Hi Jeremy,

I thought I'd write to get a discussion of a workflow for people doing RNA seq 
that I have found very useful and addresses some issues in mapping mRNA derived 
RNA-seq paired end data to the genome using tophat. Here is the approach I use 
(I have a human mRNA sample deep sequenced with a 56bp paired end read on an 
illumina generating 29 million reads):

1. Align to hg19 (in my case) using tophat and allowing up to 40 hits for each 
sequence read
2. In samtools filter for "read is unmapped", "mate is mapped" and "mate is 
mapped in a proper pair"
3. Use "group" to group the filtered sam file on c1 (which is the 
"bio-sequencer" read number) and set an operation to count on c1 as well. This 
provides a list of the reads and how many times they map to the human genome, 
because you have filtered the set for reads that have a mate pair there will be 
an even number for each read. For most of the reads the number will be 2 
(indicating the forward read maps once and the reverse read maps once and in a 
proper pair) but for reads that map ambiguously the number will be multiples of 
2. If you count these up I find that 18 million reads map once, 1.3 million map 
twice, 400,000 reads map 3 times and so on until you get down to 1 read mapping 
30 times, 1 read mapping 31 times and so on...
4. Filter the reads to remove any reads that map more than 2 times.
5. Use "compare two datasets" to compare your new list of reads that map only 
twice to pull out all the reads in your sam file that only map twice (i.e. the 
mate pairs).
6. You'll need to sort the sam file before you can use it with other 
applications like IGV.

What you end up with is a sam file where all the reads map to one site only and 
all the reads map as a proper pair. This may seem similar to setting tophat to 
ignore non-unique reads. However, it is not. This approach gives you 10-15% 
more reads. I think it is because if tophat finds (for example) that the 
forward read maps to one site but the reverse read maps to two sites it throws 
away the whole read. By filtering the sam file to restrict it to only those 
mappings that make sense you increase the number of unique reads by getting rid 
of irrational mappings.

Has anyone else found this? Does this make sense to anyone else? Am I making a 
huge mistake somewhere?

A nice aspect of this (or at least I think so!) is that by filtering in this 
manner you can also create a sam file of non-unique mappings which you can 
monitor. This can be useful if one or more genes has a problem of generating a 
lot of non-unique maps which may give problems accurately estimating its 
expression. Also, you also get a list of how many multi hits you have in your 
data so you know the scale of the problem.

Best Wishes,

Dr David A. Matthews

Senior Lecturer in Virology
Room E49
Department of Cellular and Molecular Medicine,
School of Medical Sciences
University Walk,
University of Bristol

Tel. +44 117 3312058
Fax. +44 117 3312091


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