Which Aligner Should I Use For Mapping Bacterial Rna-Seq Data

Question

[Kssr]

The reads are paired end,90 bp. I have tried using bowtie and tophat.Bowtie gave around 85 % of alignment.While tophat gave around 92 % alignment.Although Tophat calls Bowtie, any ideas why the percent alignment is higher in case of Tophat??

As bacteria do not have splice junctions,does running tophat in anyway affect the alignment.How does running tophat with GTF file affect the alignment?

I prefer using tophat since I would like to do differential analysis using tophat output with cufflinks software.Any ideas would be appreciated.

Answer 1

[Ryan Thompson]

You are probably running Bowtie in paired-end mode, which means that Bowtie requires a pair to either map together or not at all. Tophat maps each read singly because it is looking for splicing events, so it cannot assume that a pair of reads will map near each other because that pair might span an intron.

So the difference in mapping percentage is probably just because Tophat allows reads to map singly. You can do the same thing with Bowtie as well by first running in paired-end mode and using the --un option to collect unaligned pairs, and then mapping the unaligned pairs in single-end mode.

Also, I believe Cufflinks accepts any bam file as input. It is not dependent on Tophat.

[Mikael Huss]

Cufflinks does accept any bam file as input, but there is one restriction: there must be XS tags for the spliced alignments.

[Ryan Thompson]

Well, bowtie doesn't do spliced alignment, and bacteria don't splice their RNA, so I don't think that should be a problem in this case, either technically or biologically. But good point regardless, for anyone working in eukaryotes.

Answer 2

[Josh Herr]

This is a problem the authors of the Tuxedo suite have touched on, hence, this newish paper: EDGE-pro: Estimated Degree of Gene Expression in Prokaryotic Genomes

Answer 3

[Swbarnes2]

You could probably just align to a refseq file of transcripts with anything. That would be easy to count from the .bam file.