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gpertea / stringtie

Transcript assembly and quantification for RNA-Seq
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Single-end strand-specific rna-seq data parameters? #210

Open wisense opened 5 years ago

wisense commented 5 years ago

Hi, developer,

I've single-end stranded data, and specified --rna-strandness R in Hisat2 runs. So the bam alignment contain XS: tags. When running Stringtie, should I specify the --rf/--fr parameter for this type of data? It seems to me that "--rf/--fr" is designed for paired-end data.

Hope to receive your reply soon Best!

WSZ

SchulzLab commented 5 years ago

Hi, I have the same question. When I look at the assemblies, i can see that single exon genes are not assigned to a strand, so it appears to me that the strand information in the alignment records are not used automatically for single end reads. Would be great to get some feedback on this. Bests

gpertea commented 5 years ago

I don't remember writing that particular code -- but looking at the code now I can see that those options should assign the transcription strand internally for both paired and single read alignments, but only if the XS tag is missing, so they are not doing anything in the case you described, if HISAT2 already assigned the XS tags.

As for what @SchulzLab observed, single exon transcripts have nothing to do with single end reads, but if those single exon transcripts are indeed made of read alignments that do have/get an XS strand assigned, there might be an oversight somewhere in the code, because those single-exon transcripts should get a strand assigned by StringTie in that case. @SchulzLab if you have a small example (BAM) with single-end read alignments that got assembled by stringtie into a single-exon transcript without a strand assignment, despite the read alignments having an explicit (XS tag) or implicit (--fr/--rf) transcription strand assigned, I'd be interested to take a look and see if maybe there is indeed something we missed there in the code -- thank you!

piyush-jo15 commented 5 years ago

Hi. I have a similar question. I performed my alignment using HISAT2 with --rna-strandness RF option. Unfortunately I forgot to run stringtie with --rf option also. Now I am worried if stringtie assumed the library to be unstranded. Could you clarify this doubt for me? Thanks! Piyush