SEQanswers

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



Similar Threads
Thread Thread Starter Forum Replies Last Post
PE Adaptors cement_head Sample Prep / Library Generation 1 08-25-2017 11:39 AM
Wierd spikes in library prepared with NEBNext adaptors bdlarsen Illumina/Solexa 1 04-30-2016 05:08 PM
NebNext Adaptors for illumina sequencingfan Sample Prep / Library Generation 4 11-06-2014 09:13 PM
TruSeq Adaptors reported by FastQC are true adaptors? Jiafen Bioinformatics 3 11-07-2012 02:07 PM
454 Rapid Library adaptors for NEBNExt protocol Coyk Sample Prep / Library Generation 1 02-18-2012 07:49 PM

Reply
 
Thread Tools
Old 04-04-2018, 02:09 AM   #1
bsvega
Junior Member
 
Location: Madrid

Join Date: Jul 2015
Posts: 9
Default NEBNext adaptors clustering failed

Hi all,
I am having problems with the NEBnext Ultra kit for library preparation. Iīve been doing same amplicon libraries for three years with success, but since October none of my libraries seems to hybridize with the adapters of the flow cell (I got 0 clusters). It happened already with 6 different runs (Miseq v3, 600 cycles) and 240 different library preps.
The bioanalyzer pattern was good (size OK, no adapters, shape OK), final pools were quantified by Qubit, Bionalyzer and qPCR (Qiaseq). None of the quantifications worked. PhiX was loaded at 10 and 15% equimolar to pooled libraries (12pM).
NaOH and Miseq are working because other researchers are using them with no problems.
Any idea of what can be the problem?. Neither Illumina, nor NEB, are helpful.
Thanks
bsvega is offline   Reply With Quote
Old 04-04-2018, 02:59 AM   #2
Markiyan
Senior Member
 
Location: Cambridge

Join Date: Sep 2010
Posts: 115
Lightbulb Check sequencing adapters.

Try a new batch of the sequencing adapters.
Maybe one of your sequencing adapters had degraded?
Also check the thumbnail images from the run's tmp folder.

If both adapters at the end of the insert are same P5 or P7, than the qPCR would work, but clustering wouldn't.
Markiyan is offline   Reply With Quote
Old 04-04-2018, 03:18 AM   #3
bsvega
Junior Member
 
Location: Madrid

Join Date: Jul 2015
Posts: 9
Default

Hi Markiyan, thanks for your reply.
I already use two different lots of adapters (and reagents).
There are no thumbnail images, the folder is empty.
The Mise, althought it says Run completed, the runs stoped before completion (before 24h for a run of 600 cyles).
bsvega is offline   Reply With Quote
Old 04-04-2018, 04:03 AM   #4
Markiyan
Senior Member
 
Location: Cambridge

Join Date: Sep 2010
Posts: 115
Lightbulb A couple things to check:

1. Did you look into
d:\Illumina\MiSeqTemp\[your run ID]\Thumbnail_Images ?
It is available only till the start of the next run.

2. Did you use PhiX in 10-15%? - at least it should show the reads from the PhiX...

3. PS: If troubleshooting - use 300 or 500 cycles nano kits (cheaper than 600 cycles V3).
Markiyan is offline   Reply With Quote
Old 04-04-2018, 04:15 AM   #5
nucacidhunter
Jafar Jabbari
 
Location: Melbourne

Join Date: Jan 2013
Posts: 1,190
Default

Quote:
Originally Posted by bsvega View Post
The bioanalyzer pattern was good (size OK, no adapters, shape OK), final pools were quantified by Qubit, Bionalyzer and qPCR (Qiaseq). None of the quantifications worked.

if I understand correctly, individual libraries BA profile was fine but the library pool did not have any DNA according to Qubit, BA and qPCR?

If library PCR amplification post adapter ligation was succssfull, adapters are unlikely to be the cause.

You have mentioned that you are preparing amplicon libraries. It could be due to polymerase if you now using different one. More information on your library prep workflow would give clues for troubleshooting.
nucacidhunter is offline   Reply With Quote
Old 04-04-2018, 06:24 AM   #6
bsvega
Junior Member
 
Location: Madrid

Join Date: Jul 2015
Posts: 9
Default

Yes, I thought about v3-600 cycles problem, so I tried with a Miseq nano v2. Same problem, no clustering.

Idividual libraries and pool bionalyzer profiles are good. Quantification of individual libraries is done with qubit, quantification of pooled libraries is done with qubit, bioanalyzer and qPCR.

My libraries are IGH genes. I do a PCR amplification from gDNA using an international estandarized protocol (Biomed-2), so it use to work with no problems. I clean-up the PCR products with Ampure XP beads and quantitate the ammount of product used for the library normally between 100 and 500 ng. I follow the NEBNext protocol with no changes.

The bionalyzer profile between PCR products and individual libraries show a difference of around 120-130 bp. And because qPCR worked I think the libraries are OK, but I do not get any clusters.
Do not know what else to try It used to work perfectly before.
bsvega is offline   Reply With Quote
Old 04-04-2018, 07:09 AM   #7
Markiyan
Senior Member
 
Location: Cambridge

Join Date: Sep 2010
Posts: 115
Exclamation PhiX% ?

