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Forum: Sample Prep / Library Generation 03-09-2018, 12:47 PM
Replies: 3
Views: 1,439
Posted By jwfoley
No, that's just primer. An adapter dimer would be...

No, that's just primer. An adapter dimer would be ~120 bp long. This isn't long enough to contain both adapters so it won't cluster on a flow cell and therefore shouldn't affect anything.
Forum: Illumina/Solexa 01-29-2018, 02:39 PM
Replies: 2
Views: 843
Posted By jwfoley
Libraries over 2 kb don't tend to work on...

Libraries over 2 kb don't tend to work on Illumina sequencers, so do you really care what the concentration of those molecules is?

Also, are you doing a TaqMan qPCR or SYBR? If it's SYBR, you have...
Forum: Illumina/Solexa 01-29-2018, 02:34 PM
Replies: 1
Views: 686
Posted By jwfoley
If I understand your diagram correctly, and I'm...

If I understand your diagram correctly, and I'm not sure I do, the default read 1 (no custom primers) will be from the third pad leftward, into GenSpecificPrimerR and beyond, and then the default...
Forum: Bioinformatics 11-13-2017, 03:17 PM
Replies: 4
Views: 759
Posted By jwfoley
If enzymes count as chemicals, NEB has its...

If enzymes count as chemicals, NEB has its "Fragmentase" mix, which appears to be a combination of a dsDNA nicking enzyme that's had its sequence specificity engineered out of it and a second enzyme...
Forum: RNA Sequencing 11-03-2017, 03:26 PM
Replies: 4
Views: 1,665
Posted By jwfoley
No, I'm concerned that having a lot of random...

No, I'm concerned that having a lot of random bases would invite mispriming and other molecular tomfoolery, plus it's a waste of sequencing cycles if you're using short reads, as we do. Oh, and...
Forum: RNA Sequencing 11-02-2017, 07:15 AM
Replies: 4
Views: 1,665
Posted By jwfoley
Both great questions. Well, first of...

Both great questions.



Well, first of all, it has nothing to do with streptavidin, which always confuses people, and that's why our diagram (fig. S1 in Supplemental File 1) just calls it a...
Forum: RNA Sequencing 11-01-2017, 09:24 AM
Replies: 4
Views: 1,665
Posted By jwfoley
Post Smart-3SEQ: a cheap, fast protocol that is sensitive enough for FFPE single cells

Our lab has just posted a preprint (https://www.biorxiv.org/content/early/2017/10/27/207340) about our new RNA-seq protocol, Smart-3SEQ. It combines the previous 3SEQ method for 3' digital gene...
Forum: Illumina/Solexa 05-24-2017, 09:51 AM
Replies: 23
Views: 5,066
Posted By jwfoley
Question Good index sequences for unique dual indexing?

After the index-hopping scandal (http://seqanswers.com/forums/showthread.php?t=75200) (see Illumina's white paper...
Forum: Sample Prep / Library Generation 06-03-2016, 12:01 PM
Replies: 3
Views: 2,100
Posted By jwfoley
Those are the same ones in the protocol JBKri is...

Those are the same ones in the protocol JBKri is looking at, since we just copied the purchasing information when we improved on the other aspects of Faircloth & Glenn's protocol.

JBKri, why do...
Forum: Sample Prep / Library Generation 03-15-2016, 07:39 AM
Replies: 7
Views: 2,144
Posted By jwfoley
Virtually all the protocols are compatible with...

Virtually all the protocols are compatible with the Illumina HiSeq, but if you take a look at how the Clontech kit works, you'll see why it's not compatible with degraded RNA.
Forum: Sample Prep / Library Generation 03-08-2016, 05:43 PM
Replies: 270
Views: 103,977
Posted By jwfoley
No, my design was somewhat different. I remain...

No, my design was somewhat different. I remain skeptical of LNAs as discussed previously in this thread. As also discussed previously, Exiqon is the only vendor of LNAs in North America.

But if...
Forum: Illumina/Solexa 01-24-2016, 11:08 AM
Replies: 31
Views: 9,289
Posted By jwfoley
v2 chemistry or v1?

v2 chemistry or v1?
Forum: Illumina/Solexa 01-22-2016, 02:03 PM
Replies: 31
Views: 9,289
Posted By jwfoley
Okay, so basically no cost-per-read advantage...

