Analysis of Directional mRNA-seq data / Illumina

[jmtepp]

We are developing a library preparation for directional mRNA-seq. All sequences will read 5' -> 3' direction. Our protocol isolates mRNA, fragments the messages and then these are re-purified with oligodT - resulting in a library of short 3' fragments for sequencing. This eliminates the need to adjust for transcript length when doing quantitative expression analysis. We're using bowtie to align the raw reads to a (cDNA) reference transcriptome (minus introns and intergenic regions). BUT - approximately 12% of the loci in this organism (Arabidopsis) have overlapping 3' tails (Watson/Crick strands) leading to ambiguities when using BowTie ( which looks for alignments on both strands). Basically we're looking for a short read alignment algorithm that would take our directional data and align it ONLY with transcripts in the 5' -> 3' direction. Illumina is developing a directional mRNA-seq library prep method similar to the one we are using (without the purification of the 3' ends). I wonder if anyone has experience analyzing directional mRNA-seq data?

[HESmith]

Since your libraries are directional, you can use that information to resolve the ambiguity of overlapping 3' ends. The tag will be in the same 5'->3' direction as one of the two genes (conventionally indicated as +).

Harold

[Simon Anders]

Basically we're looking for a short read alignment algorithm that would take our directional data and align it ONLY with transcripts in the 5' -> 3' direction.

As you are already using Bowtie, have a look at its manual again, especially at the description of the option '--norc'. It causes Bowtie to do precisely this.

[qqtwee]

strand specific RNA-seq mapping

Does anyone know how do mapping for strand specific RNA-seq data? which tools can work well? what the parameter of tools for strand specific infomation of RNA-seq data?
Looking forward to your answer.
Thank you very much! Best wishes!

[jujubix]

Does anyone know how do mapping for strand specific RNA-seq data? which tools can work well? what the parameter of tools for strand specific infomation of RNA-seq data?
Looking forward to your answer.
Thank you very much! Best wishes!

Maybe TopHat with the '--library-type' option which will treat the reads in a strand specific manner as described in the manual?

[qqtwee]

Maybe TopHat with the '--library-type' option which will treat the reads in a strand specific manner as described in the manual?

Thank you for your reply! Do you know BWA has option that can treat reads in a strand specific ?

[jujubix]

Thank you for your reply! Do you know BWA has option that can treat reads in a strand specific ?

No, I do not know. Although the manual probably says no?

[stanikay]

Mappind strand specific RNAseq bacteria pair-end reads

Just following up on this thread, which mapper will be most ideal for mapping strand specific RNAseq bacteria pair-end reads,

I know tophat has the library type option but its mostly ideal for eukaryotic genomes.

Any thoughts will be appreciated.

Thanx