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@josquintibbitsdpivicgovau-4311
Last seen 10.4 years ago
Hi List,
In the edgeR manual it is mentioned that the package may be useful in
the
analysis of other count based studies and I am wondering if the bulk
segregation studies (based on tag counts) are such a case i.e. hoping
for
advice on the potential rightness or wrongness of doing this...
The general experimental outline is as follows:
>From parents introgressed with DNA containing allelels that are +/-
for a
trait a set of offspring is generated that segregate for the trait
(in my
case the progeny have been back-crossed to one of the parental lines
for
multiple generations so that they are effectively isogenic backgrounds
with just the QTL region segregating, while the parents can have
multiple
regions segregating from the original introgression including the QTL
region). Offspring are bulked to create pools representing + and -
bulks.
DNA is then isolated from parents and offspring and these are assayed
for
a marker set which is generated as follows:
DNA digested with a restriction enzyme, adaptor ligated to these ends
(ie
tag the restriction digestion sites). Randomly shear DNA and then
ligate
second adaptor. Enrich for fragments with two adaptors and then
sequence
fragments on Illumina HiSeq instrument. Map tags back to a reference
and
count hits. We are then looking for reference sequences that are
differentially hit in the + vs - strains in both parents and
offspring.
My thinking was that parents and offspring are effectively
replications of
the presence or absence of the QTL region and the way the counts are
obtained has similar sampling properties to when you are doing a DE
study
of gene expression. Hence I thought to use edgeR to do the analysis as
it
can normalise for both overall count numbers and the TMM normalisation
for
target tag complexity and the tagwise implementation can account for
the
technical variance arising from bulking, sampling, mapping efficieny
etc.
Looking forward to reading your comments.....
Best, Josquin
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