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Search: Posts Made By: thomasblomquist
Forum: Oxford Nanopore 12-01-2015, 09:21 AM
Replies: 6
Views: 1,887
Posted By thomasblomquist
Interesting, I tried the combined PBcR MHAP...

Interesting, I tried the combined PBcR MHAP pipeline with the oxford.spec and arrived at an assembly in 20 minutes with 98% match to the NCBI ref seq for Lambda.

The DALIGNER, POA and RunCA with...
Forum: Oxford Nanopore 11-25-2015, 04:55 AM
Replies: 6
Views: 1,887
Posted By thomasblomquist
Nanocorrect (daligner + poa), is the step...

Nanocorrect (daligner + poa), is the step preceding the celera assembly and nanopolish. This is to say, PBcR and nanopolish are next once the POA is done... When it gets done.
Forum: Oxford Nanopore 11-24-2015, 05:49 PM
Replies: 6
Views: 1,887
Posted By thomasblomquist
Partial Order Alignment Step

I'm running through Jared and Nick's Nature Methods de novo assembly approach on my Lambda burn-in FAST5 data. Just to get the pipeline up and running and some familiarity using a focused data set....
Forum: Illumina/Solexa 03-13-2015, 05:18 AM
Replies: 1
Views: 811
Posted By thomasblomquist
Depending on the type of library preparation...

Depending on the type of library preparation method you use, it could be heterodimers between non-complementary templates within your sample preparation. These heterodimers have complementarity on...
Forum: Ion Torrent 03-10-2015, 08:44 AM
Replies: 2
Views: 2,333
Posted By thomasblomquist
Ditto with the above. Is this before or after...

Ditto with the above. Is this before or after adapter and barcode trimming?

In addition: Most fragment analyzers "phorese" under non-denaturing conditions. Library preparation can lead to...
Forum: Illumina/Solexa 03-04-2015, 05:09 AM
Replies: 13
Views: 2,805
Posted By thomasblomquist
1 in a million cross-overs. The incidence of...

1 in a million cross-overs. The incidence of having one of the indexes cross-over a mispair with another index/barcode is ~1:1000 (if done optimally). The error rate is multiplicative for the dual...
Forum: Illumina/Solexa 03-03-2015, 05:10 AM
Replies: 13
Views: 2,805
Posted By thomasblomquist
Just as a follow up to the above posts. It is...

Just as a follow up to the above posts. It is certainly easy enough to dual index with single end read (just needs to be long enough to reach the other end and input the barcode). The downside to...
Forum: Illumina/Solexa 03-01-2015, 05:11 AM
Replies: 13
Views: 2,805
Posted By thomasblomquist
http://m.nar.oxfordjournals.org/content/early/2011...

http://m.nar.oxfordjournals.org/content/early/2011/10/21/nar.gkr771.full.pdf

I had always found this article a good starting point for understanding barcode cross contamination issues.

Personal...
Forum: Illumina/Solexa 02-25-2015, 09:47 AM
Replies: 5
Views: 1,661
Posted By thomasblomquist
I do it all the time. I have my target specific...

I do it all the time. I have my target specific primers with "tails on them." These tails are "universal" sequences alien to the genome your targeting. I then add on the "platform" specific P5/P7...
Forum: Illumina/Solexa 10-14-2014, 09:04 AM
Replies: 2
Views: 1,043
Posted By thomasblomquist
I recommend you peruse the findings from the FDA...

I recommend you peruse the findings from the FDA sponsored Sequencing Quality Control Consortium project http://www.nature.com/nbt/journal/v32/n9/full/nbt.3025.html .

One of the principle findings...
Forum: Ion Torrent 07-28-2014, 01:16 PM
Replies: 1
Views: 1,177
Posted By thomasblomquist
Can you give a little more description? What is...

Can you give a little more description? What is your setup, homebrew, etc? How are the libraries being made, contamination between runs, between specimens within a run, etc.

More info.

-Tom
Forum: Illumina/Solexa 06-04-2014, 10:19 AM
Replies: 2
Views: 3,370
Posted By thomasblomquist
This is indeed concerning. We synthesized our...

This is indeed concerning. We synthesized our in-line barcoding reagents to have the first 4 bases be randomized followed by our less complex sequences.

