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Forum: Sample Prep / Library Generation 05-27-2014, 08:37 AM
Replies: 4
Views: 3,857
Posted By bbeitzel
I think that we were a fairly early adopter of...

I think that we were a fairly early adopter of the Apollo, and at the time they weren't really pushing P-Suite. The main use for us is for library preps, and they already have protocols for most of...
Forum: Sample Prep / Library Generation 05-27-2014, 08:24 AM
Replies: 4
Views: 2,286
Posted By bbeitzel
Do you want to sequence RNA virus genomes out of...

Do you want to sequence RNA virus genomes out of your total nucleic acid mix? If so, you can simply DNase you total nucleic acid prep and then proceed with the RNA sample prep you are already doing.
Forum: Sample Prep / Library Generation 05-23-2014, 11:00 AM
Replies: 4
Views: 3,857
Posted By bbeitzel
We have a couple Apollo 324s, and I think all of...

We have a couple Apollo 324s, and I think all of the people in the lab really like them. We also have a Caliper SciClone, but the Apollo is much more user friendly than that (IMHO.)

One of the...
Forum: Illumina/Solexa 04-24-2014, 06:33 AM
Replies: 15
Views: 5,224
Posted By bbeitzel
We have been prepping these dual index paired end...

We have been prepping these dual index paired end libraries on our IntegenX Apollo 324, which essentially uses a TruSeq HT-like chemistry. I believe the adapters were purchased through IDT.

These...
Forum: Illumina/Solexa 04-21-2014, 09:54 AM
Replies: 15
Views: 5,224
Posted By bbeitzel
We are getting 600-700K clusters. The first read...

We are getting 600-700K clusters. The first read and index look fine, but the second index and reads fail (all Ns).
Forum: Illumina/Solexa 04-21-2014, 08:25 AM
Replies: 15
Views: 5,224
Posted By bbeitzel
HiSeq 2500 Rapid run dual index problems

Our lab is having problems getting successful dual index runs on the HiSeq 2500 rapid module (upgraded from the HiSeq 2000). Single index runs work, but on dual index runs the quality craps out...
Forum: Sanger/Dye Terminator 04-04-2014, 05:39 AM
Replies: 2
Views: 4,391
Posted By bbeitzel
You might get lower intensity fluorescence due to...

You might get lower intensity fluorescence due to loading less of the diluted DNA into the capillaries, but other than that it should be fine. We used to dry down our BigDye reactions and then...
Forum: Sample Prep / Library Generation 02-10-2014, 01:24 PM
Replies: 2
Views: 1,498
Posted By bbeitzel
Hi Gary, Do you know if it would be possible...

Hi Gary,

Do you know if it would be possible to use a denaturing loading dye to denature the samples prior to running on the Pippin Prep or ELF? I frequently denature RNA with a formalin-based...
Forum: Sample Prep / Library Generation 11-01-2013, 12:44 PM
Replies: 2
Views: 2,926
Posted By bbeitzel
.05 ng/ul is the same as .05 mg/L, not .05 kg/L ...

.05 ng/ul is the same as .05 mg/L, not .05 kg/L

.05 ng/ul works out to be 150 pM
Forum: Sample Prep / Library Generation 08-01-2013, 05:19 AM
Replies: 1
Views: 1,818
Posted By bbeitzel
In virus metagenome studies, nuclease treatment...

In virus metagenome studies, nuclease treatment is usually done prior to disrupting the virus particles. Therefore, any viral nucleic acids should be protected by the capsid.
Forum: Illumina/Solexa 05-09-2013, 01:40 PM
Replies: 18
Views: 5,981
Posted By bbeitzel
I've attached the white paper where they describe...

I've attached the white paper where they describe the kit. They've commercialized the technique described in 2 of the PNAS papers in the references.

Basically, they have a pool of 96 adapters...
Forum: Illumina/Solexa 04-09-2013, 12:57 PM
Replies: 58
Views: 39,977
Posted By bbeitzel
We usually do the post-run wash as soon as the...

We usually do the post-run wash as soon as the run finishes. If it finishes overnight, it gets washed first thing the next morning.
Forum: Illumina/Solexa 04-09-2013, 12:34 PM
Replies: 58
Views: 39,977
Posted By bbeitzel
We ran some libraries for another group that they...

