Question

resequencing RNAseq single end reads (Source: Bacteria)

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6.5 years ago

Annie • 0

Hi everyone, I am new to NGS data analysis. I have got RNAseq data of read length 50, and single end, which I need to do resequence with the reference genome of Mycobacterium tuberculosis. first thing I did is, I ran Rockhopper, which gave me transcripts.txt and operon.txt files. Now I want to do resequencing using following workflow : Alignment (by bowtie)--> Cufflinks --> Cuffmerge --> Cuffdiff. I am stuck in cufflinks step where I need to give the normalization method, which according to me should be RPKM, but in the help manual it is showing that it supports classic FPKM. Please help me with the command line and further steps...

Thanks!!

RNA-Seq Resequencing MTB Cufflinks RPKM • 1.4k views

ADD COMMENT • link 6.5 years ago by Annie • 0

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Thanks for swift reply, but my strain is H37Rv. will it be good to go with featureCounts?

ADD REPLY • link 6.5 years ago by Annie • 0

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The strain is irrelevant.

ADD REPLY • link 6.5 years ago by Devon Ryan 105k

score 2 · Answer 1 · 2018-07-06

FPKM and RPKM are the same for single end reads. However, you should not be using cufflinks, cuffmerge or cuffdiff. If there's no annotation for this species or if it's of poor quality then you can use stringTie, but otherwise you would be much better off with featureCounts and the known annotation of this species.

Note that FPKM/RPKM should never be used for anything important.