Cufflinks And Solid 5500 Rnaseq Bam Files

Question

[Ashutosh Pandey]

Hello everyone,

I have RNAseq data generated on SOLiD 5500 machine. I used Lifescope mapping tool to align the reads against the reference genome and got bam files. Now, I want to use Cufflinks for the further analysis. My RNAseq data is 50 bp fragment reads (single/unpaired) and I am not sure which library type I should use. Here are the options in Cufflinks:

Library Type

fr-unstranded (default) Standard Illumina Reads from the left-most end of the fragment (in transcript coordinates) map to the transcript strand, and the right-most end maps to the opposite strand.

fr-firststrand dUTP, NSR, NNSR Same as above except we enforce the rule that the right-most end of the fragment (in transcript coordinates) is the first sequenced (or only sequenced for single-end reads). Equivalently, it is assumed that only the strand generated during first strand synthesis is sequenced.

fr-secondstrand Directional Illumina (Ligation), Standard SOLiD Same as above except we enforce the rule that the left-most end of the fragment (in transcript coordinates) is the first sequenced (or only sequenced for single-end reads). Equivalently, it is assumed that only the strand generated during second strand synthesis is sequenced.

I assume I should be using the fr-secondstrand. But i just want to make sure?

Thanks a lot.

Answer 1

[JC]

because your data is single-ends you don't need to define the --library-type option, that's for paired-end reads.

[Damian Kao]

That is true. However SOLiD reads retain strandness throughout the library prep. Does cufflinks assume unstranded reads as default in single read assemblies? I wonder if defining a library type will allow Cufflinks to take advantage of that strandness even in single read assemblies.