RSEM - convert-sam-for-rsem

[Stuti Agrawal]

Hi,

I am trying to quantify the expression for some RNA-seq datasets using STAR and RSEM. I performed the following steps:

(a) Prepared the reference using rsem-prepare-reference. I provided the GTF file in this step.

(b) Used the ref_name.idx.fa file generate to create the genome index for STAR

(c) Aligned the RNA-seq reads to the index thus generated using star-2-pass. I used the parameters to prevent indels and perform end to end matching as mentioned in previous posts.

(d) Ran convert-sam-for-rsem on the resulting alignment file.

However, when I do that, I get the error that "Number of first and second mates in read ABC are not matched!". When I check my BAM file for the alignment of the two mates of this read, I find that there is one alignment for the first mate and two alignments for the second mate. 

Is this something that RSEM can handle? If so, how should I proceed? 

If not, is there a way to produce equal number of partially mapped reads using STAR?

Thanks,

Stuti

[Bo Li]

Hi Stuti,

Unfortunately, currently RSEM cannot handle partially mapped reads.

Best,
Bo

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[Stuti Agrawal]

Hi Bo,

Thank you for your reply, however I think these reads are not partially mapped, rather, they are multi-mapped. The first mate is not mapped (so there is one alignment entry in the BAM file) and the second mate is mapped to two different transcripts. There is one primary and one non-primary alignment for the second mate. These are Xbp reads and the CIGAR flags are XM for both the alignments for the second mate.

Stuti

[Colin Dewey]

Hi Stuti,

The preferred method of using STAR with RSEM is to map against the genome with STAR but use the "--quantMode TranscriptomeSAM” option to STAR which generates transriptome-based alignments for use with RSEM. This will likely resolve the issue you are having. See the following thread for more information:

https://groups.google.com/forum/#!searchin/rsem-users/star/rsem-users/BqXesH92tyA/hLZxFIGreDkJ

Best,
Colin

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