SEQanswers

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



Similar Threads
Thread Thread Starter Forum Replies Last Post
2x300 MiSeq read 2 failures due to reagent? ksawatzki Illumina/Solexa 68 01-19-2017 07:40 AM
MiSeq run with low Q30 and high cluster PF haroldnunez Illumina/Solexa 12 01-06-2017 06:34 AM
low Q30 in index, miseq v3 600 cnano Illumina/Solexa 2 03-14-2016 02:54 PM
MiSeq Run with low Cluster PF Help dross11 Illumina/Solexa 13 03-23-2015 08:01 AM
MiSeq v3 2x300 run, Low Q30 ReGenMC Illumina/Solexa 16 02-13-2015 03:51 PM

Reply
 
Thread Tools
Old 03-26-2018, 11:30 AM   #21
SarahAurora
Member
 
Location: Bonn Germany

Join Date: Jun 2017
Posts: 17
Default

Quote:
Originally Posted by GW_OK View Post
Onboard diagnostics are in the "System Check" icon under "Manage Instrument" from the main screen on the Miseq Control Software. The optics checks are fairly fast to run, the fluidics will take some time and babysitting (raising and lowering sippers as prompted).

Has your partner had any successful runs since your failed run?
No, we are the only one using the MiSeq. We already used it for 8 runs, but the ninth one was now the fatal one....
SarahAurora is offline   Reply With Quote
Old 03-26-2018, 11:52 AM   #22
SarahAurora
Member
 
Location: Bonn Germany

Join Date: Jun 2017
Posts: 17
Default

Quote:
Originally Posted by GenoMax View Post
If this failure is due to hardware/reagent problem (and the partner has a maintenance contract with Illumina) then Illumina generally will replace the reagents at no cost, so you should be able to re-run your samples for free (or at least no charge for the reagents).
That would be great!
SarahAurora is offline   Reply With Quote
Old 03-27-2018, 08:49 AM   #23
thermophile
Senior Member
 
Location: CT

Join Date: Apr 2015
Posts: 233
Default

I run primarily amplicons too so the metrics that I look for are a bit different than the standards that Illumina uses (our runs don't meet the base diversity that makes some of the standard number make sense).

From the %q30 it looks like something bad happened part way through R1-amplicons aren't really long enough, optics issue, fluidics issue. Whatever it was *seems* to be machine/reagent related because it didn't improve after the turnaround. Are you spiking in primers or do your amplicons use Illumina sequencing primers? If you make an error spiking in the primers, you won't see anything after the turn around.

My list to check (roughly in order)

1. %phiX aligned. is it close to what I thought I was spiking in?
2. %base does it look appropriately horrible (my 16s amplicons should be at 90% for the first ~50 bases, then decline to ~80% for the rest of the read)
3. Cluster density across flowcell, top and bottom
4. thumbnails, does the density seem about right
5. fwmh. The software is not made for 90% single base, focus issues are common, but fwmh shouldn't be too jagged
__________________
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

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 03:43 AM.


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