SEQanswers

Go Back   SEQanswers > Search Forums


Showing results 1 to 25 of 500
Search took 0.31 seconds.
Search: Posts Made By: pmiguel
Forum: Sample Prep / Library Generation 08-31-2018, 05:44 AM
Replies: 6
Views: 507
Posted By pmiguel
Thanks UCan'tBcereus, I had mis-read the PON. ...

Thanks UCan'tBcereus,
I had mis-read the PON.
You can just order oligos from IDT if you want all 384 combinations.
We already ordered a 48x48 set. Although since it is for the NovaSeq, it only...
Forum: Sample Prep / Library Generation 08-31-2018, 03:03 AM
Replies: 6
Views: 507
Posted By pmiguel
Don't forget the Picelli method: ...

Don't forget the Picelli method:
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4248319/
Forum: Sample Prep / Library Generation 08-30-2018, 08:38 AM
Replies: 6
Views: 507
Posted By pmiguel
I thought the Nextera XT kit had been...

I thought the Nextera XT kit had been discontinued in favor of the Nextera Flex kit?
Anyway, how about using Simone Picelli's Tn5 method for library prep?

Also, what about AmpliSeq? Now it is...
Forum: Sample Prep / Library Generation 08-10-2018, 08:18 AM
Replies: 2
Views: 556
Posted By pmiguel
We had some Nextera libraries that looked like...

We had some Nextera libraries that looked like this recently -- turned out they were 10X overloaded.

Also, they are a client's ATAC-seq library? We recently had a set of these where the client had...
Forum: Illumina/Solexa 07-26-2018, 08:40 AM
Replies: 14
Views: 712
Posted By pmiguel
So, I don't know if I should even bring this up....

So, I don't know if I should even bring this up. It seems to be pretty far out of most lab's comfort zone.

But native DNA electrophoresis hides a critical issue we see with some substantial...
Forum: Illumina/Solexa 07-25-2018, 06:02 AM
Replies: 14
Views: 712
Posted By pmiguel
Those mainly look like you read length was...

Those mainly look like you read length was overrunning your amplicon lengths. Did you check your libraries or the library pool on a bioanalyzer?

--
Phillip
Forum: Illumina/Solexa 07-25-2018, 04:20 AM
Replies: 14
Views: 712
Posted By pmiguel
Yeah, I've noticed with v2 chemistry 500 cycle...

Yeah, I've noticed with v2 chemistry 500 cycle runs that going above 800 K/mm2 results in higher loss of read quality towards the ends of the reads when using high bias (low complexity) samples.
I...
Forum: Illumina/Solexa 07-18-2018, 02:39 AM
Replies: 2
Views: 681
Posted By pmiguel
No idea. Maybe take a look at them untrimmed to...

No idea. Maybe take a look at them untrimmed to see what adapter type is carrying the insert.

--
Phillip
Forum: Sample Prep / Library Generation 06-25-2018, 07:18 AM
Replies: 3
Views: 1,025
Posted By pmiguel
As nucacidhunter stated, there is a bias towards...

As nucacidhunter stated, there is a bias towards shorter library fragments during clustering.

Also, keep in mind, that the bioanalyzer and (I presume) tapestation signal scale with number of...
Forum: Illumina/Solexa 06-14-2018, 12:56 PM
Replies: 3
Views: 692
Posted By pmiguel
Neither of these warnings are worrisome. BTW,...

Neither of these warnings are worrisome.
BTW, a 4 million base pair genome provides only 8 million possible places to start a sequence read. Any more reads that that and you will certainly have...
Forum: Illumina/Solexa 06-13-2018, 08:37 AM
Replies: 4
Views: 578
Posted By pmiguel
After heat denaturation you will probably still...

After heat denaturation you will probably still be able to visualize them on an RNA chip. Actually, I'm surprised the the tape station DNA fluor would be double-strand specific. The bioanalyzer DNA...
Forum: Sample Prep / Library Generation 06-08-2018, 04:11 AM
Replies: 10
Views: 1,588
Posted By pmiguel
Thanks for posting your results. I suspect that...

Thanks for posting your results.
I suspect that most of your dsDNA flourimetric signal comes from double-stranded RNA--ie, RNA folded into secondary structures. My guess is that the fluor used does...
Forum: Sample Prep / Library Generation 06-04-2018, 11:07 AM
Replies: 10
Views: 1,588
Posted By pmiguel
Okay, your minus strand reads may come from...

