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Forum: Sample Prep / Library Generation 05-17-2018, 06:56 PM
Replies: 1
Views: 268
Posted By austinso
Yes...that would be expected...can't really avoid...

Yes...that would be expected...can't really avoid it either...it depends on the coating on the bead (they have slightly different versions).

So one thing you could do is pellet the nucleic acids...
Forum: Sample Prep / Library Generation 05-13-2018, 07:06 PM
Replies: 4
Views: 479
Posted By austinso
Frankly, I think there is a typo in the protocol....

Frankly, I think there is a typo in the protocol. It shouldn't be pH 6...it really should be pH 8.

I'm not sure how you were even able to pH Tris to 6.0, since its pKa is 8.3...? i.e. it is no...
Forum: Sample Prep / Library Generation 04-01-2018, 07:12 PM
Replies: 2
Views: 998
Posted By austinso
more context is needed, but you could: 1....

more context is needed, but you could:

1. use A600 as a measure of particulate carryover within the supernatant
2. pre-bind beads with biotin-fluorescein, then use biotin to displace to get...
Forum: Illumina/Solexa 11-25-2017, 06:03 PM
Replies: 12
Views: 1,399
Posted By austinso
Doing this for a clinical lab is always a...

Doing this for a clinical lab is always a caveat...

Anyhow, I think all you would need to do is remove/comment out the lines from the Sony.xml file that is in the Exposures folder corresponding to...
Forum: Illumina/Solexa 10-18-2017, 02:31 PM
Replies: 12
Views: 1,399
Posted By austinso
Essentially, yes. Either a fixed number of random...

Essentially, yes. Either a fixed number of random bases (e.g. 5'-end of primer), or adding 0,+1, +2 bases of known composition.
Forum: Illumina/Solexa 10-18-2017, 11:38 AM
Replies: 12
Views: 1,399
Posted By austinso
Ultimately, the caveat is that the first few...

Ultimately, the caveat is that the first few bases are used as the QC step to identify successful cluster generation. So having dark cycles may not be in your best interest. But again depends on your...
Forum: Illumina/Solexa 10-18-2017, 10:19 AM
Replies: 26
Views: 4,051
Posted By austinso
For those with NovaSeqs, are you allowed to say...

For those with NovaSeqs, are you allowed to say how much the consumables are? Based on what people are charging for a lane, it looks like about $40K per run...
Forum: Illumina/Solexa 10-18-2017, 10:09 AM
Replies: 12
Views: 1,399
Posted By austinso
Been a while since I've done this on the MiSeq,...

Been a while since I've done this on the MiSeq, and I know they've consistently updated these files to make it harder to modify...

All the protocols have traditionally been in XML files. So if you...
Forum: Sample Prep / Library Generation 05-16-2017, 08:50 AM
Replies: 12
Views: 6,843
Posted By austinso
Just to add clarification, usually the loss is...

Just to add clarification, usually the loss is associated with the "dead volume" (i.e. the liquid between beads) and sometimes the wetting of the plastic surface. So the more beads you use, the...
Forum: Sample Prep / Library Generation 05-09-2017, 07:44 AM
Replies: 5
Views: 799
Posted By austinso
I second the Meyer protocol. In fact he alludes...

I second the Meyer protocol. In fact he alludes to that application in that protocol. Just remember to scale according to molarity and not to mass. Works well if everything being cobbled together is...
Forum: Illumina/Solexa 04-18-2017, 06:16 PM
Replies: 19
Views: 4,262
Posted By austinso
Makes me wonder if single molecule consensus is...

Makes me wonder if single molecule consensus is out of the question, since it would seem that the mitigation strategy requires defined barcode sequences.
Forum: Illumina/Solexa 04-01-2017, 11:49 AM
Replies: 113
Views: 22,311
Posted By austinso
Re: 3000/4000 From what I could glean, based on...

Re: 3000/4000
From what I could glean, based on the published specs (which are really vague, perhaps on purpose), the amount of library loaded ranges between 3-9 billion.

The yield is 0.75...
Forum: Illumina/Solexa 04-01-2017, 11:13 AM
Replies: 113
Views: 22,311
Posted By austinso
Apparently for all of them. And that is the lower...