We ran some libraries for another group that they had prepared in a totally separate lab. They picked up reads from our previous run in their data, and we picked up reads from their libraries in our...
Forum: Illumina/Solexa 04-09-2013, 04:47 AM
Replies: 58
Views: 39,977
Posted By bbeitzel
We see the carryover as well at around the same %...

We see the carryover as well at around the same % you are seeing it. We just updated to the most recent RTA/MCS, and in the new manual they change the wash to 0.5% Tween instead of water. We'll see...
Forum: Illumina/Solexa 03-13-2013, 05:08 AM
Replies: 4
Views: 2,031
Posted By bbeitzel
We've done several 1x300 runs with 300 cycle...

We've done several 1x300 runs with 300 cycle kits, and there is a noticeable dropoff in quality after ~base 250 on good runs. I can't imagine that a 1x500 run would give you more than ~300 bases of...
Forum: Illumina/Solexa 01-22-2013, 07:24 AM
Replies: 17
Views: 11,906
Posted By bbeitzel
We are seeing the same thing on our MiSeq runs. ...

We are seeing the same thing on our MiSeq runs. We were doing some pathogen identification runs, and were seeing cross-contamination in demultiplexed reads (ie. reads from "known" samples run on the...
Forum: Illumina/Solexa 01-02-2013, 07:55 AM
Replies: 9
Views: 3,283
Posted By bbeitzel
I'm not 100% sure, but I don't think the newly...

I'm not 100% sure, but I don't think the newly synthesized read 1 complementary strand is stripped away prior to starting the index and read 2 reads. If it was being stripped away, I would assume...
Forum: Illumina/Solexa 01-02-2013, 07:35 AM
Replies: 9
Views: 3,283
Posted By bbeitzel
The read 1 primer will only anneal to one of the...

The read 1 primer will only anneal to one of the strands, and the read 2 (and index) primer will only anneal to the other strand.
Forum: Illumina/Solexa 10-31-2012, 12:57 PM
Replies: 37
Views: 21,266
Posted By bbeitzel
We've been getting low cluster densities since...

We've been getting low cluster densities since our MiSeq was upgraded. I used to load at 8 pM (by Kapa qPCR) and get ~500-600K / mm2. Our last 2 runs were loaded at 15 pM and only gave ~150-175K /...
Forum: Sample Prep / Library Generation 09-26-2012, 04:46 AM
Replies: 3
Views: 2,206
Posted By bbeitzel
I routinely do this for sequencing viral genomes...

I routinely do this for sequencing viral genomes out of complex mixtures. I PCR ~6-10 amplicons spanning the genome (12kb) with ~300-500 bp overlap between the amplicons. I then mix the amplicons...
Forum: Bioinformatics 09-17-2012, 07:50 AM
Replies: 18
Views: 3,112
Posted By bbeitzel
Have you tried mapping integration sites as in: ...

Have you tried mapping integration sites as in:

http://www.ncbi.nlm.nih.gov/pubmed/19038346

Basically, you fragment the DNA, end repair, add adapter, and then PCR with a virus-specific primer...
Forum: Bioinformatics 09-17-2012, 07:30 AM
Replies: 3
Views: 2,338
Posted By bbeitzel
We had a similar issue with some metagenomics...

We had a similar issue with some metagenomics samples that we were doing for a collaborator, and if I recall correctly, we could identify the Sigma primers by examining a small number of the reads. ...
Forum: The Pipeline 08-08-2012, 05:22 AM
Replies: 103
Views: 142,546
Posted By bbeitzel
I saw a while back that ONT is hiring FASs...

I saw a while back that ONT is hiring FASs (http://www.nanoporetech.com/careers/job-centre/view/144). I took that as a sign that they were getting close to commercial launch, but maybe I am just...
Forum: Illumina/Solexa 08-06-2012, 12:10 PM
Replies: 83
Views: 27,688
Posted By bbeitzel
Or the right tool doesn't exist yet, so you make...

Or the right tool doesn't exist yet, so you make do with what you have...
Forum: Illumina/Solexa 08-06-2012, 12:08 PM
Replies: 20
Views: 20,158
Posted By bbeitzel
groad, If you have incorporated the...

groad,

If you have incorporated the Illumina-specific sequences (sequence read primer binding sites, bridge PCR sequences, index read primer site) into your primers, then the 12 Ns would come...
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