SEQanswers

Go Back   SEQanswers > Bioinformatics > Bioinformatics



Similar Threads
Thread Thread Starter Forum Replies Last Post
Low cluster Passing Filter Percentage justinjun Illumina/Solexa 2 06-01-2015 07:23 AM
viewing read map data from Subread/Rsubread akh22 Bioinformatics 5 04-29-2014 05:28 PM
Low mapping percentage with TopHat2 DerSeb RNA Sequencing 3 06-05-2012 06:35 AM
the low percentage of mapped reads wangli RNA Sequencing 2 04-04-2012 06:56 AM
low percentage of reads mapped rahilsethi SOLiD 3 09-13-2010 07:01 AM

Reply
 
Thread Tools
Old 03-06-2016, 04:42 AM   #1
kajot
Member
 
Location: Germany

Join Date: Dec 2013
Posts: 16
Default Subread low percentage of assigned fragments in ENCODE data

Hi there guys!


I am using Subread v1.5.0-p1 to get tables of counts for RNAseq data I got from ENCODE (https://www.encodeproject.org/experiments/ENCSR000BYX/).
What I am trying to get are counts for genes (using reads assigned to exons) since I want to ultimately turn the numbers into FPKM or TPM.

However, I am getting around 13% of successfully assigned reads:

|| Total fragments : 72502756 ||
|| Successfully assigned fragments : 9324091 (12.9%) ||
|| Running time : 2.20 minutes ||

This doesn't sound right to me, I am used to getting 50-75% of reads assigned in RNAseq, using HTseq + DESeq2. I don't expect ENCODE data to be of poor quality so I think it's something on my side.

I tried all the combinations of fr-firststrand, fr-secondstrand, rf-firststrand, rf-secondstrand etc. Nothing gives me higher values than those 13%.
Using GENCODE M3 annotation gtf since the data was originally aligned to mm9.
This is a paired-end library from Illumina GAIIx sequencer.

Any tips ?
kajot is offline   Reply With Quote
Old 03-06-2016, 02:40 PM   #2
shi
Wei Shi
 
Location: Australia

Join Date: Feb 2010
Posts: 235
Default

Could you show the content of the ".summary" file which was part of featureCounts output?
shi is offline   Reply With Quote
Old 03-07-2016, 06:40 AM   #3
kajot
Member
 
Location: Germany

Join Date: Dec 2013
Posts: 16
Default

I found the problem, I was using wrong GTF annotation, should've used GENCODE mouse M1 since vM3 is built on mm10...
kajot is offline   Reply With Quote
Reply

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 01:39 AM.


Powered by vBulletin® Version 3.8.9
Copyright ©2000 - 2022, vBulletin Solutions, Inc.
Single Sign On provided by vBSSO