I'm working on an RNAseq experiment in a non-model species. I have a de novo transcriptome i'm using as my reference to align paired end illumina reads since we do not yet have a working genome.
I've done an initial alignment of my reads using both Bowtie2, but upon further consideration I thought that RSEM may be a better system due to its ability to more accurately give read counts for multiple-mapping reads. my alignment rate dropped significantly with the RSEM alignment, which I hypothesize is due to mainly to the fact that "Since currently RSEM does not handle indel, local and discordant alignments, the Bowtie2 parameters are set in a way to avoid those alignments".
I see a trade-off: higher alignment with Bowtie2, but randomly assign multireads (which could skew my analysis) or account for multireads with RSEM, but let my alignment suffer because indels and discordant alignments are not allowed.
1. Is RSEM capable of properly handing a bowtie2 alignment (with "-a" option to report all alignments) as input, producing counts for each transcript which account for these multireads, while taking advantage of an excellent bowtie2 alignment?
2. Is it better to randomly assign multireads for an RNA-seq experiment using Bowtie2 alignment or more accurately quantify mutlireads with RSEM (w/ Bowtie2, excluding indels and discordant alignments)?
thanks in advance.