SEQanswers

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



Similar Threads
Thread Thread Starter Forum Replies Last Post
MiSeq v3 2x300 run, Low Q30 ReGenMC Illumina/Solexa 16 02-13-2015 03:51 PM
MiSeq Reagent kit v3 exo Illumina/Solexa 15 01-14-2014 06:42 PM
Dual-index barcode read failures james hadfield Illumina/Solexa 12 12-16-2013 09:57 AM
MiSeq Reagent Kit v3 paolo.kunder Illumina/Solexa 4 09-22-2013 11:02 PM
miSeq failures during cluster generation greenhilly Illumina/Solexa 10 07-19-2012 08:25 AM

Reply
 
Thread Tools
Old 04-08-2016, 12:55 AM   #61
DRYTCYV
Member
 
Location: Portugal

Join Date: Apr 2011
Posts: 50
Default

Quote:
Originally Posted by lac302 View Post
My last run worked fine even overclustered at 1800k/mm2, 85%PF.
I got one of that cartridges

Our FAS said that Illumina wants to release "the new" V3 kit after Summer.
The last V3 kit we used as a V2 2x250 cycles, the difference is that the V3 got more tiles, so, more data even with the 2x250.

V2 kit its the best kit, in terms of quality and less sensible to overclustering. If Illumina change the V2 kit tiles from 28 to 38 tiles, it would be the best of both worlds.
DRYTCYV is offline   Reply With Quote
Old 04-08-2016, 07:09 AM   #62
dannyhi321
Member
 
Location: uk

Join Date: Jul 2014
Posts: 51
Default

Quote:
Originally Posted by lac302 View Post
Is this only an issue for amplicon sequencing? I haven't run a V3 600 cycle kit in a few months but have a library to sequence next week...WGS, FastQ only.

My last run worked fine even overclustered at 1800k/mm2, 85%PF.

I typically run the 600 cycle kits at 2x275. Quality over quantity is fine for my needs.
I think you will find your logic is unsound, a 2x300 kit that only "works" at 2x275 "sometimes" is not quality!

Do you think you could share the Q Score graphs? would be interesting!
dannyhi321 is offline   Reply With Quote
Old 01-06-2017, 09:28 PM   #63
Shimul
Junior Member
 
Location: Brisbane

Join Date: Jan 2017
Posts: 8
Default

Hello there,

I am running my bacterial library using Miseq V3. The Q30 is now 83.1% after 250 cycles, yesterday it was 96% after 70 cycles. But rest of the parameters are consistent: clusters PF 91.4%, cluster density 1092K/mm^2, which are the same as yesterday. Do you speculate anything? DO you think that the run will be still okay?

Many thanks.

Shimul
Shimul is offline   Reply With Quote
Old 01-06-2017, 09:56 PM   #64
Brian Bushnell
Super Moderator
 
Location: Walnut Creek, CA

Join Date: Jan 2014
Posts: 2,637
Default

Illumina's "Q30" values are completely fabricated; I suggest you ignore them. If you want to know the actual quality of your reads, you need to map them. For example:

bbmap.sh in=reads.fq ref=ref.fa mhist=mhist.txt qhist=qhist.txt qahist=qahist.txt

This will give you histograms of the actual quality of your reads.
Brian Bushnell is offline   Reply With Quote
Old 01-06-2017, 10:51 PM   #65
nucacidhunter
Senior Member
 
Location: Iran

Join Date: Jan 2013
Posts: 1,013
Default

Quote:
Originally Posted by Shimul View Post
Hello there,

I am running my bacterial library using Miseq V3. The Q30 is now 83.1% after 250 cycles, yesterday it was 96% after 70 cycles. But rest of the parameters are consistent: clusters PF 91.4%, cluster density 1092K/mm^2, which are the same as yesterday. Do you speculate anything? DO you think that the run will be still okay?

Many thanks.

Shimul
Run is fine and you would expect Q30 drop a bit more as cycle number increases. Read 2 quality will be lower than read 1 as well. Cluster density and PF% is calculated early in sequencing cycles and will not change.
nucacidhunter is offline   Reply With Quote
Old 01-10-2017, 12:17 PM   #66
jlli2000
Junior Member
 
Location: california

Join Date: Jan 2010
Posts: 2
Default Mixed different 16S metagenomics run

Does anyone have the experience of mixing different 16S 806R , 16S 926R and 18S libraries into one MiSeq run? How about mixing 16S onestep PCR libraries with two step PCR libraries?

Thanks,

Jin
jlli2000 is offline   Reply With Quote
Old 01-10-2017, 12:33 PM   #67
d00b
Member
 
Location: CA

Join Date: Jan 2009
Posts: 17
Default you can mix multiple amplicons

Hi Jin,

we do run multiple amplicons that labeled with different index primers.
However, we do always see index contamination in very low level.

We do two step library preparation.
1) amplification with Miseq adapter
2) Index amplification with different index combination

pool library based on realtime PCR result.

Last edited by d00b; 01-10-2017 at 12:35 PM.
d00b is offline   Reply With Quote
Old 01-11-2017, 05:58 AM   #68
thermophile
Senior Member
 
Location: CT

Join Date: Apr 2015
Posts: 192
Default

The biggest issue when mixing amplicons is the lenghts. I've successfully mixed 16s v4 (~300bp) with amplicons that are as small as 190bp and as large as 450 and run on 2x250. Your q30 plot will look weird, but I haven't had any of these mixed runs fail in a way that seems dependent on the mixing. Remember to calculate nM for each length and that the shorter frags will bind better than the longer ones.
__________________
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 01-19-2017, 07:40 AM   #69
Jessica_L
Senior Member
 
Location: Washington, D.C. metro area

Join Date: Feb 2010
Posts: 116
Default

My lab is looking at bringing in the TruSight Tumor 15 assay. It seems like most of the v3 issues are related to low complexity libraries (16s RNA-seq reads, etc.) so I'm assuming that we'll be okay with the TST15? I'm wondering if we'll run into problems with some of our other methods, though. We also run the Archer Dx FusionPlex for RNA Fusion detection, and the molecular barcodes in assay have a low complexity region in read 1.

Does anyone have any experience with TST15 or other assays (aside from 16s rRNA) on the v3 chemistry?

Thanks!
Jessica_L 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 03:49 PM.


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