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Forum: Sample Prep / Library Generation 08-23-2019, 09:12 AM
Replies: 2
Views: 810
Posted By GW_OK
Something (Tape/BioA/FragA) for size estimation...

Something (Tape/BioA/FragA) for size estimation followed by KAPA qPCR for absolute quantification. That's the gold standard in my opinion. If it hasn't been qPCR'd we don't run it!
Forum: Illumina/Solexa 08-22-2019, 01:38 PM
Replies: 1
Views: 636
Posted By GW_OK
Short read 1 on a Nova

Anyone tried a read 1 less than 25 cycles on a Nova? We've done it before on Miseq and Nextseq but not on Nova. I'm wondering if there's anything weird about the patterned flowcell that would make...
Forum: Illumina/Solexa 08-22-2019, 01:25 PM
Replies: 3
Views: 1,207
Posted By GW_OK
A bit late but here's a reagent price list I...

A bit late but here's a reagent price list I found from an email on 11/24/2014

2019 is here:
https://mkt.illumina.com/rs/600-XEX-927/images/q1-2019-amr-price-book-usd.pdf
Forum: Illumina/Solexa 02-04-2019, 09:59 AM
Replies: 1
Views: 922
Posted By GW_OK
Too many to list. How about posting the...

Too many to list.

How about posting the %base graph, %Q30 graph, run metrics, and the bionanalyzer/tapestation/gel image for the libraries?
Forum: Illumina/Solexa 01-08-2019, 07:59 AM
Replies: 5
Views: 872
Posted By GW_OK
Might be due to the decrease in base diversity at...

Might be due to the decrease in base diversity at the beginning of the reads. Maybe you've got some primer dimer in there?
Forum: Illumina/Solexa 01-07-2019, 12:41 PM
Replies: 5
Views: 872
Posted By GW_OK
What'd the metrics graphs (intensity, %base,...

What'd the metrics graphs (intensity, %base, %Q30, error rate) look like?
Forum: Illumina/Solexa 12-13-2018, 11:41 AM
Replies: 1
Views: 762
Posted By GW_OK
Comparing A260 vs picogreen/Qubit should tell you...

Comparing A260 vs picogreen/Qubit should tell you if there's a large amount of ssDNA present.
Forum: Illumina/Solexa 11-08-2018, 08:47 AM
Replies: 1
Views: 1,319
Posted By GW_OK
I'm surprised you haven't had at least one visit...

I'm surprised you haven't had at least one visit from an FSE if this is a recurrent problem. How did you confirm that this was caused by bubbles?
Forum: Illumina/Solexa 11-08-2018, 08:39 AM
Replies: 8
Views: 2,596
Posted By GW_OK
One thing worth keeping in mind as well is the...

One thing worth keeping in mind as well is the difference in clustering chemistries between the two instruments. With the Nova using ExAmp and the Miseq using the "classic" bridge amplification the...
Forum: Service Providers 05-31-2018, 10:51 AM
Replies: 2
Views: 5,481
Posted By GW_OK
Hi Eric, We're CAP/CLIA and interested in...

Hi Eric,
We're CAP/CLIA and interested in collaborating.
Forum: Illumina/Solexa 04-06-2018, 01:24 PM
Replies: 14
Views: 2,030
Posted By GW_OK
If there's no mention of custom primers in the...

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...
Forum: Illumina/Solexa 04-04-2018, 09:53 AM
Replies: 14
Views: 2,030
Posted By GW_OK
If you have a shift in peak size of ~125bp...

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...
Forum: Illumina/Solexa 03-26-2018, 12:25 PM
Replies: 22
Views: 2,674
Posted By GW_OK
Onboard diagnostics are in the "System Check"...

Onboard diagnostics are in the "System Check" icon under "Manage Instrument" from the main screen on the Miseq Control Software. The optics checks are fairly fast to run, the fluidics will take some...
Forum: Illumina/Solexa 03-26-2018, 11:58 AM
Replies: 22
Views: 2,674
Posted By GW_OK
What's troubling to me is that you don't have any...

What's troubling to me is that you don't have any intensity in read 2, which should always show if you have at least a read 1, regardless of index.

Have you run any of the onboard diagnostic...
Forum: Illumina/Solexa 03-26-2018, 11:09 AM
Replies: 22
Views: 2,674
Posted By GW_OK
That's intensity. I was talking about the graph...

That's intensity. I was talking about the graph that is from % Base, which is selectable in the menu from the gray button under Intensity.

Still, though, in looking at this graph it appears you...
Forum: Illumina/Solexa 03-26-2018, 10:48 AM
Replies: 22
Views: 2,674
Posted By GW_OK
Can you post the %base graph?

Can you post the %base graph?
Forum: Illumina/Solexa 03-19-2018, 07:34 AM
Replies: 7
Views: 2,152
Posted By GW_OK
It might be a thermal issue. Edit: But on...

It might be a thermal issue.

Edit: But on two different machines?
Forum: Illumina/Solexa 03-13-2018, 07:11 AM
Replies: 3
Views: 1,617
Posted By GW_OK
I do know Hiseq 2500's have two cameras. Here's a...

I do know Hiseq 2500's have two cameras. Here's a picture of the optical deck in case anyone is interested. You can see the green and red lasers at the left and top and the two cameras at angles on...
Forum: Illumina/Solexa 03-12-2018, 06:48 AM
Replies: 7
Views: 1,727
Posted By GW_OK
Good to know. I suppose the iSeq is single...

Good to know.

I suppose the iSeq is single color, since it's got the CMOS in the flowcell.
Forum: Illumina/Solexa 03-09-2018, 10:17 AM
Replies: 7
Views: 1,727
Posted By GW_OK
The lasers excite at fixed wavelengths. The...

The lasers excite at fixed wavelengths. The fluorophores emit at different respective wavelengths. The example given by kc3228 is for the dual color chemistries of the NextSeq and NovaSeq. The...
Forum: Illumina/Solexa 02-09-2018, 08:47 AM
Replies: 3
Views: 1,324
Posted By GW_OK
Well, yeah, but that's some serious capex burn.

Well, yeah, but that's some serious capex burn.
Forum: Sample Prep / Library Generation 02-09-2018, 07:43 AM
Replies: 2
Views: 1,146
Posted By GW_OK
Is there a reason you can't draw into a PAXgene...

Is there a reason you can't draw into a PAXgene tube?
Forum: Illumina/Solexa 02-09-2018, 07:17 AM
Replies: 3
Views: 1,324
Posted By GW_OK
Wow. I wonder who surplused $5M in...

Wow. I wonder who surplused $5M in instrumentation?
Forum: Illumina/Solexa 02-09-2018, 06:42 AM
Replies: 6
Views: 1,068
Posted By GW_OK
Custom primers have nothing to do with generation...

Custom primers have nothing to do with generation of clusters. Both ends of the library adapters must have the requisite P5 and P7 sites for proper bridge amplification on the flowcell.

And you...
Forum: Illumina/Solexa 02-08-2018, 11:54 AM
Replies: 6
Views: 1,068
Posted By GW_OK
But you can spike in custom primers, yeah? ...

But you can spike in custom primers, yeah?

And single read or paired-end, it doesn't really matter on the NextSeq? If it'll cluster you can just set it to do read 1.

We do single reads all the...
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