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

Similar Threads
Thread Thread Starter Forum Replies Last Post
Miseq v3 600 cycle kit - poor read 1 quality incognitos Illumina/Solexa 3 11-09-2016 10:46 AM
Index read Q30 problems on NextSeq Vesperholly2 Illumina/Solexa 5 11-06-2015 04:32 AM
MiSeq: Sudden drop in %> Q30 halfway read 1 vehuardo Illumina/Solexa 3 09-18-2015 06:08 AM
Poor Nextera XT index read quality on Miseq ustar Illumina/Solexa 2 07-22-2014 08:12 AM
Illumina MiSeq read orientiation Puhekupla Bioinformatics 4 06-19-2014 04:08 AM

Thread Tools
Old 10-03-2017, 02:16 PM   #1
Junior Member
Location: houston

Join Date: Oct 2014
Posts: 2
Default Illumina MiSeq Poor Q30 scores in Read 1 relative to Read 2

Hi everyone,
We are consistently seeing an issue on our MiSeq where Read 1 has very poor Q30 scores, followed by a successful sequencing of Read 2 with Q30 scores above 80%. We have had successful runs where we didn't see this trend but it seems about every other run we see this happening. We have talked with Illumina Tech Support and they don't seem to know what is going on. They adjusted the temperature of the instrument (set it a few degrees lower) but this didn't seem to fix the issue. Has anyone else had similar issues? Any insight would be much appreciated.
Thanks! See below for run specifics:

Chemistry: Miseq v3 600 cycle kit.
Read configurations: R1 = 150 cycles, Index = 16 cycles, R2 = 150 cycles.

The library is a DNA hybridization capture, with avg library length ~320 bases

The library was run with 10% PhiX spike-in.

Custom primers were NOT used.
Attached Images
File Type: png V3 poor Q30 scores in R1.png (23.0 KB, 18 views)
jreuther is offline   Reply With Quote
Old 10-03-2017, 06:30 PM   #2
Senior Member
Location: US

Join Date: Dec 2010
Posts: 318

Indeed very weird. Fortunately we have not seen this problem so far.
Are you using custom sequencing primers?
luc is offline   Reply With Quote
Old 10-03-2017, 10:18 PM   #3
Senior Member
Location: Iran

Join Date: Jan 2013
Posts: 1,096

Two possible causes:
1- Instrument related
2- Library P5 adapter

In case2 the adapter sequences could have variations from standard sequences which affect R1 primer annealing or binding stability resulting in low quality. Paying attention to phasing/prephasing in good and bad runs should highlight some differences. This case is more prevalent in PCR amplicon based library preps that adapters are added by PCR because of the possibility that some oligos will have indels or base substitutions resulting from low quality synthesis or storage.
nucacidhunter is offline   Reply With Quote
Old 10-24-2017, 04:39 PM   #4
Junior Member
Location: california

Join Date: Jan 2010
Posts: 4

Does anyone have experience to run Miseq V3 600 kit with uneven r1 and r2 cycles? i.e. r1 400 and r2 200?


jlli2000 is offline   Reply With Quote

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 07:38 PM.

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