Let us all know if you have any issues...
Forum: Illumina/Solexa 06-02-2014, 10:11 PM
Replies: 14
Views: 4,641
Posted By thomasblomquist
http://m.pnas.org/content/111/5/1891.full ...

http://m.pnas.org/content/111/5/1891.full

See above. Careful what you assume is a "duplicate."

There are fragmentation and ligation hotspots that may mimic duplicate reads of an amplified...
Forum: Illumina/Solexa 04-15-2014, 07:13 PM
Replies: 58
Views: 34,376
Posted By thomasblomquist
Hi Kristen, Some time back you stumbled...

Hi Kristen,

Some time back you stumbled across our paper on targeted RNA seq: http://seqanswers.com/forums/showthread.php?t=35379

The use of the competitive internal standard molecules...
Forum: Illumina/Solexa 02-21-2014, 10:45 AM
Replies: 3
Views: 1,098
Posted By thomasblomquist
Post-PCR cleanup of multiplex amplicon library...

Post-PCR cleanup of multiplex amplicon library prep is almost a given. Ampure beads, gel electrophoresis and band cut out, column exclusions, etc, are all decent options for Illumina library prep...
Forum: Illumina/Solexa 02-07-2014, 08:39 AM
Replies: 6
Views: 2,341
Posted By thomasblomquist
The issue with not having biological/library...

The issue with not having biological/library replicates, aside, yes, you can trim the 3' 100 bases from your reads to achieve a pseudo-50 base read length.

I've done this for comparison purposes...
Forum: Ion Torrent 01-29-2014, 09:19 AM
Replies: 14
Views: 6,448
Posted By thomasblomquist
Heterodimerization is the primary cause of...

Heterodimerization is the primary cause of polyclonal bead population. Which is to say denaturation of complementary homodimer DNA strands, and subsequent reannealing with a somewhat complementary,...
Forum: Ion Torrent 12-12-2013, 08:04 AM
Replies: 6
Views: 5,873
Posted By thomasblomquist
Just to add some hear-say to the pile. But the...

Just to add some hear-say to the pile. But the delay in increased density may be a function of bleed-over from adjacent beads pumping out H+ ions during sequencing. The signal-noise ratio is...
Forum: Illumina/Solexa 11-29-2013, 01:46 PM
Replies: 2
Views: 4,063
Posted By thomasblomquist
QIIME pipeline a subset of your reads. A few...

QIIME pipeline a subset of your reads. A few "OTU" consensus sequences will come from this. Blast then against the NCBI nucleotide database and you'll have a general idea if it's technical...
Forum: Literature Watch 11-26-2013, 01:48 PM
Replies: 3
Views: 5,319
Posted By thomasblomquist
As to comment on why we moved away from...

As to comment on why we moved away from traditional RNA-seq. Some preliminary data from colleagues using unsupervised biomarker identification in RNA seq libraries from different clinical samples...
Forum: Literature Watch 11-26-2013, 01:38 PM
Replies: 3
Views: 5,319
Posted By thomasblomquist
Hi Kristen, You are correct in your...

Hi Kristen,

You are correct in your statement that we can provide back either median normalized abundance, reference gene normalized abundance, or absolute copies based on the internal standard...
Forum: Ion Torrent 11-14-2013, 07:20 AM
Replies: 14
Views: 6,448
Posted By thomasblomquist
Just finished developing our method for targeted...

Just finished developing our method for targeted quantitative RNA sequencing. Our studies use Ion torrent platform, but this approach works well on Illumina as well. Our unpublished data shows our...
Forum: Literature Watch 11-13-2013, 05:28 PM
Replies: 3
Views: 5,319
Posted By thomasblomquist
Targeted quantitative RNA-sequencing with interlibrary concordance and reduced cost

http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0079120

Hello SEQanswers Forum. Long time follower, occasional thread responder. I have been in the qPCR world for some years...
Forum: Illumina/Solexa 10-15-2013, 01:02 PM
Replies: 2
Views: 1,915
Posted By thomasblomquist
This is a research design specific question, and...

This is a research design specific question, and entails understanding whether your library is targeted or not. If targeted, and the gDNA targets are different than the cDNA targets, then yes these...
Forum: Illumina/Solexa 10-15-2013, 12:55 PM
Replies: 12
Views: 5,698
Posted By thomasblomquist
This is one reason why we now have placed our own...

This is one reason why we now have placed our own barcodes inline flanking our sequence of interest. They are read out in read 1 and 2. And based on these reads we demultiplex the sequences into...
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