SEQanswers

Go Back   SEQanswers > Sequencing Technologies/Companies > Illumina/Solexa



Similar Threads
Thread Thread Starter Forum Replies Last Post
tRNA and rRNA depletion ramsemm RNA Sequencing 1 06-15-2018 01:43 AM
RiboCop - An efficient way of rRNA depletion by Lexogen lexogen Vendor Forum 1 08-07-2016 11:13 PM
Best method/kit for rRNA depletion memento Illumina/Solexa 0 07-31-2013 06:49 PM
rRNA depletion samples partially degradated ValentinadM RNA Sequencing 1 02-01-2013 03:26 AM
tRNA/5S rRNA depletion mateo Sample Prep / Library Generation 4 01-26-2012 02:48 PM

Reply
 
Thread Tools
Old 05-07-2016, 04:59 PM   #1
chenyudr
Junior Member
 
Location: China

Join Date: Feb 2016
Posts: 7
Default a lot of multiple alignment reads after rRNA depletion

I met the situation of high % reads with multiple alignment sites. Our procedure is removing rRNA by Ribozero kit from ~1 microgram RNA isolated from rat liver tissue followed by standard RNA seq library construction. In some samples, we found a lot of reads, ~40-50%, has high similarity with rRNA and other ~20% reads with multiple alignment sites, of them most belong to 7sRNA.
We also checked bioanalyzer map of RNA after rRNA removal and found a ~140bp peak with with high FU value ~200. However we didn't check peaks around 18s and 28s positions in the map.
We speculated that the tissue has kind of specific features that Ribozero kit can't remove 7s RNA or SnRNA thus result in this multi-alignment situation.

Has this happened in your experiment? Could you give me some suggestion?

Many thanks,

Yuk
chenyudr is offline   Reply With Quote
Old 05-07-2016, 10:20 PM   #2
dpryan
Devon Ryan
 
Location: Freiburg, Germany

Join Date: Jul 2011
Posts: 3,480
Default

The ribozero kits are only ever "OK". We pretty commonly still have up to 20% rRNA (rarely higher, though it happens on occasion) and this always causes the multimapping issues you're seeing.
dpryan is offline   Reply With Quote
Old 05-08-2016, 09:31 PM   #3
chenyudr
Junior Member
 
Location: China

Join Date: Feb 2016
Posts: 7
Default

Yes, but we didn't find similar situation in samples from other tissues.
chenyudr is offline   Reply With Quote
Old 05-08-2016, 11:21 PM   #4
dpryan
Devon Ryan
 
Location: Freiburg, Germany

Join Date: Jul 2011
Posts: 3,480
Default

Then perhaps the kit is indeed missing something. Some tissues do have different rRNA composition (e.g., sperm), so it can happen.
dpryan is offline   Reply With Quote
Reply

Tags
multiple alignment, ribo-zero, rnaseq

Thread Tools

Posting Rules
You may not post new threads
You may not post replies
You may not post attachments
You may not edit your posts

BB code is On
Smilies are On
[IMG] code is On
HTML code is Off




All times are GMT -8. The time now is 05:37 PM.


Powered by vBulletin® Version 3.8.9
Copyright ©2000 - 2020, vBulletin Solutions, Inc.
Single Sign On provided by vBSSO