SEQanswers

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



Similar Threads
Thread Thread Starter Forum Replies Last Post
MiSeq Run Fail - Microsoft Issue? Olivia16 Illumina/Solexa 10 03-09-2015 07:13 AM
MiSeq gDNA reads still fail "Kmer content" and "per base seq content" after trimming" ysnapus Illumina/Solexa 4 11-12-2014 07:25 AM
Fail Lane :( tcw General 0 07-23-2014 06:30 AM
Best way to trim MiSeq - NEBNext Multiplex Oligos for Illumina Library foolishbrat Illumina/Solexa 1 01-27-2014 03:10 AM
cuffmerge fail papori Bioinformatics 0 07-31-2011 03:01 PM

Reply
 
Thread Tools
Old 07-05-2016, 06:57 AM   #21
GenoMax
Senior Member
 
Location: East Coast USA

Join Date: Feb 2008
Posts: 6,766
Default

@vanessamata: I am not an experimental expert so take this with a grain of salt until someone confirms it but have you thought about heating your sample before loading? That should help break up any strange structures that may be forming. I remember this working for some difficult to sequence samples in past.
GenoMax is offline   Reply With Quote
Old 07-06-2016, 10:19 AM   #22
Jessica_L
Senior Member
 
Location: Washington, D.C. metro area

Join Date: Feb 2010
Posts: 116
Default

That's a good point, GenoMax-- for the TruSeq Amplicon v1 that I've used, the library that's eluted off beads post-normalization is single stranded in dilute NaOH and that requires a short heating followed by snap cooling on ice prior to loading. It's not a bad idea to try. The Illumina protocol for the TruSeq Amplicon calls for 96 degrees C for 2 minutes, followed by 5 minutes in an ice water bath, if you wanted to try something similar.
Jessica_L is offline   Reply With Quote
Old 07-06-2016, 01:55 PM   #23
nucacidhunter
Senior Member
 
Location: Melbourne

Join Date: Jan 2013
Posts: 1,166
Default

Considering discussion so far the most likely cause would be errors in overhang primer design or synthesis such as a 3' single base mismatch which prevents priming sequencing. I would check sequences on the oligo tubes to see if it matches with the intended sequences. Also 5 out of 6 unsequenced tags start with T but it would less likely be the cause.
nucacidhunter is offline   Reply With Quote
Old 07-07-2016, 03:49 AM   #24
vanessamata
Member
 
Location: Porto

Join Date: Mar 2014
Posts: 11
Default

@GenoMax and Jessica-L

I think you might be right!

The 16S metagenomics illumina protocol also mentions this step! For some reason the technician responsible for preparing the libraries to load into MiSeq was not doing it!

Next time I will try to heat up the library and see if this still happens or not!

Thanks a lot!
vanessamata is offline   Reply With Quote
Reply

Tags
custom tag, illumina, multiplex fail

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 07:24 PM.


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