1. Do you use any custom sequencing primers (esp R1)? If yes - more detail where and for which reads.

2. HOW MUCH PhiX % did you load!?? - I hope that at least 10%?

PS: there are some GC stretches in those reads - so please also check the thermal cycling performance of the instrument during the cluster generation.
Markiyan is offline   Reply With Quote
Old 04-04-2018, 09:53 AM   #8
GW_OK
Senior Member
 
Location: Oklahoma

Join Date: Sep 2009
Posts: 405
Default

If you have a shift in peak size of ~125bp between your initial amplicon and the final library you make from it then it should have the appropriate adapters ligated to each end.

If your qPCR of these libraries is "not working" you should stop immediately and investigate. I think (at least for the Kapa kit I use) the qPCR primers are based off of the p5 and p7 sites and if you cannot qPCR it then you have no hope of generating clusters.

If you are loading 10% PhiX (as mentioned in the first post) it is very odd that you are seeing a failure in clustering. Two things that might cause this are accidentally telling the Miseq to use a custom primer when you don't want it to (leading to lack of priming and nothing detected) or overclustering (as too many clusters may look like no clusters if focus fails).
GW_OK is offline   Reply With Quote
Old 04-04-2018, 02:47 PM   #9
nucacidhunter
Jafar Jabbari
 
Location: Melbourne

Join Date: Jan 2013
Posts: 1,190
Default

Good points have been raised in posts above. A run with 40% PhiX spike in should clear whether your library has any issues or if fail is due to preparation and sequencing set up.
nucacidhunter is offline   Reply With Quote
Old 04-06-2018, 02:08 AM   #10
bsvega
Junior Member
 
Location: Madrid

Join Date: Jul 2015
Posts: 9
Default

Thank you for your input. I wil try to answer everything.

- I donīt use custom primers because NebNext does not need them.
- qPCR, bionalyzer and Qubit work for individual libraries and also for pooled libraries. qPCR quantification used to give a slighty higher concentration (I guess because of heteroduplex from these are low diversity libraries). I run the same pool twice according to qPCR and Qubit quantifications with same results.
- I used to load 5% Phix with these libraries and worked fine. Since the failed runs I am loading 10 and 15%. I dont get anyclusters, neither from PhiX. As GW_OK said are accidentally telling the Miseq to use a custom primer when you don't want it to (leading to lack of priming and nothing detected). How can I check this?. The samplesheet does not specify to use custom primers.

" Markiyan : PS: there are some GC stretches in those reads - so please also check the thermal cycling performance of the instrument during the cluster generation.". How can I check this?.

- Because the clustering has failed, there are no thumbnails to review, except for one run with 64 k/mm2. The thumbanails shows almost no signal. Overclustering is not the issue.

Any thoughts are wellcome, and thank you so much for your help.
bsvega is offline   Reply With Quote
Old 04-06-2018, 02:29 AM   #11
josdegraaf
Member
 
Location: Germany

Join Date: Mar 2010
Posts: 33
Default

did you consider the naoh you use? is it fresh and < 1mM final concentration in your pool?
it could be (but does not explain no phix clusters) that the denaturation is insufficient, try heat denaturation in that case (high GC amplicons).
josdegraaf is offline   Reply With Quote
Old 04-06-2018, 03:21 AM   #12
Markiyan
Senior Member
 
Location: Cambridge

Join Date: Sep 2010
Posts: 115
Lightbulb

rkiyan : PS: there are some GC stretches in those reads - so please also check the thermal cycling performance of the instrument during the cluster generation.". How can I check this?.

Basically one need to check for 2 things:

1. The flowcell makes a good thermal contact with the Miseq's thermal block during the thermal cycling (no gaps between flowcell glass and thermal block!)

2. Get a thermocouple to the side of the thermal block (or a thermal testing flow cell) and log the temperature changes during the cluster generation.

(Similar to: http://jamimmunology.blogspot.co.uk/...le-logger.html)

Assuming that the instrument is on the service contract - the Illumina's FAS should do the above steps.

- Because the clustering has failed, there are no thumbnails to review, except for one run with 64 k/mm2. The thumbanails shows almost no signal. Overclustering is not the issue.

Any thoughts are welcome, and thank you so much for your help.[/QUOTE]

And if you set up a pure PhiX run, does it work as expected?

If yes - something in your library is inhibiting the cluster formation...

PS: Any attempts to use a heat treatment step for the library denaturation before loading?
Markiyan is offline   Reply With Quote
Old 04-06-2018, 01:24 PM   #13
GW_OK
Senior Member
 
Location: Oklahoma

Join Date: Sep 2009
Posts: 405
Default

If there's no mention of custom primers in the sample sheet then that's not something to worry about.

My next thought would be to check the concentration of your NaOH. A heat denaturation attempt might be worthwhile, as several others have previously mentioned.

You could also just assume that there's some sort of inhibitor in there and do another cleanup of your library to try and remove whatever it is.
GW_OK is offline   Reply With Quote
Old 04-08-2018, 09:23 AM   #14
bsvega
Junior Member
 
Location: Madrid

Join Date: Jul 2015
Posts: 9
Default

The libraires have two rounds of ampure XP cleaning. We also try the Generead size selection columns.

We check NaOH (same NaOH vial is working with other 4nM libraries). Maybe try higher concentrations of NaOH (need to neutralize before loading) and also heat denaturation.

Meanwhile I am going to try the Kappa kit with some samples. I am trying to solve the issue because there is no more DNA for some samples to repeat library (need to rescue them).

Thanks for all your thoughts.
bsvega is offline   Reply With Quote
Reply

Tags
clustering failed, miseq amplicon, nebnext

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 03:38 PM.


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