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Forum: RNA Sequencing 06-21-2018, 01:23 PM
Replies: 2
Views: 657
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: 268
Views: 98,883
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: 600
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: 911
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: 934
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: 847
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: 2,024
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: 827
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: 815
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: 815
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: 1,235
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, 11:50 AM
Replies: 6
Views: 972
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, 09:13 AM
Replies: 6
Views: 972
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, 02:30 PM
Replies: 9
Views: 1,474
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, 10:34 AM
Replies: 2
Views: 685
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, 10:26 AM
Replies: 1
Views: 691
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, 09:18 AM
Replies: 4
Views: 1,390
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, 01:47 PM
Replies: 4
Views: 1,978
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, 08:53 AM
Replies: 4
Views: 1,256
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, 08:42 AM
Replies: 1
Views: 844
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, 12:41 PM
Replies: 4
Views: 1,256
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...
Forum: Bioinformatics 10-25-2016, 09:31 AM
Replies: 10
Views: 6,126
Posted By cmbetts
Generally, you can't just drop a linux binary...

Generally, you can't just drop a linux binary into a Cygwin environment and expect it to run. As you alluded to, you almost certainly have to use MinGW to compile your own binaries. As someone...
Forum: RNA Sequencing 10-25-2016, 09:17 AM
Replies: 5
Views: 1,822
Posted By cmbetts
I've never seen any tissue dependence on rRNA...

I've never seen any tissue dependence on rRNA depletion. It's all happening on purified RNA, so unless there's a wacky rRNA isoform not covered by the kit's probe set, it should work fine.
I've...
Forum: Bioinformatics 10-17-2016, 04:34 PM
Replies: 2
Views: 773
Posted By cmbetts
I can't speak for why Illumina hasn't done it,...

I can't speak for why Illumina hasn't done it, but at the companies I've worked at, we kept it at six or eight for Illumina compatibility.
Since almost all of Illumina's kits use six or eight, it's...
Forum: General 10-17-2016, 10:04 AM
Replies: 6
Views: 2,065
Posted By cmbetts
I would venture to guess that it's probably got...

I would venture to guess that it's probably got something to do with the buffer used in the digest. Several years ago, I was screening polymerases for library amplification and found that the...
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