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3 votes

Discrepancy with featurecounts analysis using a forward stranded and reverse stranded protocol

This kit uses the dUTP method to preserve strandedness. That (to my knowledge) always produces a reverse library. Not sure where you have your information of a forward library from, but we used this ...
ATpoint's user avatar
  • 1,430
3 votes
Accepted

Why is there antisense sequence in RNAseq data

Yes, the mRNA results should include CDSs (not only, don't forget you also have UTR sequences which are non-coding so are not part of the CDS), but there is no reason why they should all be from genes ...
terdon's user avatar
  • 10.2k
3 votes
Accepted

How to detect bedtools version used by pybedtools (in order to correctly preserve strand information when merging gtf records)

pybedtools assumes that bedtools is in your path and bedtools itself will return the version with bedtools --version. So: ...
Devon Ryan's user avatar
  • 19.7k
2 votes

HISAT2: RNA strandedness

According to the help produced when I type hisat2 --help, the default strandedness when running HISAT2 is unstranded: ...
gringer's user avatar
  • 14.3k
2 votes

At what processing step should library strandedness type be taken into account?

I usually take into account library strandness during the mapping to the reference step (but I never worked with bigwig files myself). Assuming RNA-seq data from Illumina, you can use Hisat2 for ...
aechchiki's user avatar
  • 2,676
2 votes

At what processing step should library strandedness type be taken into account?

If your aligner has a strandedness option then go ahead and use it. The general idea here being that you'll preferentially align correctly to genes. Whether to bother with strandedness here depends on ...
Devon Ryan's user avatar
  • 19.7k
1 vote

At what processing step should library strandedness type be taken into account?

Firstly, right at the start if the experiment it's important that your RNA samples are processed with a strand-specific protocol (e.g. Illumina's TruSeq Stranded) in order to produce stranded ...
ithinkiam's user avatar
  • 356
1 vote

Does rRNA depletion protocol give higher number of mapped reads in Intronic regions?

It's not so much that you have "intronic contamination" or "genomic contamination", rather you're not selecting explicitly for full-length mature transcripts with rRNA depletion. ...
Devon Ryan's user avatar
  • 19.7k
1 vote

At what processing step should library strandedness type be taken into account?

Strandedness should be taken into account at the mapping stage. If you are presented with only a BAM file, ask for the raw reads. If that's not possible, you can recover the reads (as fastq sequences)...
gringer's user avatar
  • 14.3k

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