Entering edit mode
Nicole Ertl
▴
10
@nicole-ertl-6570
Last seen 10.2 years ago
Dear Bioconductor users,
I'm working on a novel organism (no genome, only a reference
transcriptome I had prepared with Trinity) and I have to do some
differential gene expression analysis, using RNA-Seq data, produced
with the Illumina TruSeq (non directional) kit. Most of my experiments
have 2 conditons: control & treatment, one experiment has 4
conditions: control & 3 treatments. I have 6 biological replicates
each per control/treatment.
I've seen quite a few publications (BMC, PLOS one and others) that
have aligned their reads to a reference transcriptome and then used
RSEM or eXpress (+ sometimes FPKM/RPMK) to produce the count table
which they then used with DESeq to analyse their data. Most don't
really go into any sort of detail, so it's hard to follow what has
been done. I've seen the "Count-based differential expression analysis
of RNA sequencing data using R and Bioconductor" publication online
and in it is mentioned that in the case of no genome, a reference
transcriptome can be built, reads aligned to it and counted and then
the standard pipeline for differential analysis used. The
documentation for DESeq (and DESeq2), says to use raw counts, and
nothing (rounded) normalised or counts of covered base pairs. I had a
look at the RSEM and eXpress documentation and both seem to do some
kind of estimation due to the isoforms inherent in a transcriptome? On
the RSEM website it mentions that "popular differential expression
(DE) analysis tools such as edgeR and DESeq do not take variance due
to read mapping uncertainty into consideration. Beacause read mapping
ambiguity is prevalent among isoforms and de novo assembled
transcripts, these tools are not ideal for DE detection in such
conditions." They suggest to use EBSeq, but I found max a handful of
papers on google scholar that actually used RSEM-EBSeq. I'm new to all
this and it's getting quite confusing. Could you please help? What
would I have to do with my data and/or my reference transcriptome to
be able to use eg the RSEM - DESeq (maybe DESeq2) pipeline? Is there a
pipeline that you could recommend in my situation?
Thank you so much for your time.
Kind Regards,
Nicole
University of the Sunshine Coast, Locked Bag 4, Maroochydore DC,
Queensland, 4558 Australia.
CRICOS Provider No: 01595D
Please consider the environment before printing this email.
This email is confidential. If received in error, please delete it
from your system.
[[alternative HTML version deleted]]