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Search: Posts Made By: pmiguel
Forum: General 11-10-2017, 05:29 AM
Replies: 7
Views: 5,424
Posted By pmiguel
I would speculate as follows: High or low %GC...

I would speculate as follows: High or low %GC will increase the chances of stable single-stranded structures (EG stem loops) forming. In vivo, polymerases will act in concert with a host of other...
Forum: Illumina/Solexa 10-20-2017, 09:39 AM
Replies: 1
Views: 330
Posted By pmiguel
TruSeq® Stranded mRNA LT (Illumina) is designed...

TruSeq® Stranded mRNA LT (Illumina) is designed to fragment long RNAs using heat and divalent cations and then prime 1st and 2nd strand synthesis using hexamer primers.

miRNAs are a specific type...
Forum: Illumina/Solexa 10-18-2017, 08:38 AM
Replies: 10
Views: 1,269
Posted By pmiguel
For Nextera XT, we have always used the Illumina...

For Nextera XT, we have always used the Illumina primers.
Your logic (above) looks sound to me.

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Phillip
Forum: Sample Prep / Library Generation 10-18-2017, 07:15 AM
Replies: 3
Views: 416
Posted By pmiguel
Thanks for that follow-up. Interesting. -- ...

Thanks for that follow-up. Interesting.

--
Phillip
Forum: Sample Prep / Library Generation 10-17-2017, 09:43 AM
Replies: 3
Views: 416
Posted By pmiguel
Hi patkrat, Yes, the apparently longer products...

Hi patkrat,
Yes, the apparently longer products are likely "bubble products". It is hypothesized that bubble products form during PCR when the concentration of denatured products is high enough to...
Forum: Sample Prep / Library Generation 10-13-2017, 08:48 AM
Replies: 10
Views: 711
Posted By pmiguel
Hi Emil, I would strongly recommend that you...

Hi Emil,
I would strongly recommend that you verify this yourself by aligning your p5 and the reverse (in the 3' - 5' direction) of your p7 sequence. You will see the terminal 12 bases on one side...
Forum: Illumina/Solexa 10-13-2017, 08:40 AM
Replies: 26
Views: 2,155
Posted By pmiguel
Well, not as different as you might think. Please...

Well, not as different as you might think. Please see the attachment.

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Phillip
Forum: Sample Prep / Library Generation 10-11-2017, 02:09 PM
Replies: 10
Views: 711
Posted By pmiguel
Hi Emil, Most TruSeq Illumina kits use the...

Hi Emil,
Most TruSeq Illumina kits use the Y-adapters. The common "TruSeq" DNA and RNAseq ones will offer a normal single index kit option (usually going up to 24 indexes) or a "high thoughput" one...
Forum: Sample Prep / Library Generation 10-11-2017, 10:01 AM
Replies: 10
Views: 711
Posted By pmiguel
Is this a Y-adapter design? If so, the P7 and P5...

Is this a Y-adapter design? If so, the P7 and P5 need to anneal over the last 12 bases of the P5/first 12 bases of the P7, with a 3' "T" overhang to work with many Illumina work flows. When you add 8...
Forum: Illumina/Solexa 10-10-2017, 12:00 PM
Replies: 26
Views: 2,155
Posted By pmiguel
Did you see much phiX index hopping? Searching...

Did you see much phiX index hopping? Searching the "undetermined" fastq for index hops involving one of i7/i5 GGGGGGGG/TCGTAGTG I tentatively identified a fair number. Of course they may just be the...
Forum: Illumina/Solexa 10-10-2017, 11:28 AM
Replies: 26
Views: 2,155
Posted By pmiguel
You can buy the UDI 96 and/or the UDI 384 kit...

You can buy the UDI 96 and/or the UDI 384 kit from IDT as a custom order. Just contact their sales. We bought the UDI 96. Made libraries (both no Amp DNA and RNAseq libraries) from them. Ran them on...
Forum: Illumina/Solexa 10-09-2017, 11:59 AM
Replies: 10
Views: 1,269
Posted By pmiguel
Yeah, good question since we will likely need to...

Yeah, good question since we will likely need to know this if we need to run more than 16 samples dual unique. I'd just check with a nanodrop first, see if the acetate salt is too high to allow you...
Forum: Illumina/Solexa 10-09-2017, 11:16 AM
Replies: 26
Views: 2,155
Posted By pmiguel
Seems unlikely. That aspect was only introduced...

