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Forum: Sample Prep / Library Generation 12-19-2018, 03:49 PM
Replies: 15
Views: 12,799
Posted By cmbetts
Any polymerase with 3' Exonuclease activity can...

Any polymerase with 3' Exonuclease activity can do end repair in the presence of nucleotides, which would include many PCR polymerases. Here's a paper using Pfu for old school cloning...
Forum: Bioinformatics 11-26-2018, 04:47 PM
Replies: 2
Views: 306
Posted By cmbetts
The way I handled it in the past was to build my...

The way I handled it in the past was to build my STAR index with both genomes/annotations simultaneously making sure to rename the chromosomes such that they're unique ("human_chr1" and "mouse_chr1"...
Forum: Bioinformatics 11-01-2018, 05:21 PM
Replies: 1
Views: 386
Posted By cmbetts
It looks like the BAM Analysis Kit software is...

It looks like the BAM Analysis Kit software is trying to do a whole lot more than generating a VCF file (replicating commercial genealogy reports). If what you need is a VCF file, there are many...
Forum: Sample Prep / Library Generation 11-01-2018, 05:10 PM
Replies: 3
Views: 650
Posted By cmbetts
Probably fine. DNA is pretty stable at RT in...

Probably fine. DNA is pretty stable at RT in standard storage buffers. You can always verify the accuracy by rerunning some previously quanted samples.
Forum: RNA Sequencing 06-21-2018, 02:23 PM
Replies: 2
Views: 1,135
Posted By cmbetts
Have you verified with your sequencing provider...

Have you verified with your sequencing provider that they gave you the right data back? I've previously had a vender return another user's data to me. Luckily, we had used a custom protocol and it...
Forum: Sample Prep / Library Generation 06-08-2018, 09:59 AM
Replies: 270
Views: 109,701
Posted By cmbetts
There's tons of low MW products there indicating...

There's tons of low MW products there indicating massive RNA degradation, which you have in both your cell and purified RNA samples. Did you check the RIN of your pure RNA? If it's high, then...
Forum: Sample Prep / Library Generation 05-16-2018, 03:44 PM
Replies: 4
Views: 975
Posted By cmbetts
That protocol looks like a scaled down version of...

That protocol looks like a scaled down version of the various homebrew AmpureXP recipes out there. It's probably a cost savings measure to avoid the premium on the real deal. I'd agree with the...
Forum: Sample Prep / Library Generation 10-03-2017, 01:15 PM
Replies: 6
Views: 1,136
Posted By cmbetts
Each gene is considered separately in regards to...

Each gene is considered separately in regards to the UMIs and the UMIs will be incorporated in a Poisson distribution (a la digital PCR). In the context of single cells, there's never going to be...
Forum: RNA Sequencing 08-30-2017, 11:04 AM
Replies: 6
Views: 1,099
Posted By cmbetts
1) These methods typically have 10-20% overall...

1) These methods typically have 10-20% overall efficiency. If your transcript of interest has low expression levels, it will frequently drop out. This is purely binomial statistics and can't be...
Forum: Sample Prep / Library Generation 08-07-2017, 10:33 AM
Replies: 2
Views: 995
Posted By cmbetts
There's a couple reasons not to worry about it,...

There's a couple reasons not to worry about it, but the primary ones are:
1) The denaturation temperature before RT isn't high enough to melt high MW gDNA
2) Even if gDNA was primed, the template...
Forum: Bioinformatics 06-01-2017, 01:50 PM
Replies: 4
Views: 2,825
Posted By cmbetts
Generally once you're >30 cells the average...

Generally once you're >30 cells the average expression starts to converge on bulk sequencing (Prepared by the same protocol). The issue here is that you can't recreate the 10X protocol on your bulk...
Forum: Illumina/Solexa 05-24-2017, 11:00 AM
Replies: 6
Views: 997
Posted By cmbetts
If there isn't anything sensitive, you could post...

If there isn't anything sensitive, you could post your adapter/primer sequences and sample sheet and have another set of eyes make sure everything is right on that end before trying to diagnose what...
Forum: Sample Prep / Library Generation 05-08-2017, 04:09 PM
Replies: 5
Views: 946
Posted By cmbetts
The Meyer lab protocol for ancient DNA would also...

