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Forum: Sample Prep / Library Generation 10-08-2019, 02:47 PM
Replies: 3
Views: 227
Posted By cmbetts
By bad annotation, I mean that the...

By bad annotation, I mean that the polyadenylation sites in RefSeq/Gencode are often incorrect, which can make it difficult to amplify the 3'UTR (Refseq in particular tends to choose the longest 3'...
Forum: Sample Prep / Library Generation 10-07-2019, 03:33 PM
Replies: 3
Views: 227
Posted By cmbetts
1. Sure, you can swap in different length RT...

1. Sure, you can swap in different length RT primers without a noticeable impact on performance
2. SSII is definitely better than SSIII, but I don't know about IV
3. I personally like Takara's...
Forum: General 10-01-2019, 12:57 PM
Replies: 4
Views: 464
Posted By cmbetts
Again, it's highly dependent on your situation. ...

Again, it's highly dependent on your situation.

You'll need to either buy a ssDNA library kit (usually a minimum 12rxn size) or put something homebrew together based on a published protocol. which...
Forum: General 09-30-2019, 11:48 AM
Replies: 4
Views: 464
Posted By cmbetts
Yes, but with levels of difficulty ranging from...

Yes, but with levels of difficulty ranging from trivial to very difficult. If they're for an NGS related application, there's a good chance they're just common primers you can pick from a catalogue...
Forum: Ion Torrent 08-02-2019, 08:22 AM
Replies: 19
Views: 2,209
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: 818
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: 1,606
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: 544
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: 1,158
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: 670
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: 1,158
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: 1,158
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: 1,113
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: 1,208
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: 17,041
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: 783
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: 611
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: 937
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: 1,407
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: 124,647
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: 1,384
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: 1,534
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: 1,362
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: 1,251
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: 3,631
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...
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