SEQanswers

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



Similar Threads
Thread Thread Starter Forum Replies Last Post
Error while running bcl2fastq saupra Bioinformatics 9 12-13-2016 07:22 AM
Comparing Cassava1.8.2 to bcl2fastq DrWorm Bioinformatics 5 03-30-2016 06:32 PM
bcl2fastq on windows? jdb Illumina/Solexa 4 10-13-2015 04:25 AM
How filter bcl2fastq reads ? ClemBuntu Bioinformatics 2 01-29-2015 11:54 PM
install bcl2fastq-1.8.4 min1204 Bioinformatics 17 07-23-2014 04:37 AM

Reply
 
Thread Tools
Old 12-13-2016, 07:39 AM   #1
thermophile
Senior Member
 
Location: CT

Join Date: Apr 2015
Posts: 233
Default amplicon runs using bcl2fastq v217

Has anyone noticed issues with quality scores on amplicon runs since the new version of bcl2fastq came out? I've had a number of runs be questionable since late Oct, but I also changed machines at the same time. Trying to figure out if I need to adjust wetlab conditions to new machine or if it's related to the software update.
__________________
Microbial ecologist, running a sequencing core. I have lots of strong opinions on how to survey communities, pretty sure some are even correct.
thermophile is offline   Reply With Quote
Old 12-13-2016, 07:46 AM   #2
GenoMax
Senior Member
 
Location: East Coast USA

Join Date: Feb 2008
Posts: 6,764
Default

bcl2fastq v.2.x uses binned Q-scores. I assume you are referring to that here as "issues"? Have you seen this paper?

@Brian has a way to re-calibrate those using BBMap. There should be a post on here that describes how to do that.
GenoMax is offline   Reply With Quote
Old 12-13-2016, 07:49 AM   #3
thermophile
Senior Member
 
Location: CT

Join Date: Apr 2015
Posts: 233
Default

The beginning of R2 qscores are really low. This last run, R1 looks great with ~10% phiX aligned. R2 is horrible, <1% phiX aligned. I'm not sure how this would be software related but the thumbnails look fine-the machine didn't lose focus, clusters are there.

oh hadn't seen that paper, thanks. Haven't used bbmap but know it's on one of our clusters
__________________
Microbial ecologist, running a sequencing core. I have lots of strong opinions on how to survey communities, pretty sure some are even correct.
thermophile is offline   Reply With Quote
Old 12-13-2016, 07:59 AM   #4
thermophile
Senior Member
 
Location: CT

Join Date: Apr 2015
Posts: 233
Default

The qscore profiles look similar to what I was seeing at the beginning of the year. But the machine that I'm currently using is the one that actually lead us to blame the TCM of my old machine
http://seqanswers.com/forums/showthread.php?t=65825
__________________
Microbial ecologist, running a sequencing core. I have lots of strong opinions on how to survey communities, pretty sure some are even correct.
thermophile is offline   Reply With Quote
Old 12-13-2016, 08:40 AM   #5
kmcarr
Senior Member
 
Location: USA, Midwest

Join Date: May 2008
Posts: 1,147
Default

Quote:
Originally Posted by thermophile View Post
Has anyone noticed issues with quality scores on amplicon runs since the new version of bcl2fastq came out? I've had a number of runs be questionable since late Oct, but I also changed machines at the same time. Trying to figure out if I need to adjust wetlab conditions to new machine or if it's related to the software update.
Are you sure the issue is with bcl2fastq? QScores are assigned (binned or not) by RTA. bcl2fastq merely extracts the scores already stored in the BCL file by RTA.
kmcarr is offline   Reply With Quote
Old 12-13-2016, 08:41 AM   #6
thermophile
Senior Member
 
Location: CT

Join Date: Apr 2015
Posts: 233
Default

not sure of anything. so maybe rerunning older version of bcl2fastq isn't going to change anything?
__________________
Microbial ecologist, running a sequencing core. I have lots of strong opinions on how to survey communities, pretty sure some are even correct.
thermophile is offline   Reply With Quote
Old 12-13-2016, 12:53 PM   #7
thermophile
Senior Member
 
Location: CT

Join Date: Apr 2015
Posts: 233
Default

talked to tech support, bcl2fastq is very unlikely to be the issue.
__________________
Microbial ecologist, running a sequencing core. I have lots of strong opinions on how to survey communities, pretty sure some are even correct.
thermophile is offline   Reply With Quote
Reply

Tags
amplicon, bioinformatics, microbiome, miseq

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 07:13 PM.


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