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Forum: Illumina/Solexa 04-05-2016, 01:58 PM
Replies: 2
Views: 1,099
Posted By bilyl
What is your total elution volume? What kind of...

What is your total elution volume? What kind of total ng output are you getting from the first round of PCR? How many cycles of PCR are you using? Are you doing size selection? What is your shear...
Forum: Illumina/Solexa 04-05-2016, 01:53 PM
Replies: 7
Views: 1,969
Posted By bilyl
Do you mean it failed at the 300th cycle; eg at...

Do you mean it failed at the 300th cycle; eg at the end of one side of a 2x300 run?

You have to be more aggressive with the tech support if they aren't helpful. Ask for a PhiX run using a 2x300...
Forum: Illumina/Solexa 03-17-2016, 01:49 PM
Replies: 12
Views: 2,421
Posted By bilyl
Sorry, I meant the actual sequencer itself had...

Sorry, I meant the actual sequencer itself had problems with the pumps and Peltier. The easiest way to test this would be to do a PhiX run on a preclustered and R1-primed flowcell that was generated...
Forum: Illumina/Solexa 03-14-2016, 02:08 PM
Replies: 12
Views: 2,421
Posted By bilyl
How do the intensities scale on the flow cell?...

How do the intensities scale on the flow cell? Has Illumina tried a PhiX run?

We had an issue similar to this and I believe it turned out to be a combination of flowrate and Peltier problems.
Forum: Illumina/Solexa 03-01-2016, 02:37 PM
Replies: 15
Views: 8,341
Posted By bilyl
Dumb question, but how is it possible that the...

Dumb question, but how is it possible that the HiSeq 4000 is not robust to low diversity? Isn't the whole point of patterned flow cells to get good registration of clusters, and then from there get...
Forum: Illumina/Solexa 09-17-2015, 05:59 PM
Replies: 12
Views: 5,276
Posted By bilyl
Is Bustard still being used on current Illumina...

Is Bustard still being used on current Illumina systems? I had thought that they are using something else.
Forum: Illumina/Solexa 09-10-2015, 08:32 PM
Replies: 12
Views: 5,276
Posted By bilyl
Interesting... I wonder if you could implement...

Interesting... I wonder if you could implement your own basecaller with the CIFs, especially for specialized assays? Or are these files already too far along in the processing?
Forum: Illumina/Solexa 07-17-2015, 02:16 PM
Replies: 13
Views: 4,383
Posted By bilyl
Actually, I'm not sure if anyone has noticed this...

Actually, I'm not sure if anyone has noticed this but I've observed a lot of off-target "hits" when using KAPA HiFi for 16S amplification, especially when there is human host DNA present. Q5/Phusion...
Forum: Illumina/Solexa 06-07-2015, 02:44 PM
Replies: 6
Views: 3,775
Posted By bilyl
Commerically available V4 kits play well with...

Commerically available V4 kits play well with 2x150. You could do a V1-V2 amplicon but you'll need longer reads to do that.

As for the NextSeq, you'll have a really bad time unless you can base...
Forum: Sample Prep / Library Generation 06-06-2015, 01:48 PM
Replies: 17
Views: 12,503
Posted By bilyl
Why not a Labchip GX? It can run 96-well plates...

Why not a Labchip GX? It can run 96-well plates of samples and can perform regular NGS quant as well as genomic DNA assays.
Forum: Illumina/Solexa 05-29-2015, 10:05 AM
Replies: 17
Views: 2,738
Posted By bilyl
This can probably work, as long as you have the...

This can probably work, as long as you have the P5 and P7 sequences surrounding the sequencing primer. That is, it's not exactly an inverted repeat.

The only issue you will have is competition for...
Forum: Illumina/Solexa 05-23-2015, 03:07 PM
Replies: 9
Views: 2,146
Posted By bilyl
Somewhat relevant: I have seen the occasional...

Somewhat relevant: I have seen the occasional improperly made buffers. Things like 10mM Tris Hcl pH 8.5 suddenly became 100mM because someone was tired when dispensing from stock. If that's the case...
Forum: Illumina/Solexa 05-21-2015, 10:25 PM
Replies: 9
Views: 2,146
Posted By bilyl
Have you thought about doing a bead cleanup of...

