RNA-seq library prep without poly-A selection

turnersd · 01-31-2012, 07:51 AM

Hello. I'm working on a project to examine gene expression in a system where RNA-processing has gone awry. I expect to see aberrant splicing, alternative transcription start site usage, aberrant poly-adenylation and capping, etc. RNA-seq is the perfect tool for looking at both differential expression and strange RNA-structural events.

But I don't want to do a poly-A selection because I'll probably lose all these odd transcripts that I'm most interested in that might not be properly polyadenylated, spliced, capped, etc. Yet I don't want to sequence a ton of rRNAs and tRNAs.

What kind of library preparation techniques should I investigate that don't require a poly-A selection?

Many thanks.

gringer · 01-31-2012, 08:35 AM

Quote:

Yet I don't want to sequence a ton of rRNAs and tRNAs.

What kind of library preparation techniques should I investigate that don't require a poly-A selection?

Given that RNA selection can be biased and will not necessarily remove all rRNAs, and current NGS sequencers give you oodles of data to play around with, I don't see a problem with sequencing the rRNA as well as other RNA.

Let's say you have 80% rRNA in your sample, and 80% mappable reads. The amount of mappable non-rRNA sample will be about 16% of the original reads, so if you've got 100M single-end reads then that's 16M non-rRNA reads. With 50bp reads, that's a mean coverage of ~7x for a 120Mb transcriptome (vs ~33x for a "perfect" rRNA extraction).

apratap · 01-31-2012, 09:27 AM

One way to do it is two extract RNA in two steps. 1.) Capture Poly-A tail 2.) Perform ribosomal depletion on the leftover sample and the leftover should contain Poly A-.

When we do this we still see about 70-80% of the #2 sample still contain rRNA/tRNA.

I would be interested in knowing if others can get a cleaner sample and methods used.

Thanks!
-Abhi

Gustavo · 01-31-2012, 11:21 PM

Consider using duplex-specific nuclease (DSN):
http://www.ncbi.nlm.nih.gov/pubmed?t...fic%20nuclease

pbluescript · 02-01-2012, 04:51 AM

If you don't get rid of the rRNA, you might miss some of the rare/odd transcripts you mentioned you wanted to look for. Size selection is enough to get rid of tRNAs, which are generally under 100bp long. For rRNAs, I would use a ribosome depletion kit. Getting rid of those reads would be very useful in getting the depth you might need to see aberrant transcripts.

mediator · 02-01-2012, 09:48 AM

You can just do two rounds of rRNA removal. I've used invitrogen's ribominus, cleared more than 90% of rRNA in first round (confirmed by real time PCR of 18s).

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01-31-2012, 07:51 AM	#1
turnersd Senior Member Location: Charlottesville, VA Join Date: May 2011 Posts: 112	RNA-seq library prep without poly-A selection Hello. I'm working on a project to examine gene expression in a system where RNA-processing has gone awry. I expect to see aberrant splicing, alternative transcription start site usage, aberrant poly-adenylation and capping, etc. RNA-seq is the perfect tool for looking at both differential expression and strange RNA-structural events. But I don't want to do a poly-A selection because I'll probably lose all these odd transcripts that I'm most interested in that might not be properly polyadenylated, spliced, capped, etc. Yet I don't want to sequence a ton of rRNAs and tRNAs. What kind of library preparation techniques should I investigate that don't require a poly-A selection? Many thanks.

01-31-2012, 09:27 AM	#3
apratap Member Location: Bay Area Join Date: Jan 2009 Posts: 58	One way to do it is two extract RNA in two steps. 1.) Capture Poly-A tail 2.) Perform ribosomal depletion on the leftover sample and the leftover should contain Poly A-. When we do this we still see about 70-80% of the #2 sample still contain rRNA/tRNA. I would be interested in knowing if others can get a cleaner sample and methods used. Thanks! -Abhi

01-31-2012, 11:21 PM	#4
Gustavo Junior Member Location: Seattle Join Date: Jun 2010 Posts: 4	Consider using duplex-specific nuclease (DSN): http://www.ncbi.nlm.nih.gov/pubmed?t...fic%20nuclease

02-01-2012, 04:51 AM	#5
pbluescript Senior Member Location: Boston Join Date: Nov 2009 Posts: 224	If you don't get rid of the rRNA, you might miss some of the rare/odd transcripts you mentioned you wanted to look for. Size selection is enough to get rid of tRNAs, which are generally under 100bp long. For rRNAs, I would use a ribosome depletion kit. Getting rid of those reads would be very useful in getting the depth you might need to see aberrant transcripts.

02-01-2012, 09:48 AM	#6
mediator Member Location: New England Join Date: Nov 2010 Posts: 27	You can just do two rounds of rRNA removal. I've used invitrogen's ribominus, cleared more than 90% of rRNA in first round (confirmed by real time PCR of 18s).