SEQanswers

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



Similar Threads
Thread Thread Starter Forum Replies Last Post
is sequencing cost still dropping? xyzzy123 General 20 10-19-2015 07:59 AM
Cufflinks Dropping Transcripts ercfrtz Bioinformatics 6 11-12-2012 09:35 AM
SHRiMP with FASTAQ input CS Student Bioinformatics 1 03-15-2012 12:39 PM
please help with .fastaq and .sam format rnaeye Bioinformatics 6 02-01-2012 08:58 AM
fastaq to csfasta and associated QVs Sheila SOLiD 2 04-06-2010 12:15 AM

Reply
 
Thread Tools
Old 01-24-2017, 10:26 AM   #1
s_farraj
Junior Member
 
Location: Saudi Arabia

Join Date: May 2012
Posts: 3
Default Dropping in QC of FASTAQ

Hi all,
We have MiseqDX and we upgraded it to be compatible with 600cycles kit.
We made after that 2 runs:
The first one was for 16srRNA library following the recommended protocol from illumina (It doesn't depend on any library preparation kit). We used 600cycles kit.
The second run is for whole genome sequencing for different species (viruses & bacteria). We used Nextera XT kit with 300cycle kit.
In both runs when we check the QC for the fastaq files we found a systematic dropping in all samples. I mean by dropping, the quality score reduced to near 2 then returns back to 37 in all the samples in the run (the pictures attached for the QC for one of the samples in each run, the rest samples are the same).
The dropping in the first run was in R1 from cycle 5 to cycle 20.
In the second run, The dropping was in R2 from cycle 30 to 75 cycle.

Did anyone face problem like this?
Is there any explanation for this situation?

Thanks
Attached Images
File Type: png 16s R2.png (14.7 KB, 9 views)
File Type: png 16s R1.png (14.5 KB, 6 views)
File Type: png S4 R1.png (11.2 KB, 3 views)
File Type: png S4 R2.png (11.4 KB, 5 views)
s_farraj is offline   Reply With Quote
Old 01-24-2017, 10:32 AM   #2
GenoMax
Senior Member
 
Location: East Coast USA

Join Date: Feb 2008
Posts: 6,574
Default

You must have a region of low nucleotide diversity in that region in the middle (e.g. A's in majority of clusters at that cycle) which leads to these drops in qualities. Can you post nucleotide distribution plot from FastQC?

What version chemistry is this? What is cluster density and PF%? Was phiX spiked in and at what concentration?

Last edited by GenoMax; 01-24-2017 at 10:47 AM.
GenoMax is offline   Reply With Quote
Old 01-24-2017, 11:07 AM   #3
s_farraj
Junior Member
 
Location: Saudi Arabia

Join Date: May 2012
Posts: 3
Default

Thanks for your quick response.
I attached the Fasta QC report for the sample for the second run and pic for its data.
This run didn't have Phix.
But like I said in the previous post, this was for whole genome sequences for different species.
This one was V2 but the first run was with V3.
Attached Images
File Type: png Picture1.png (101.6 KB, 14 views)
Attached Files
File Type: pdf S4 R2.pdf (1.08 MB, 12 views)
s_farraj is offline   Reply With Quote
Old 01-24-2017, 11:21 AM   #4
thermophile
Senior Member
 
Location: CT

Join Date: Apr 2015
Posts: 201
Default

check your thumbnails. I'd guess that your density is actually much higher than 1300-genomes should have very high PF
__________________
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 01-25-2017, 06:22 AM   #5
s_farraj
Junior Member
 
Location: Saudi Arabia

Join Date: May 2012
Posts: 3
Default

Thanks for your answer
Today the engineering come and check these runs and he said its over clustered
I think I will reduce the conc. of pooling library from 10pM to 6pM hoping avoid this dropping issue.
s_farraj 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 10:02 AM.


Powered by vBulletin® Version 3.8.9
Copyright ©2000 - 2017, vBulletin Solutions, Inc.
Single Sign On provided by vBSSO