RSEM vs. samtools vs. other for expression?

[yeaman]

Hello all,

Apologies if these are stupid questions...I'm still a bit of a noob.

1. Does anyone know if it is possible to run RSEM to use rsem-calculate-expression on .fasta files along with qual scores? It seems that it uses qual scores in fastq files only.

2. What is the best way to estimate expression for various different HiSeq transcriptome runs based on comparison to a reference transcriptome assembled de novo in Trinity? I need to be mindful of the fact that the Trinity build will contain several closely related sequences due to sequencing errors/alternative splicing, and I want to restrict the alignments to only the best-hits (using the qual scores). I have been told that BWA+samtools is one approach, RSEM another...any thoughts on the best way?

Thanks in advance!

[yeaman]

Apparently it isn't possible to use fasta+qual files in RSEM, but there isn't much difference in alignments between fasta without qual vs. fastq. I haven't tested this myself though.