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

Similar Threads
Thread Thread Starter Forum Replies Last Post
low Q30 in index, miseq v3 600 cnano Illumina/Solexa 3 06-05-2019 01:43 PM
Tandem dual index- low Q30 for index 2 juliar Illumina/Solexa 1 02-18-2019 09:25 AM
Illumina MiSeq Poor Q30 scores in Read 1 relative to Read 2 jreuther Illumina/Solexa 4 02-23-2018 05:45 AM
Index read Q30 problems on NextSeq Vesperholly2 Illumina/Solexa 5 11-06-2015 04:32 AM
MiSeq v3 2x300 run, Low Q30 ReGenMC Illumina/Solexa 16 02-13-2015 04:51 PM

Thread Tools
Old 11-21-2019, 12:59 AM   #1
Junior Member
Location: South Africa

Join Date: Jan 2016
Posts: 3
Default MiSeq - issue with index and read 2 (Low Q30)

I recently had a MiSeq run fail and am not 100% sure what caused it.

Here are some details:
Amplicon sequencing run of RNA FFPE samples (125 genes) using a MiSeq V3 (600 cycle) kit loaded at 14 pM. Cluster Density is 909 K/mm2.

Phix spiked in at 15% and sequencing run configured to 131 x 2 paired-end.

SAV Data by Cycle, %Q30:

SAV Run summary:

I am leaning towards reagent failure, incorrect run parameters or an issue with the indices.

I would appreciate any help.

Ross_Mcf is offline   Reply With Quote
Old 11-21-2019, 04:59 AM   #2
Junior Member
Location: Slovenia

Join Date: Sep 2019
Posts: 1

Seeing as Read 1 was OK (not great, not terrible) and the issues started after that, it might be reagent related or an issue with the system fluidics/optics.

I do not think indexes or run parameters might be a factor here. Just from the Q30 plot the libraries seem to be fine, but %Base from SAV or Bioanalyzer images might help to confirm that.

I think it might be best to show the run to Illumina, a competent support might look at the SAV data (%Base, FWHM) and figure out the problem quickly.
sifekonja is offline   Reply With Quote
Old 11-21-2019, 06:09 AM   #3
Junior Member
Location: South Africa

Join Date: Jan 2016
Posts: 3

Thanks for the answer @sifekonja!

I initially thought the failure might be due to low library or barcode diversity. This is copied from the library prep protocol: "The Illumina sequencers will work best when index diversity within a run is high. For example, if eight samples are included in a run, and the user chooses to use only one MBC Adapter paired with eight different MiSeq Index 1 Primers, the run may fail due to low barcode diversity. In this example it is best to use eight different Archer MBC Adapters paired with eight different MiSeq Index 1 Primers"

My question then would be why is the reverse read so badly affected?

It definitely also looks like it could be a reagent issue.
Ross_Mcf is offline   Reply With Quote
Old 11-21-2019, 06:17 AM   #4
Senior Member
Location: UK

Join Date: Jan 2010
Posts: 390

With a 15% PhiX spike in, the last thing it can be described as is 'low diversity'. In fact that's a very high spike in level for more recent MiSeq sequencing protocols for low diversity libraries. Cluster density is low, but in general providing it isn't 'too low' then you should have pretty good quality data. Index diversity I'm not so sure about index diversity, normally you should just follow the guides to add the appropriate diversity in your indexes.. quite spelled out in the Illumina protocols at least.

Contact Illumina support as suggested. If it's a reagent failure, then depending on the nature of the MiSeq support contract, you'll get free reagents to run again.

With regards to why the second read might be affected, remember the order that things are actually sequenced in and what they represent. Read 1, Read 2, Read 3 and Read 4 are in your metrics. Your sequencing reads are Reads 1 and 4 in this context. It does look like the quality tanks on the second index read in particular.

Last edited by Bukowski; 11-21-2019 at 06:24 AM.
Bukowski is offline   Reply With Quote
Old 11-21-2019, 06:22 AM   #5
Junior Member
Location: South Africa

Join Date: Jan 2016
Posts: 3

Hi @Bukowski, thanks for your feedback.

While I fully agree regarding the 15% PhiX and low diversity library I was more concerned with the statement from the protocol about the indices. I will be getting in touch with Illumina.
Ross_Mcf is offline   Reply With Quote
Old 11-25-2019, 09:46 AM   #6
Location: Atlanta

Join Date: Nov 2018
Posts: 16

I had a issue with the NextSeq where the Q30 score for read 2 looked horrible and it was determined to be a pump failure.
itstrieu 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 07:43 PM.

Powered by vBulletin® Version 3.8.9
Copyright ©2000 - 2019, vBulletin Solutions, Inc.
Single Sign On provided by vBSSO