Question: converting RF strand-specific mRNA sequencing data to FR stand-specific
0
gravatar for upadhyay.maulik
4 months ago by
upadhyay.maulik0 wrote:

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?

rna-seq • 156 views
ADD COMMENTlink modified 3 months ago by swbarnes27.5k • written 4 months ago by upadhyay.maulik0

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?

ADD REPLYlink written 4 months ago by genomax80k

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.

ADD REPLYlink written 4 months ago by upadhyay.maulik0
1

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.
ADD REPLYlink modified 4 months ago • written 4 months ago by genomax80k
0
gravatar for swbarnes2
3 months ago by
swbarnes27.5k
United States
swbarnes27.5k wrote:

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

ADD COMMENTlink written 3 months ago by swbarnes27.5k
Please log in to add an answer.

Help
Access

Use of this site constitutes acceptance of our User Agreement and Privacy Policy.
Powered by Biostar version 2.3.0
Traffic: 1181 users visited in the last hour