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Old 02-24-2016, 03:24 AM   #1
Alex852013
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Location: Germany

Join Date: Jan 2013
Posts: 17
Default Up to date ERCC spike ins RNA seq analysis

Hello everybody,

I'm expecting to get my ERCC spiked RNA seq sequencing files soon.
Therefore i would like to find out what's the best way to analyze them.
There are several post where people ask some specific questions to parts of the analysis, but i couldn't find a beginning to end thing.

My idea is to
- map to data with TopHat2
- find the number of reads per gene in the bam file from TopHat2 with HTSeq count
- normalize these reads to the ERCCs. But i have no idea how to do that part

I found a post suggesting to use loess normalization
https://www.biostars.org/p/81337/

But there is also a paper, which claims that loess normalization is not a really good way to go. Additionally, i don't get how they run their suggested solution.
http://www.nature.com/nbt/journal/v3.../nbt.2931.html

So maybe someone has a suggestion how to do the ERCC normalization in 2016, what program i can use and what file or format one has to use. Maybe someone knows a thread, where it is written down how exactly to use the code.

I guess, finally i would need to use DESeq count to compare my triplicates within different time points.

Thanks a lot, Alex
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