SEQanswers

Go Back   SEQanswers > Applications Forums > RNA Sequencing



Similar Threads
Thread Thread Starter Forum Replies Last Post
Brainstorming "ddRAD-BiS" lukeSeq Sample Prep / Library Generation 4 11-07-2015 06:03 PM
Error in installation of MAP assembler amitbik Bioinformatics 0 08-05-2014 08:26 PM
Sources of error in NGS and importance of replicates Genohub Service Providers 0 12-12-2013 10:14 PM
Maq map error fc35802 Bioinformatics 0 03-21-2012 02:47 AM

Reply
 
Thread Tools
Old 07-22-2016, 02:31 AM   #1
Thias
Member
 
Location: Münster, Germany

Join Date: Mar 2013
Posts: 42
Default Brainstorming: Error sources for Cuffquant failure: Map Mass: 0.00

Hello folks,

I have an issue with Cuffquant v2.2.1, which fails to quantify the expression (create .cxb files) of the current RNA-seq I am working on:

Code:
 
Inspecting maps and determining fragment length distributions.
Map Properties:
Normalized Map Mass: 0.00
Raw Map Mass: 0.00
Number of Multi-Reads: 0 (with 0 total hits)
Fragment Length Distribution: Truncated Gaussian (default)
Default Mean: 200
Default Std Dev: 80
This is the first paired-end sequenced RNA-seq, which I try to process, however I have a working pipeline for some older single-end Illumina data.

I have doublechecked / considered those error sources and would like to ask you, if you are aware of other possible reasons:
  • Reads: Initially quality trimmed, but after I read here in the forum, that this might confuse cufflinks when determining insert sizes, I also aligned the untrimmed reads (adaptors cut) with no luck either.
  • Aligner: bbmap 34.41, but I set the options xstag=T and xmtag=T, which is supposed to generate bamfiles suitable for Cufflinks.
  • Alignments are indexed. There are clear signals at the positions of the transcripts visible in bedgraph.
  • Reference genome and GTF-file: Both have been used for multiple alignments and correctly quantified SE-RNA-seqs. Chromosome names do match. I also unsuccessfully tried another cufflinks-usable GTF-file from a collaborating bioinformatician.
  • Although I believe fr-unstranded is the correct library type for the experiment, I tried all three one by one.
  • I installed and tried cufflinks v. 2.2.0 instead of v.2.2.1. Both versions successfully were able to requantify a previous RNA-seq and are hence working (same aligner, reference genome and gtf file used)

I would appreciate any suggestions, what I could still try. I am out of ideas at the moment...

Thanks for your help
Matthias

Last edited by Thias; 07-22-2016 at 02:33 AM.
Thias is offline   Reply With Quote
Reply

Tags
cufflinks, cuffquant, map mass, paired-end, paired-end sequencing

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 05:49 PM.


Powered by vBulletin® Version 3.8.9
Copyright ©2000 - 2018, vBulletin Solutions, Inc.
Single Sign On provided by vBSSO