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Forum: Illumina/Solexa 03-23-2015, 08:01 AM
Replies: 13
Views: 7,956
Posted By esherman
Here are the instructions from an email chain...

Here are the instructions from an email chain with Illumina tech support detailing how to do a fluidics test on the MiSeq:



The volume (fluidics) test takes about an hour or so to complete and...
Forum: Illumina/Solexa 03-20-2015, 08:48 AM
Replies: 13
Views: 7,956
Posted By esherman
Thanks for the images! I'm a bit concerned...

Thanks for the images!

I'm a bit concerned by the intensities, especially in read 1. Your S/N ratio is surprisingly ok; however, in my talks with Illumina tech support, they say that they never...
Forum: Illumina/Solexa 03-20-2015, 08:11 AM
Replies: 13
Views: 7,956
Posted By esherman
We PCR'd an aliquot of standard from the Kapa...

We PCR'd an aliquot of standard from the Kapa qPCR kit for an even cheaper option. We now have 5 liters of standard for less than $20. Buying this from Kapa would cost over $100 million :p

As...
Forum: Illumina/Solexa 02-23-2015, 08:53 AM
Replies: 3
Views: 1,956
Posted By esherman
MiSeq photobleaching

I've noticed that even when accounting for kit expiration date, cluster density, %PF, phasing, and prephasing, some of our runs have excellent Q-scores all the way until the end of the read...
Forum: Illumina/Solexa 02-19-2015, 06:55 AM
Replies: 1
Views: 1,565
Posted By esherman
Which instrument was used to sequence your...

Which instrument was used to sequence your samples? The MiSeq, NextSeq (I think), and HiSeq (on rapid-run mode) all perform on-board cluster generation, which means that each library is pumped...
Forum: Illumina/Solexa 02-05-2015, 06:03 AM
Replies: 12
Views: 2,288
Posted By esherman
I had heard from tech support that the base...

I had heard from tech support that the base caller can only handle 400-500K clusters/mm^2 for each of the 4 channels (A, T, G, and C). If there are more clusters per channel than that, it...
Forum: Sample Prep / Library Generation 12-16-2014, 05:41 AM
Replies: 4
Views: 2,078
Posted By esherman
Thanks for the info, nucacidhunter. I did a...

Thanks for the info, nucacidhunter. I did a trial with non-precious gDNA and I got equal shearing using samples containing 1ug and 5ug of DNA (as quantified by picogreen). However, samples...
Forum: Sample Prep / Library Generation 12-13-2014, 02:12 PM
Replies: 4
Views: 2,078
Posted By esherman
Covaris shearing concentrated DNA

I know that one of Covaris' claims to fame is that their AFA shearing is DNA concentration independent. They have literature showing bioanalyzer traces of identical size distributions obtained from...
Forum: Illumina/Solexa 12-02-2014, 11:27 AM
Replies: 2
Views: 2,928
Posted By esherman
MiSeq v3 Reagents with v2 flowcell

I remember seeing that the MiSeq v2 and v3 flow cells are exactly the same, and that the main difference between the kits is the chemistry (in the reagent cartridge), recipes, and physical area of...
Forum: Illumina/Solexa 11-28-2014, 12:04 PM
Replies: 3
Views: 2,339
Posted By esherman
How long is the amplicon that had poor quality...

How long is the amplicon that had poor quality scores? My guess is that you're sequencing the entire amplicon, through the Illumina p5/p7/primer landing sequences, and into the flow cell. I think...
Forum: Illumina/Solexa 11-15-2014, 12:44 PM
Replies: 1
Views: 1,475
Posted By esherman
Given there's a bunch of bases that start with...

Given there's a bunch of bases that start with low Q-scores and continue with low Q-scores, I wonder if there's a particular tile on the flow cell that was dirty or otherwise gave bad data. On...
Forum: Illumina/Solexa 11-13-2014, 11:08 AM
Replies: 18
Views: 8,030
Posted By esherman
'Assembly PCR' for homemade amplicon library prep

I'm trying to make homemade amplicon library preps for the Illumina MiSeq, and my fusion primers always end up being around 100bp (if not more!) by the time I add all the necessary sequences (p5/p7,...
Forum: Illumina/Solexa 11-03-2014, 12:06 PM
Replies: 5
Views: 2,363
Posted By esherman
Thanks for the input, everyone! I did a...

Thanks for the input, everyone!

I did a quick test where I amplified a single region of the genome using PCR primers containing the Illumina sequences and 8 N's immediately downstream of the R1...
Forum: Illumina/Solexa 10-23-2014, 10:10 AM
Replies: 5
Views: 2,363
Posted By esherman
MiSeq Low Diversity - N's in primer solution

My lab does a lot of MiSeq sequencing of low-diversity amplicon libraries and we've been using a standard 10% PhiX spike-in with reasonable results. I just heard about the idea of adding a few N's...
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