Seems unlikely. That aspect was only introduced to the thread after Brian's post.

Brian actually praises the accuracy of the NovaSeq--which is what is claimed later is an issue.

I don't think...
Forum: Illumina/Solexa 10-09-2017, 10:26 AM
Replies: 1
Views: 519
Posted By pmiguel
I always read it as "primer". -- ...

I always read it as "primer".

--
Phillip
Forum: Illumina/Solexa 10-09-2017, 10:20 AM
Replies: 26
Views: 2,155
Posted By pmiguel
Actually you can purchase full (96 or 384) UDI...

Actually you can purchase full (96 or 384) UDI adapter sets from IDT. Although last I checked they were still a custom synthesis order. Which means you get more adapter than you are likely to use at...
Forum: Illumina/Solexa 10-09-2017, 10:12 AM
Replies: 26
Views: 2,155
Posted By pmiguel
Brian, "HiSeq" doesn't denote a single kind of...

Brian,
"HiSeq" doesn't denote a single kind of Illumina instrument. HiSeq 2000/2500 are quite different from HiSeq 3000/4000/X. Which type do you mean?

--
Phillip
Forum: Sample Prep / Library Generation 10-04-2017, 10:37 AM
Replies: 2
Views: 438
Posted By pmiguel
No reads, or no clusters? Nextera sequencing...

No reads, or no clusters?
Nextera sequencing primers aren't included in HiSeq High Output chemstry reagents. But they are in MiSeq/HiSeq Rapid chemsitry and so should be in the NextSeq reagent kits....
Forum: Illumina/Solexa 09-25-2017, 12:10 PM
Replies: 7
Views: 1,193
Posted By pmiguel
Wait bbp. You are sure this is really Illumina...

Wait bbp. You are sure this is really Illumina who gave you that answer? Because Illumina usually sounds just as portrayed by GenoMax above.
And if it isn't Illumina, then who is it?
I didn't mean...
Forum: Illumina/Solexa 09-25-2017, 09:25 AM
Replies: 7
Views: 1,193
Posted By pmiguel
I doubt that this is the case. The limitation is...

I doubt that this is the case. The limitation is more likely phasing/prephasing -- which has to do with the ensemble of product molecule containing members slightly behind or slightly ahead of the...
Forum: RNA Sequencing 09-18-2017, 08:12 AM
Replies: 4
Views: 1,331
Posted By pmiguel
Usually it either means your library is...

Usually it either means your library is "bottomed-out" or you have some spike-in controls of fixed length. By "bottomed-out" I mean amplified from a very limited pool of input amplicons.

As to...
Forum: Bioinformatics 09-14-2017, 01:37 PM
Replies: 7
Views: 825
Posted By pmiguel
Must. Not. Write. Perl. One. Liner. -- ...

Must. Not. Write. Perl. One. Liner.

--
Phillip
Forum: Genomic Resequencing 09-12-2017, 01:07 PM
Replies: 4
Views: 1,452
Posted By pmiguel
Would your answer change if the WGS coverage was...

Would your answer change if the WGS coverage was 30X, instead of "low"?

--
Phillip
Forum: Illumina/Solexa 09-12-2017, 06:33 AM
Replies: 4
Views: 441
Posted By pmiguel
Usually when the percentage of undetermined is...

Usually when the percentage of undetermined is that high, it means the wrong index sequence was used. (Unless you had 50% phiX spiked in, as nucacidhunter said.) It is possible to determine if you...
Forum: Illumina/Solexa 09-11-2017, 01:08 PM
Replies: 1
Views: 387
Posted By pmiguel
One read pair will be the reverse-complement of...

One read pair will be the reverse-complement of the other read-pair. So they will not be considered "duplicates".
Also, even if your software did consider these to be "duplicates" not all amplicon...
Forum: Sample Prep / Library Generation 08-21-2017, 12:08 PM
Replies: 10
Views: 1,145
Posted By pmiguel
If you are running these on a...

If you are running these on a non-patterned-flowcell Illumina sequencer, ignore the primer peaks. If you are running them on a patterned-flowcell Illumina sequencer (HiSeq 3000/4000/X and NovaSeq)...
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