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Forum: Illumina/Solexa 05-08-2019, 01:51 AM
Replies: 0
Views: 1,048
Posted By vanessamata
overclustered run?

Hi all,

I sent a custom made library pool consisting of 3 amplicons (nextera like adaptors with 7bp indexes) - 80% of the pool - and genomic library of birds (trueseq single index adaptors) - 20%...
Forum: Illumina/Solexa 07-07-2016, 03:49 AM
Replies: 23
Views: 5,302
Posted By vanessamata
@GenoMax and Jessica-L I think you might be...

@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...
Forum: Illumina/Solexa 07-02-2016, 12:17 PM
Replies: 23
Views: 5,302
Posted By vanessamata
Yes. 6,8,9, and 10 failed on both runs. And 7...

Yes. 6,8,9, and 10 failed on both runs. And 7 worked on both runs.

Yes, the only thing that makes sense to me is that for some (molecular) reason the structure of these primers/tags are not...
Forum: Illumina/Solexa 07-02-2016, 10:47 AM
Replies: 23
Views: 5,302
Posted By vanessamata
The samples that ran on this flowcell were of...

The samples that ran on this flowcell were of different sources, but yes, they were different from these ones with tags...
Forum: Illumina/Solexa 06-30-2016, 01:59 PM
Replies: 23
Views: 5,302
Posted By vanessamata
Tags 1,2,3,4,5, and 7 worked Tags 6,8,9,10,11,...

Tags 1,2,3,4,5, and 7 worked
Tags 6,8,9,10,11, and 12 failed

I can't really see any pattern...:confused:
Forum: Illumina/Solexa 06-30-2016, 01:54 PM
Replies: 23
Views: 5,302
Posted By vanessamata
Hi everyone! Just to make you a quick...

Hi everyone!

Just to make you a quick update.

I took 3 PCR products amplified with some Tags that failed (6,8,9,10) and one that didn't (7) and I re-did the Indexing PCR, this time in separate...
Forum: Illumina/Solexa 05-19-2016, 11:19 AM
Replies: 23
Views: 5,302
Posted By vanessamata
I know I know... I just can't think of any...

I know I know... I just can't think of any mistake that would lead to these kind of results... :rolleyes: I was expecting some of the samples to eventually fail/be over-represented, after pooling 12...
Forum: Illumina/Solexa 05-19-2016, 11:04 AM
Replies: 23
Views: 5,302
Posted By vanessamata
Yes, what I'm going to try to do next is sequence...

Yes, what I'm going to try to do next is sequence in separate some samples of those plates that failed and see whether the problem persists or not... but I was hoping someone would have had the same...
Forum: Illumina/Solexa 05-19-2016, 09:23 AM
Replies: 23
Views: 5,302
Posted By vanessamata
Yeah, we've tried techsupport and they have no...

Yeah, we've tried techsupport and they have no idea. Since the problem is plate specific, they say that there seems to be no problem with the run it self, but with our library prep. :(
Forum: Illumina/Solexa 05-19-2016, 08:16 AM
Replies: 23
Views: 5,302
Posted By vanessamata
Update: I tested doing the indexing pcr...

Update: I tested doing the indexing pcr independently (before pooling) for 3 primer combinations that failed sequencing. They all incorporated the index, as they went from ~280bp to ~380bp. So it's...
Forum: Illumina/Solexa 05-18-2016, 12:33 PM
Replies: 23
Views: 5,302
Posted By vanessamata
I checked every index pcr reaction on agarose gel...

I checked every index pcr reaction on agarose gel and they all gained ~100bp.
However, since every reaction had 12 different samples in it, it's impossible to know if all samples were able to...
Forum: Illumina/Solexa 05-18-2016, 08:09 AM
Replies: 23
Views: 5,302
Posted By vanessamata
miseq multiplex fail

Hi,

I'm doing metabarcoding of bat diet by amplifying a 157bp region of CO1.

I'm using illumina protocol for 16s metagenomic libraries, but with tag modified primers. This means that the PCR...
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