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Forum: Illumina/Solexa 01-08-2019, 09:29 AM
Replies: 5
Views: 614
Posted By hoytpr
Must just be primer dimers. I talked with...

Must just be primer dimers. I talked with Illumina today and they looked at it. Basically said it's normal for a NextSeq to have these runs of "N" (they call "N-masking when multiplexing") when...
Forum: Illumina/Solexa 01-07-2019, 12:03 PM
Replies: 5
Views: 614
Posted By hoytpr
The % NoCall looks a little weird: ...

The % NoCall looks a little weird:

https://www.dropbox.com/s/osm8higjbe93460/No-Calls.png?dl=0
Forum: Illumina/Solexa 01-07-2019, 11:54 AM
Replies: 5
Views: 614
Posted By hoytpr
Most everything looked okay. There is a little...

Most everything looked okay. There is a little glitch at 35bp in the data by Cycle Error rate

https://www.dropbox.com/s/ijb1ikhr9xahv1t/metrics.png?dl=0
Forum: Illumina/Solexa 01-07-2019, 09:26 AM
Replies: 5
Views: 614
Posted By hoytpr
High % "N" in first 35bp using MultiQC

Hi all,
Just did some sequencing for a client and got a little odd plot using MultiQC for the "FastQ per base N-Content". It looks like the first 35 bp had problems with "N" then suddenly stopped....
Forum: Core Facilities 11-29-2018, 10:16 AM
Replies: 1
Views: 2,458
Posted By hoytpr
Ramiro, you're gonna need a query-able database....

Ramiro, you're gonna need a query-able database. If MySql is not on your favorites list, Microsoft Access has a business template that can be hacked to run a core facility. It's a pain to transfer...
Forum: Illumina/Solexa 11-29-2018, 10:07 AM
Replies: 2
Views: 1,266
Posted By hoytpr
Hi cmccabe, I know this is a late reply, but cat9...

Hi cmccabe, I know this is a late reply, but cat9 is pretty much how any Nextseq500 is hooked up if you aren't using BaseSpace. Our server was set up with an Windows VM and them mapped as a drive to...
Forum: Illumina/Solexa 11-20-2018, 07:32 AM
Replies: 1
Views: 606
Posted By hoytpr
Rolling circle dsDNA vs ssDNA experience?

Does anyone have experience with samples using rolling circle amplification that can tell me whether they have relatively high or low amounts of ssDNA? The client is not local so it would help to...
Forum: Sample Prep / Library Generation 09-13-2018, 06:05 AM
Replies: 3
Views: 1,363
Posted By hoytpr
We just did a Zoom call with them, and I was very...

We just did a Zoom call with them, and I was very impressed. We will order one of their kits when we get in a large number of bacterial DNAs. They are working on RNA kits, but don't offer them yet....
Forum: Illumina/Solexa 08-27-2018, 08:37 AM
Replies: 6
Views: 1,399
Posted By hoytpr
Right, I suspect the final data might be...

Right, I suspect the final data might be affected, but in this case, the project was probably at least a year old. We just want to help.

We have both a Tapestation and 2100 and they tend to agree...
Forum: Illumina/Solexa 08-27-2018, 05:55 AM
Replies: 6
Views: 1,399
Posted By hoytpr
UCan'tBcereus: Your post could not have been...

UCan'tBcereus: Your post could not have been better timed. We have a client with some very precious samples that show both peaks, but the RIN scores are very low. We were considering turning down the...
Forum: Illumina/Solexa 07-25-2018, 06:28 AM
Replies: 6
Views: 1,399
Posted By hoytpr
Thanks for the great advice Jakob. Pete

Thanks for the great advice Jakob.

Pete
Forum: Illumina/Solexa 07-03-2018, 12:54 PM
Replies: 6
Views: 1,399
Posted By hoytpr
Opinion needed on Truseq vs Scriptseq

So quite simply, when doing the math, 48 bacterial RNA-seq samples (libraries) would cost over a thousand bucks more using Truseq + Ribozero, versus using ScriptSeq "complete" + Scriptseq "barcodes"...
Forum: Illumina/Solexa 02-13-2018, 07:53 AM
Replies: 5
Views: 1,026
Posted By hoytpr
So, you'd have to quantify by QPCR then, as...

