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Forum: Illumina/Solexa 07-25-2018, 06:28 AM
Replies: 2
Views: 465
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: 2
Views: 465
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: 860
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: 860
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: 860
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: 767
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: 767
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: 767
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: 767
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: 2,072
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: 529
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: 529
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: 1,246
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: 1,246
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: 1,246
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...
Forum: Illumina/Solexa 10-26-2017, 08:13 AM
Replies: 13
Views: 1,246
Posted By hoytpr
FYI: I wanted to post back about an error we got...

FYI: I wanted to post back about an error we got running 384 samples. We'd done 224 before, but going over 250 might give you an error like:

ERROR: bcl2fastq::common::Exception: 2017-Oct-25...
Forum: Illumina/Solexa 10-26-2017, 06:59 AM
Replies: 13
Views: 1,246
Posted By hoytpr
Thanks Jakob, I did NOT see that link up at the...

Thanks Jakob, I did NOT see that link up at the top right, and had manually yanked those sequences out from the DemuxSummaryF1Ln.txt files.

At least three and probably five of the samples have...
Forum: Illumina/Solexa 10-25-2017, 06:05 AM
Replies: 13
Views: 1,246
Posted By hoytpr
Thanks, seems so logical in retrospect. I...

Thanks, seems so logical in retrospect. I appreciate the help and will set it up today.
There are likely some failed samples also, according to the client. But hopefully we can get most of the 43...
Forum: Illumina/Solexa 10-24-2017, 02:48 PM
Replies: 13
Views: 1,246
Posted By hoytpr
Client sends wrong index. Run is done.

We had a client send us a set of 43 multiplexed samples. Although the NextSeq500 run went well, only 54% of the reads passed filter (PF). We normally get in the 93%+ range, so we dug but couldn't...
Forum: Illumina/Solexa 07-24-2017, 06:26 AM
Replies: 8
Views: 1,060
Posted By hoytpr
Thumbs up Just wanted to thank y'all for your help. All...

Just wanted to thank y'all for your help. All 200+ samples processed perfectly on the first try.
Forum: Illumina/Solexa 07-20-2017, 12:00 PM
Replies: 8
Views: 1,060
Posted By hoytpr
Tried both ways. Nope. I can't figure it out. IEM...

Tried both ways. Nope. I can't figure it out. IEM accepts the SamplePrepKit file with 384 wells. But when I try to load a plate with 384 wells I get an error saying "Index was outside the bounds of...
Forum: Illumina/Solexa 07-20-2017, 09:09 AM
Replies: 8
Views: 1,060
Posted By hoytpr
Hmmm, Actually, the IEM only gives me the...

Hmmm,
Actually, the IEM only gives me the option of 96-well plates. Do we need to manually create a new 384-well plate file first? Or use a HiSeq option?
Forum: Illumina/Solexa 07-20-2017, 09:03 AM
Replies: 8
Views: 1,060
Posted By hoytpr
Great, thanks. We have IEM and should be able to...

Great, thanks. We have IEM and should be able to handle the index names.
Forum: Illumina/Solexa 07-20-2017, 08:44 AM
Replies: 8
Views: 1,060
Posted By hoytpr
Question Any tricks? 244 sample SampleSheet.csv using NextSeq?

We are sequencing a pool of 244 samples on our new (now obsolete?) NextSeq500. First time with over 96 samples. Can we just make one SampleSheet.csv with the 244 samples and their indecies...
Forum: Illumina/Solexa 04-20-2017, 11:57 AM
Replies: 5
Views: 965
Posted By hoytpr
Thanks very much for the links and clarification....

Thanks very much for the links and clarification.

-p
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