Okay, so basically no cost-per-read advantage over the MiSeq either. I don't mind paying an extra $50 per run to double my read lengths. But if I were buying a new machine, that wouldn't be worth the...
Forum: Illumina/Solexa 01-21-2016, 11:41 AM
Replies: 31
Views: 9,289
Posted By jwfoley
If we're comparing reads to reads, the more apt...

If we're comparing reads to reads, the more apt comparison is 150 cycles and 25M reads for a MiSeq v3 kit ($850). Does anyone know the pricing for the MiniSeq's 75-cycle kit?
Forum: Illumina/Solexa 12-02-2015, 09:14 AM
Replies: 12
Views: 2,103
Posted By jwfoley
Yes, it is the reverse complement of the Nextera...

Yes, it is the reverse complement of the Nextera read 1 adapter in the Illumina customer letter: http://support.illumina.com/downloads/illumina-customer-sequence-letter.html
Forum: Sample Prep / Library Generation 12-02-2015, 07:22 AM
Replies: 23
Views: 4,394
Posted By jwfoley
I've always just run it immediately, so I don't...

I've always just run it immediately, so I don't know what happens if you wait.
Forum: Sample Prep / Library Generation 12-02-2015, 06:32 AM
Replies: 23
Views: 4,394
Posted By jwfoley
Is this a typo? Even starting from a single...

Is this a typo? Even starting from a single molecule, 70 PCR cycles would give you about 21 g of DNA, according to a quick back-of-the-envelope calculation, though of course you'll exhaust the...
Forum: Sample Prep / Library Generation 12-02-2015, 06:14 AM
Replies: 23
Views: 4,394
Posted By jwfoley
The graph is consistent with my experience; 0.4X...

The graph is consistent with my experience; 0.4X isn't going to work and even 0.5X is dodgy. For the record, you really should recalibrate your SPRI conditions for your specific reaction mix, because...
Forum: Illumina/Solexa 12-02-2015, 06:02 AM
Replies: 12
Views: 2,103
Posted By jwfoley
Your .docx protocol says you can follow the...

Your .docx protocol says you can follow the standard MiSeq protocol, so I would do that. Illumina's .pdf protocol has a few special instructions to watch out for. I'm fairly certain the v2 kits...
Forum: Illumina/Solexa 12-01-2015, 09:47 AM
Replies: 12
Views: 2,103
Posted By jwfoley
The protocol you linked has another round of PCR...

The protocol you linked has another round of PCR after the target enrichment, so you'll still come out of it with double-stranded libraries.
Forum: Illumina/Solexa 12-01-2015, 07:20 AM
Replies: 12
Views: 2,103
Posted By jwfoley
This is just the older version of the standard...

This is just the older version of the standard protocol (for step 8 above). For the current MiSeq v3 kits, you start with 5 uL @ 4 nM, then add NaOH to denature it, then dilute this mix to 1000 uL...
Forum: Illumina/Solexa 12-01-2015, 06:13 AM
Replies: 12
Views: 2,103
Posted By jwfoley
Oh, so you're asking about a third thing, the...

Oh, so you're asking about a third thing, the pooling after library prep but before target enrichment.

The steps are, roughly:

Isolate 50 ng gDNA from each sample
Normalize each gDNA sample...
Forum: Illumina/Solexa 12-01-2015, 04:49 AM
Replies: 12
Views: 2,103
Posted By jwfoley
Are you asking about the amount of DNA you use...

Are you asking about the amount of DNA you use for library prep, or the amount of library you use for sequencing?

The final amount of library you use for sequencing is 5 uL @ 4 nM, at least on a...
Forum: Core Facilities 11-30-2015, 01:27 PM
Replies: 10
Views: 4,905
Posted By jwfoley
I can't help you, but can you tell us what issues...

I can't help you, but can you tell us what issues you had and whether you'd already tried the same libraries on a different machine?
Forum: Illumina/Solexa 11-30-2015, 01:13 PM
Replies: 13
Views: 3,365
Posted By jwfoley
Yes, N + 25 is the pattern we found in the...

Yes, N + 25 is the pattern we found in the manual, though it looks like they might be removing that information in more recent editions. And regarding the software, it seems to be getting more...
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