the JACUSA pileup.

2nd question: https://www.biostars.org/p/169942/ It mainly boils down?

• Lib stranded:
• no -> UNSTRANDED
• yes -> FIRST or SECOND? You can check this in IGV: Orientation of reads and gene annotation are inverse -> FIRST; same orientation -> SECOND

[aayushraman]

Thank you for answering my questions!