So, you'd have to quantify by QPCR then, as fluorescence or Bioanalyzers won't tell you the number of correctly formatted sequences with adapters?
Forum: Illumina/Solexa 02-12-2018, 03:47 PM
Replies: 5
Views: 1,026
Posted By hoytpr
You're right, that does resemble over...

You're right, that does resemble over amplification. I was going to run the samples on a denaturing gel but that protocol says not to. Can it be fixed? I think the protocol was mostly custom. I'm not...
Forum: Illumina/Solexa 02-12-2018, 02:18 PM
Replies: 5
Views: 1,026
Posted By hoytpr
Small RNA library with 200bp extra peak

I just looked at our new small RNA library submitted by a respected lab on the Bioanalyzer. The small RNA library should be about 178, and it's there, but most of the samples have a broader but...
Forum: Illumina/Solexa 02-09-2018, 02:40 PM
Replies: 6
Views: 904
Posted By hoytpr
Just to put a tack in this thread. ...

Just to put a tack in this thread.
Communication in cores is always a key to success. The client created a perfectly compatible small RNA Illumina library. They just didn't know it.

Edit: They...
Forum: Illumina/Solexa 02-08-2018, 12:14 PM
Replies: 6
Views: 904
Posted By hoytpr
Hadn't thought of custom primer spike-ins. What a...

Hadn't thought of custom primer spike-ins. What a cool idea!

Just add in the same number of pMoles... as long as one end of the library attaches to the flowcell, and the custom primers make it to...
Forum: Illumina/Solexa 02-08-2018, 07:45 AM
Replies: 6
Views: 904
Posted By hoytpr
Hi GW, Yes I mean cBot (:o). But after digging...

Hi GW,
Yes I mean cBot (:o). But after digging deep in the protocol I saw this:

# During cluster generation on the cBot, it is necessary to select
the ‘‘SR_TubeStripHyb’’ recipe because the...
Forum: Illumina/Solexa 02-06-2018, 02:05 PM
Replies: 6
Views: 904
Posted By hoytpr
PARE sequencing; single-read cluster generation

We got a request to do PARE sequencing (J. Zhai et al. / Methods 67 (2014) 84–90, http://dx.doi.org/10.1016/j.ymeth.2013.06.025) but someone correct me if I'm wrong.

The "single-read cluster...
Forum: Illumina/Solexa 01-10-2018, 11:20 AM
Replies: 13
Views: 2,568
Posted By hoytpr
Turnaround is really important. The market for...

Turnaround is really important. The market for these machines is the ~$100 per bacterial genome smaller groups. 1.2 Gbp is about 8-10 bacteria at 30X coverage. If the libraries are cheap enough...
Forum: Illumina/Solexa 01-10-2018, 05:10 AM
Replies: 2
Views: 627
Posted By hoytpr
Yes, and it was confirmed yesterday to be a bug...

Yes, and it was confirmed yesterday to be a bug in IEM 1.14 and 1.15 (just released), but not in 1.11. I have not tested 1.12, or 1.13.
Forum: Illumina/Solexa 01-09-2018, 02:03 PM
Replies: 2
Views: 627
Posted By hoytpr
If you use IEM, watch out for A027

We just completed a sequencing run that used the Illumina A027 (or the AR027) index on one of the samples. Setting it up on the NEWER version of IEM incorrectly assigns it the sequence of ATTCTT, but...
Forum: Illumina/Solexa 10-26-2017, 12:37 PM
Replies: 13
Views: 1,535
Posted By hoytpr
Thanks. Yes, I learned a lesson today. The run...

Thanks. Yes, I learned a lesson today. The run was only slightly overclustered and the 384-index run had 90% PF (all the bad PF were labeled default or unknown). Our PhiX was only 1.2%.

After the...
Forum: Illumina/Solexa 10-26-2017, 11:21 AM
Replies: 13
Views: 1,535
Posted By hoytpr
I wrote a response earlier, but I was timed out...

I wrote a response earlier, but I was timed out and then the message must have gotten lost. I'll write another and try to cut/paste.
-pete
Forum: Illumina/Solexa 10-26-2017, 09:38 AM
Replies: 13
Views: 1,535
Posted By hoytpr
Seems to be entirely an error on the client's...

Seems to be entirely an error on the client's end. Apparently uses a lot of undergrads (which I fully support) but there apparently some students remarking about "problems" and "mistakes" the others...
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