Okay, your minus strand reads may come from genomic DNA. But you have to treat DNA pretty roughly to fragment it down to a size (less than 500 bp) that could be amplified by PCR after ligation of...
Forum: Sample Prep / Library Generation 06-04-2018, 07:58 AM
Replies: 10
Views: 1,588
Posted By pmiguel
Since Ribo-Zero depleted RNA will include...

Since Ribo-Zero depleted RNA will include non-poly A+ messages, then it isn't clear to me that they would be expected to be on the same strand as the cDNA. Certainly it is possible that various sorts...
Forum: Sample Prep / Library Generation 05-23-2018, 11:00 AM
Replies: 5
Views: 977
Posted By pmiguel
We run qc chips for people doing ATACseq with...

We run qc chips for people doing ATACseq with some frequency. I'm never sure what a "good" result is.
I guess you want a large percentage of your DNA to be in the nucleosome monomer size range? But...
Forum: Bioinformatics 05-10-2018, 06:14 AM
Replies: 1
Views: 312
Posted By pmiguel
Unlikely that they derive from RNA. My guess...

Unlikely that they derive from RNA. My guess would be that if you took a few kb of sequence from the largest contig and did a blastn to nt at genbank you would get a hit to chloroplast. Speculation...
Forum: Illumina/Solexa 04-25-2018, 08:48 AM
Replies: 6
Views: 966
Posted By pmiguel
Although, a little weirdly, the Epicentre...

Although, a little weirdly, the Epicentre "scriptseq" kit is sold by Illumina and that uses step-out PCR to add the indexes, etc.

Well, not indexes but "index"-- I don't think ScriptSeq uses dual...
Forum: Illumina/Solexa 04-23-2018, 06:40 AM
Replies: 7
Views: 1,365
Posted By pmiguel
Illumina instruments preferentially cluster...

Illumina instruments preferentially cluster shorter amplicons. The Agilent chip you include shows visible material below 600 bp. Since this lower molecular weight material is present, it will...
Forum: Metagenomics 04-18-2018, 10:47 AM
Replies: 13
Views: 1,812
Posted By pmiguel
Yes, we tried it, although not extensively. It...

Yes, we tried it, although not extensively. It worked, just no better than without denaturation.

Remember there is nothing to stop the denatured molecules from re-annealing--especially at the...
Forum: Metagenomics 04-18-2018, 06:45 AM
Replies: 13
Views: 1,812
Posted By pmiguel
Just keep in mind you would likely need to run...

Just keep in mind you would likely need to run the samples after heat denaturation/snap cooling on a denature chip -- eg, an RNA chip.

--
Phillip
Forum: Metagenomics 04-17-2018, 12:42 PM
Replies: 13
Views: 1,812
Posted By pmiguel
Mostly from amplicon libraries submitted by...

Mostly from amplicon libraries submitted by customer labs. I'm speculating that they are primers and primer-dimers. But who knows? We have seen them in some Nextera XT libraries. And to less extents...
Forum: Metagenomics 04-17-2018, 08:13 AM
Replies: 13
Views: 1,812
Posted By pmiguel
Those are just 16S sequences. Since FastQC only...

Those are just 16S sequences. Since FastQC only appears to be looking at the first 50 bases, I don't think it is a good assessment of the presence of primer dimers in your libraries. You could follow...
Forum: Metagenomics 04-16-2018, 04:50 AM
Replies: 13
Views: 1,812
Posted By pmiguel
The length of the reads in your .fastq file...

The length of the reads in your .fastq file likely does not include low-quality base clipping. The MiSeq will happily call bases on background noise. So the length of the reads is not diagnostic for...
Forum: Illumina/Solexa 04-05-2018, 10:43 AM
Replies: 1
Views: 700
Posted By pmiguel
Not clear what this is. Or what "minimizing index...

Not clear what this is. Or what "minimizing index hopping" means. What fold reduction are they seeing with use of this reagent?

More info, including the manual here...
Forum: RNA Sequencing 04-03-2018, 08:38 AM
Replies: 7
Views: 1,769
Posted By pmiguel
Okay, then my hypothesis is that the reverse read...

Okay, then my hypothesis is that the reverse read is always reading 5' in the cDNA of the forward read. So that elevated AT% is just polyA tail. Or, since you mention hits to "predicted genes", the...
Showing results 1 to 25 of 500

 


All times are GMT -8. The time now is 11:35 PM.


Powered by vBulletin® Version 3.8.9
Copyright ©2000 - 2018, vBulletin Solutions, Inc.
Single Sign On provided by vBSSO