This is a test version of Biostars. For the public version, visit https://www.biostars.org.
What is the best way to handle unmapped reads from RNA-Seq data

I have used tophat2 to map rna-seq reads to a draft genome. The alignment percentage is around 75-80% for all samples. When I take the unmapped reads and blast them, they hit the same organism, indicating the unmapped reads might have potential information. How do I deal with the unmapped reads and include them in DE analysis or any other downstream analysis ? Should I go with entirely different pipeline like trinity ?

rna-seq tophat2 trinity
• 6,772 views • link
updated by Ram Ram • written by GouthamAtla GouthamAtla
maybe allow a few more mismatches with tophat?
• 0 views • link
written by Martombo Martombo

thanks. but I thinks Its more about incomplete genome rather than alignment problem.

• 0 views • link
written by GouthamAtla GouthamAtla

2 answers

I have tried STAR and the mapping percentage increased up to 90-92% ( with tophat2, it was only up to 75-85%). I will try BBMap soon.

• 0 views • link
written by GouthamAtla GouthamAtla

I suggest using a more sensitive aligner (BBMap), so you have fewer unmapped reads and thus less bias.

• 0 views • link
written by Brian Bushnell Brian Bushnell

Okay. I will try that.

• 0 views • link
written by GouthamAtla GouthamAtla

Note that BBMap has a parameter "maxindel" which defaults to "maxindel=16000". This is fine for plants, fungi, and microbes, but if you are sequencing vertebrates (or anything else with introns longer than ~16kbp) you should increase it to about the 98th percentile of intron length in that organism (in mammals this means around 100kbp to 200kbp). All other parameters can be left as default.

• 0 views • link
updated by Ram Ram • written by Brian Bushnell Brian Bushnell

Hi, is BBMap output comparable with cufflinks/StringTie ?

• 0 views • link
written by GouthamAtla GouthamAtla

It's sam, so you can conver it and sort it with bam. By the way, did you trim your reads for quality?

• 0 views • link
written by apelin20 apelin20

For Cufflinks, you should add the flag xs=firststrand or whatever because Cufflinks needs that, and intronlen=10 to make introns in cigar strings printed as 'N' instead of 'D'. If samtools is installed, BBMap can directly output bam files rather than sam files, if you name the output file something.bam.

• 0 views • link
updated by Ram Ram • written by Brian Bushnell Brian Bushnell

Log in to answer this question.

More posts like this