The Meyer lab protocol for ancient DNA would also work http://www.nature.com/nprot/journal/v8/n4/abs/nprot.2013.038.html if you want to go full homebrew on your library prep
Forum: Sample Prep / Library Generation 05-08-2017, 10:57 AM
Replies: 5
Views: 946
Posted By cmbetts
One of the miRNA methods would probably work. ...

One of the miRNA methods would probably work. The RNA ligase used in most preps can also act on ssDNA. You might need to do a PNK treatment first. I haven't looked at the library prep chemistry in...
Forum: RNA Sequencing 04-21-2017, 09:45 AM
Replies: 4
Views: 1,557
Posted By cmbetts
You should expect to see considerably lower...

You should expect to see considerably lower counts to exons, mostly replaced by intron derived reads when comparing a ribo depletion to dT purified.
I'm surprised that your FF has lower exon counts...
Forum: Introductions 04-07-2017, 12:50 PM
Replies: 6
Views: 1,135
Posted By cmbetts
Are you sequencing mRNA or miRNA? I think it...

Are you sequencing mRNA or miRNA? I think it would be easier to give a suggestion if you described your experiment more.

I've never actually used the ERCCs for normalization. Most of the DE...
Forum: Introductions 04-06-2017, 10:13 AM
Replies: 6
Views: 1,135
Posted By cmbetts
The most commonly used exogenous spike in for...

The most commonly used exogenous spike in for RNA-Seq is the ERCCs sold through Thermo. Their user manuals have guidelines for adding the appropriate amount to different sample types to get the...
Forum: Illumina/Solexa 03-29-2017, 03:30 PM
Replies: 9
Views: 1,739
Posted By cmbetts
I think that 25% is a pretty good starting...

I think that 25% is a pretty good starting number. I used to regularly sequence libraries that looked like (8bp diversity + 15bp linker) x 3 on a NextSeq, and things tended to go badly when we...
Forum: Bioinformatics 03-20-2017, 11:34 AM
Replies: 2
Views: 855
Posted By cmbetts
Somebody more knowledgeable may correct me, but...

Somebody more knowledgeable may correct me, but the statistical methods used by DESeq2 rely on having the raw read counts to calculate power and significance, and therefore can't use normalized...
Forum: Sample Prep / Library Generation 03-20-2017, 11:26 AM
Replies: 3
Views: 1,561
Posted By cmbetts
1) If I remember right, they mention in the paper...

1) If I remember right, they mention in the paper that they got a statistically significant yield increase adding dNTPs at that step. It definitely would still work, just with somewhat lower...
Forum: Sample Prep / Library Generation 02-07-2017, 10:18 AM
Replies: 4
Views: 1,825
Posted By cmbetts
That thread specifically covers the SMART-Seq2...

That thread specifically covers the SMART-Seq2 protocol, so there might be other considerations depending on what method you're planning on using. The recommendations there will be appropriate for...
Forum: Sample Prep / Library Generation 12-04-2016, 02:47 PM
Replies: 4
Views: 2,169
Posted By cmbetts
I haven't done it for that kit specifically, but...

I haven't done it for that kit specifically, but I like putting the ERCCs into the lysis mix. That way the performance of the ERCCs can be a better reflection of RNases coming in with the cells and...
Forum: RNA Sequencing 11-14-2016, 09:53 AM
Replies: 4
Views: 1,429
Posted By cmbetts
The important thing is to not have any more...

The important thing is to not have any more library or NaOH than used when running a single library. The way that I typically do it is to mix an equal volume of the 4nM libraries together, just...
Forum: Bioinformatics 11-11-2016, 09:42 AM
Replies: 1
Views: 961
Posted By cmbetts
You might have luck using tools designed for...

You might have luck using tools designed for ChIP-Seq experiments. What you're trying to do with this RNA binding enzyme has a very similar objective, finding coverage peaks and binding motifs
Forum: RNA Sequencing 11-04-2016, 01:41 PM
Replies: 4
Views: 1,429
Posted By cmbetts
1) I'm not totally clear by what you mean by...

1) I'm not totally clear by what you mean by library normalization here, but since you reference the Nextera XT beads I'm assuming you mean standardizing the concentrations of multiple libraries so...
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