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Forum: Ion Torrent 08-02-2019, 08:22 AM
Replies: 16
Views: 835
Posted By cmbetts
Is there a reason you need your own sequencer? ...

Is there a reason you need your own sequencer? You're highly unlikely to ever break even vs making homebrew Illumina libraries and sending them to a service facility that will pool them onto a...
Forum: Sample Prep / Library Generation 05-22-2019, 09:19 AM
Replies: 2
Views: 667
Posted By cmbetts
The FA/BA is probably a more accurate measurement...

The FA/BA is probably a more accurate measurement (although I trust qubit more for concentrations >1ng/ul). It's easy to get background readings of 0.1-0.3ng/ul by qubit. Did you run a negative...
Forum: General 05-14-2019, 04:05 PM
Replies: 1
Views: 1,110
Posted By cmbetts
Well that sure cleared up how to translate from...

Well that sure cleared up how to translate from one programming language from another...

Your PI probably wants it in Perl because they can read/write Perl, but not Python and want to double check...
Forum: Sample Prep / Library Generation 04-05-2019, 01:41 PM
Replies: 3
Views: 464
Posted By cmbetts
Take a page out of old school RACE (or...

Take a page out of old school RACE (or Tang/Quartz scRNA-Seq for a more modern example). Add a homopolymer tail using TdT, perform second strand with a complementary primer also with a defined tag,...
Forum: Sample Prep / Library Generation 04-05-2019, 01:21 PM
Replies: 10
Views: 1,031
Posted By cmbetts
Template switching definitely doesn't need a 5'...

Template switching definitely doesn't need a 5' cap to work. Clontech's (Takara) SMARTer stranded kits all use template switching on chemically fragmented RNA with random priming. The internal...
Forum: Bioinformatics 04-01-2019, 11:22 AM
Replies: 7
Views: 604
Posted By cmbetts
That sequence isn't derived from the fragment...

That sequence isn't derived from the fragment you're trying to sequence. 100% of it was chemically synthesized by your oligo synthesis company. It match close enough to your insert of interest to...
Forum: Sample Prep / Library Generation 03-01-2019, 09:31 AM
Replies: 10
Views: 1,031
Posted By cmbetts
I'd start with as SMARTseq2 like protocol as fits...

I'd start with as SMARTseq2 like protocol as fits with your design and optimize from there.
I can't go too much in the black magic parts (former Clontech/Takara employee, and still friendly with...
Forum: Sample Prep / Library Generation 03-01-2019, 09:10 AM
Replies: 10
Views: 1,031
Posted By cmbetts
Are you using default SSII buffer? MgCl2 levels...

Are you using default SSII buffer? MgCl2 levels are very important for TS, and higher than most standard buffers.
Forum: RNA Sequencing 02-28-2019, 04:51 PM
Replies: 2
Views: 985
Posted By cmbetts
I'd be careful about using them for Prokaryotes. ...

I'd be careful about using them for Prokaryotes. Many of them are actually bacterial genes, mostly B.subtilis if I remember correctly, but also some antibiotic resistance genes descended from Affy...
Forum: General 02-14-2019, 12:52 PM
Replies: 1
Views: 1,061
Posted By cmbetts
Very doable, although random priming isn't...

Very doable, although random priming isn't necessary for second strand if you're using standard Gubler-Hoffman, the RNaseH handles the priming. This Nature Methods paper from the Broad should give...
Forum: Sample Prep / Library Generation 12-19-2018, 02:49 PM
Replies: 15
Views: 16,184
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, 03:47 PM
Replies: 2
Views: 670
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, 04:21 PM
Replies: 1
Views: 557
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, 04:10 PM
Replies: 3
Views: 887
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, 01:23 PM
Replies: 2
Views: 1,370
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, 08:59 AM
Replies: 278
Views: 121,173
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, 02:44 PM
Replies: 4
Views: 1,303
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, 12:15 PM
Replies: 6
Views: 1,441
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, 10:04 AM
Replies: 6
Views: 1,314
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, 09:33 AM
Replies: 2
Views: 1,191
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, 12:50 PM
Replies: 4
Views: 3,523
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, 10:00 AM
Replies: 6
Views: 1,214
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, 03:09 PM
Replies: 5
Views: 1,084
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, 09:57 AM
Replies: 5
Views: 1,084
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, 08:45 AM
Replies: 4
Views: 2,040
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...
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