converting RF strand-specific mRNA sequencing data to FR stand-specific

Question

[upadhyay.maulik]

Dear all,

We have carried out stand-specific mRNA sequencing (using the Illumina paired end protocol) of five tissues. For four tissues, we used FR strand specific library, while for the remaining tissue we used RF strand specificity, now my query is how can I change the strand specificity of the sample from RF to FR? Can I just replace the tag "/2" to "/1" and vice-versa to convert data obtained from RF library? Or should I also carry out the reverse complement of the reads obtained using RF library? Moreover, we also have single-end mRNA sequencing data from the same tissues. And I plan to merge this paired-end and single-end sequencing data to carry out transcriptome assembly. So following is the approach that I am planning to apply: 1). Carry out separate filtering of paired-end and single-end sequencing data, 2). Reverse complement the reads with tag "/2" after which merge all the paired-end and single-end data and run the trinity algorithm assuming single end data with strand information as F. Is this the right approach?

[GenoMax]

For four tissues, we used FR strand specific library, while for the remaining tissue we used RF strand specificity

Can you clarify what you mean by this?

[upadhyay.maulik]

For four tissues we used strand specific protocol that generated FR orientation for mRNA seq reads, while for one tissue we used the protocol that generated RF orientation for mRNA seq reads.

[GenoMax]

You actually used a different kit/protocol for one sample. That is puzzling and is likely to cause a batch effect. If you know what you need to do (just reverse or reverse complement the read) then you can use reformat.sh from BBMap suite to change the orientation of your reads.

rcomp=f                 (rc) Reverse-complement reads.
rcompmate=f             (rcm) Reverse-complement read 2 only.
comp=f                  (complement) Reverse-complement reads.

Answer 1

[swbarnes2]

Don't do that. Just process each sample properly.