SEQanswers

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



Similar Threads
Thread Thread Starter Forum Replies Last Post
RNA-seq in the NovaSeq scrosby RNA Sequencing 2 03-09-2018 08:19 AM
NovaSeq Experience nangel Illumina/Solexa 26 11-02-2017 09:15 PM
NovaSeq from Illumina GenoMax Illumina/Solexa 113 08-03-2017 03:51 AM
Puzzing MiSeq run (failure) Vesperholly2 Illumina/Solexa 1 11-15-2014 12:44 PM
MiSeq run failure - inhibition? firefly2280 Illumina/Solexa 24 11-14-2014 07:13 PM

Reply
 
Thread Tools
Old 11-01-2018, 02:18 AM   #1
Tcom111
Junior Member
 
Location: Israel

Join Date: May 2012
Posts: 3
Default Novaseq vs Miseq failure

Hi all,

This is the first time I'm trying to run a MIP (molecular inversion probe) library of ~800 genomic targets, dual index library in Novaseq.
The primers we use are custom primers for all reads (R1, R2, Ind). The Miseq run of the same pool looked like this (Miseq1, Miseq2 figures), while there was a fall in the first 50 bases of both R1 and R2 in the Novaseq run (Novaseq1, Novaseq2). Has anyone had any encounter with such phenomena and can suggest how to solve it? I heard an explanation of low hybridization efficiency of the custom primers in the Novaseq, but how can this explain the rest of the sequencing process?

Also, I'll be glad to hear from satisfied/non-satisfied Novaseq users general experience compared to other machines (namely Hiseq).

Many thanks!
Attached Images
File Type: gif Miseq1.gif (66.2 KB, 39 views)
File Type: gif Miseq2.gif (30.8 KB, 21 views)
File Type: gif Novaseq1.gif (94.4 KB, 37 views)
File Type: gif Novaseq2.gif (79.2 KB, 27 views)
Tcom111 is offline   Reply With Quote
Old 11-01-2018, 09:07 AM   #2
pmiguel
Senior Member
 
Location: Purdue University, West Lafayette, Indiana

Join Date: Aug 2008
Posts: 2,313
Default

The new version of SAV has a metric "% occupancy" for each tile. You should check that.
It looks like you either had very low occupancy (very under-clustered) or very massive over-clustering. (Actually I think the NovaSeq is pretty resistant to over-clustering.)

I personally would be terrified to use custom primers on an S4 flowcell. Actually we never used them even once on our previous instrument (HiSeq 2500).

--
Phillip
pmiguel is offline   Reply With Quote
Old 11-02-2018, 03:27 AM   #3
GenoMax
Senior Member
 
Location: East Coast USA

Join Date: Feb 2008
Posts: 6,972
Default

MiSeq is the champ of sequencing difficult libraries. I would not compare success on a MiSeq to a different sequencer for odd libraries. If you are running an un-supported application then you are on your own when you push the bounds of technology.

When you stay within bounds of supported applications NovaSeq performs very well.
GenoMax is offline   Reply With Quote
Old 11-05-2018, 10:13 PM   #4
mama
Junior Member
 
Location: paris

Join Date: Sep 2008
Posts: 7
Default

Which version of SAV enables to see the % occupancy per tile. How can you check if the loading concentration looks good?
Many thanks!
mama is offline   Reply With Quote
Old 11-05-2018, 10:19 PM   #5
Tcom111
Junior Member
 
Location: Israel

Join Date: May 2012
Posts: 3
Default

Thanks for your replies!
Quote:
I personally would be terrified to use custom primers on an S4 flowcell. Actually we never used them even once on our previous instrument (HiSeq 2500).
and
Quote:
When you stay within bounds of supported applications NovaSeq performs very well.
Novaseq and other machines have a specific protocol for custom primers, although I understand the fear.. I believe that it may have to do with the machine different temperature differences.

I was told that the intensities are low, which may resulted in faulty results.
Any thoughts about that?
Tcom111 is offline   Reply With Quote
Old 11-06-2018, 06:22 AM   #6
pmiguel
Senior Member
 
Location: Purdue University, West Lafayette, Indiana

Join Date: Aug 2008
Posts: 2,313
Default

Quote:
Originally Posted by mama View Post
Which version of SAV enables to see the % occupancy per tile. How can you check if the loading concentration looks good?
Many thanks!
SAV 2.4.5 includes an "occupied count" and "% occupied" metric in the drop down list of the Flow Cell Chart pane of the Analysis tab.

--
Phillip
pmiguel is offline   Reply With Quote
Old 11-08-2018, 07:39 AM   #7
GW_OK
Senior Member
 
Location: Oklahoma

Join Date: Sep 2009
Posts: 411
Default

One thing worth keeping in mind as well is the difference in clustering chemistries between the two instruments. With the Nova using ExAmp and the Miseq using the "classic" bridge amplification the same library may (and probably will) cluster differently between them. We were actually warned by our FAS during our Nova install to not QC pools on a Miseq Nano prior to a large Nova flowcell and instead run on an iSeq since it also uses ExAmp.

I'm wondering if your poor Nova run was due to presence of adapter/primer dimer, which can easily take over with ExAmp. Do you have an electropherogram of the library and/or FastQC results showing adapter dimer or insert size metrics?
GW_OK is offline   Reply With Quote
Old 12-10-2018, 02:31 AM   #8
Tcom111
Junior Member
 
Location: Israel

Join Date: May 2012
Posts: 3
Default Followup - Successful Novaseq run

Hi all,

As a followup on this issue (I owe it to the generous people here).

I ran the same library in Miniseq which has similar temperature properties and it looked really good. As a final step, I noticed the sequencing provider had some technical issues, including not supplying coherent answers as for what they did, and moreover, were unclear of what they suggest to do (at the end, they said they will rerun with addition of Phix and mix standard and custom primers). I chose to run in a different facility which supplies excellent support even before sending samples. The run had great QC, similar to the Miseq data (!) with S2 chip, yielding 3.7B reads.

Bottom line, Novaseq can do custom primers. And sometimes you should be lucky when selecting the provider (both sequencing provider have reputation and certificates etc.).

See attached figures for the successful Novaseq run (see posts before to compare).

Best.
Attached Images
File Type: png Nova2.png (31.6 KB, 16 views)
File Type: png Nova1.png (105.9 KB, 19 views)
Tcom111 is offline   Reply With Quote
Old 12-10-2018, 03:01 AM   #9
GenoMax
Senior Member
 
Location: East Coast USA

Join Date: Feb 2008
Posts: 6,972
Default

I am glad things worked to your satisfaction. Thanks for providing a final update.
GenoMax is offline   Reply With Quote
Reply

Tags
miseq, novaseq

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 12:24 AM.


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