miseq multiplex fail - Page 2

GenoMax · 07-05-2016, 06:57 AM

@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.

Jessica_L · 07-06-2016, 10:19 AM

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.

nucacidhunter · 07-06-2016, 01:55 PM

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.

vanessamata · 07-07-2016, 03:49 AM

@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!

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07-05-2016, 06:57 AM	#21
GenoMax Senior Member Location: East Coast USA Join Date: Feb 2008 Posts: 6,696	@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.

07-06-2016, 10:19 AM	#22
Jessica_L Senior Member Location: Washington, D.C. metro area Join Date: Feb 2010 Posts: 116	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.

07-06-2016, 01:55 PM	#23
nucacidhunter Senior Member Location: Melbourne Join Date: Jan 2013 Posts: 1,137	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.

07-07-2016, 03:49 AM	#24
vanessamata Member Location: Porto Join Date: Mar 2014 Posts: 11	@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!