Have you thought about doing a bead cleanup of the prepared library before sequencing? Maybe something is carrying over.

Alternatively, perhaps you can mix in 50/50 the library that just worked,...
Forum: Illumina/Solexa 05-11-2015, 04:36 PM
Replies: 10
Views: 2,764
Posted By bilyl
If you read papers carefully enough, you can...

If you read papers carefully enough, you can always make your own homebrew master mixes for library prep. It will always be more expensive to buy from a vendor.

That being said, you could spend...
Forum: Illumina/Solexa 04-06-2015, 07:43 PM
Replies: 10
Views: 3,162
Posted By bilyl
I haven't read the method from the paper, but did...

I haven't read the method from the paper, but did you use custom primers? It is a common mistake to use the wrong primer blend for read 2. PhiX uses the "PE Read 2" primer, which is different from...
Forum: Sample Prep / Library Generation 03-04-2015, 03:34 PM
Replies: 6
Views: 3,645
Posted By bilyl
Why not just randomly fragment the amplicon with...

Why not just randomly fragment the amplicon with fragmentase and then make a library with barcodes corresponding to each one? You could assemble them afterward. You would get around using the...
Forum: Illumina/Solexa 02-19-2015, 08:07 PM
Replies: 2
Views: 1,687
Posted By bilyl
That's interesting -- do you have any...

That's interesting -- do you have any before/after specs?

We noticed a huge jump in cluster densities after the 1.3 update, but the PF and Q30 scores weren't looking so hot. Do you know what the...
Forum: Illumina/Solexa 02-18-2015, 10:24 PM
Replies: 8
Views: 3,553
Posted By bilyl
I've seen this several times. In my experience...

I've seen this several times. In my experience it's almost certainly due to overclustering so you should ask the staff. The reads will look fine in R1, but after the turnaround step the quality gets...
Forum: Illumina/Solexa 02-12-2015, 12:55 PM
Replies: 2
Views: 1,175
Posted By bilyl
It depends on your downstream processes. If you...

It depends on your downstream processes. If you are simply calling SNVs then it doesn't really matter. But for other applications (eg. CNVs), many programs prefer to have similar coverage between...
Forum: Sample Prep / Library Generation 02-11-2015, 02:57 PM
Replies: 20
Views: 23,468
Posted By bilyl
We have a fragment analyzer and a labchip GX. I...

We have a fragment analyzer and a labchip GX. I haven't seen the former being used very much in our facility but the GX gets a lot of play. Personally I like it a lot because the assays are very...
Forum: Sample Prep / Library Generation 12-28-2014, 08:43 PM
Replies: 16
Views: 5,842
Posted By bilyl
Just use the Kapa Hyper Prep kit with your own...

Just use the Kapa Hyper Prep kit with your own adapters. Crank up the adapter concentration and you can even go PCR-free for downstream assays.
Forum: Sample Prep / Library Generation 11-29-2014, 01:10 PM
Replies: 4
Views: 2,145
Posted By bilyl
What kind of samples will you be processing? For...

What kind of samples will you be processing? For many applications NEB Fragmentase could be your best bet even though sometimes it is a bit finicky.

Alternatively, you should look around your...
Forum: Illumina/Solexa 11-25-2014, 12:07 AM
Replies: 6
Views: 3,063
Posted By bilyl
Anyone tried the Rapid Run v2 reagents?

Has anyone tried out the new HiSeq Rapid Run v2 reagents? They are compatible with 500 cycle runs but are also offered in 50 and 200 cycle.

The user manual says the supported cluster density is...
Forum: Illumina/Solexa 10-23-2014, 12:52 PM
Replies: 5
Views: 2,038
Posted By bilyl
The Miseq is pretty robust with high diversity. I...

The Miseq is pretty robust with high diversity. I doubt you'll get that much better signal with playing around with Ns versus the time you put in.

FYI, the Hiseq has a new software update that...
Forum: Sample Prep / Library Generation 10-19-2014, 12:10 AM
Replies: 2
Views: 1,095
Posted By bilyl
I routinely shear a couple hundred ng of DNA with...

I routinely shear a couple hundred ng of DNA with a Covaris then concentrate it down to 20ul with an Amicon Ultra-0.5 10k. From there I can scale down my Kapa Hyper Prep reagents to 0.4x of what they...
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