Apparently for all of them. And that is the lower end (attached see pg. 16).
Forum: Illumina/Solexa 03-22-2017, 04:11 PM
Replies: 113
Views: 22,311
Posted By austinso
On another note: 150 uL of a 1 nM library...

On another note:

150 uL of a 1 nM library (~90 billion molecules) minimum for loading is a lot of library when you consider you can get by with 1.4 billion for the NextSeq and 7 billion for the...
Forum: Illumina/Solexa 03-10-2017, 08:29 PM
Replies: 0
Views: 665
Posted By austinso
Lane area versus tile area

Anyone know where to find data on the fractional area imaged versus the flow cell area for a MiSeq vs. NextSeq500. Thanks.
Forum: Illumina/Solexa 03-16-2016, 09:40 AM
Replies: 6
Views: 2,143
Posted By austinso
Huh...interesting! Very much appreciated! Do...

Huh...interesting! Very much appreciated!

Do you have an idea of what those 2 really high cluster density runs/samples were?

Best

Austin
Forum: Illumina/Solexa 03-16-2016, 07:16 AM
Replies: 6
Views: 2,143
Posted By austinso
Thanks! Interesting that it seems that q30 seems...

Thanks! Interesting that it seems that q30 seems to collapse at ~200K/mm2 with the exception of a couple of "outliers".

Question: There seems to be two kinds of sequencing kits or 2 library...
Forum: Illumina/Solexa 03-15-2016, 07:53 PM
Replies: 6
Views: 2,143
Posted By austinso
NextSeq500 optimal cluster density?

Starting using our NextSeq500, and trying to gauge what the consensus is for ideal cluster density. FAE recommends 150-200K/mm2.

More importantly, I wanted to know what people's experiences are...
Forum: Sample Prep / Library Generation 03-08-2016, 02:03 PM
Replies: 28
Views: 9,644
Posted By austinso
Within chemistries, pretty good. We can certainly...

Within chemistries, pretty good. We can certainly dial in 1000K/mm2 when the libraries are produced with a standardized workflow.



I think that within libraries from a given chemistry, you...
Forum: Sample Prep / Library Generation 03-07-2016, 09:18 PM
Replies: 28
Views: 9,644
Posted By austinso
Yes. That is a huge barrier. That being said,...

Yes. That is a huge barrier. That being said, cost per sample will be less than KAPA, since you only need to dilute and measure the sample at 1e-6 to get number of molecules. No standards required....
Forum: Illumina/Solexa 02-16-2016, 10:44 PM
Replies: 50
Views: 9,596
Posted By austinso
So...did you do the shipping yourself and not pay...

So...did you do the shipping yourself and not pay the fees...?



You didn't have an external back up of your sequencing data...? Or are you referring to Basespace? You certainly have access to...
Forum: Illumina/Solexa 02-13-2016, 10:08 AM
Replies: 50
Views: 9,596
Posted By austinso
Post wasn't in reply to you. And I wasn't...

Post wasn't in reply to you. And I wasn't questioning the legality of your purchase. But it did remind me to post what I learned recently from our reps in the Bay area.

And for clarifications...
Forum: Illumina/Solexa 02-12-2016, 12:18 PM
Replies: 50
Views: 9,596
Posted By austinso
FWIW Illumina instituted a policy this year...

FWIW

Illumina instituted a policy this year where they will now officially recognize third-party purchased equipment and allow it to be put on a service contract with the following caveats:

1....
Forum: Core Facilities 01-18-2016, 06:25 PM
Replies: 5
Views: 3,006
Posted By austinso
MiSeqDx

Just wondering how many are out there in the field that people are willing to admit they have...? Or is it like Bigfoot, rumors of their presence in the wild but not actually seeing them firsthand.
...
Forum: Sample Prep / Library Generation 01-18-2016, 06:07 PM
Replies: 5
Views: 4,105
Posted By austinso
Probably more important for PacBio, but azide is...

Probably more important for PacBio, but azide is present (as a preservative), and that can affect polymerase activity.
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