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Forum: Metagenomics 04-18-2018, 10:47 AM
Replies: 13
Views: 558
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: 558
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: 558
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: 558
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: 558
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: 323
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: 1,108
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...
Forum: RNA Sequencing 03-30-2018, 01:07 PM
Replies: 7
Views: 1,108
Posted By pmiguel
How were the libraries constructed? What average...

How were the libraries constructed? What average insert size did they have? Were the libraries stranded?

--
Phillip
Forum: RNA Sequencing 03-30-2018, 08:40 AM
Replies: 7
Views: 1,108
Posted By pmiguel
RNAseq? Tell us more about the libraries. Are...

RNAseq?
Tell us more about the libraries.
Are you say the forward reads show this bimodal GC distribution but the reverse reads do not? Or does "_1" and "_2" mean something else.
--
Phillip
Forum: Illumina/Solexa 03-19-2018, 05:56 AM
Replies: 7
Views: 1,321
Posted By pmiguel
How did your phiX run look? -- Phillip

How did your phiX run look?

--
Phillip
Forum: Core Facilities 03-15-2018, 08:33 AM
Replies: 7
Views: 993
Posted By pmiguel
We just create a pool using 1 ul of each library...

We just create a pool using 1 ul of each library to be tested. But when we analyze the results we are looking at relative clusters/ul from libraries of the same type. That info is used to make a pool...
Forum: Core Facilities 03-15-2018, 08:10 AM
Replies: 7
Views: 993
Posted By pmiguel
Hi Sergio, I agree. I would not expect it to...

Hi Sergio,
I agree. I would not expect it to work, but it does. We've tried a denature+snap cool prior to ampure and it performs the same as a non-pre-denatured ampure.
I have no explanation at the...
Forum: Illumina/Solexa 03-14-2018, 11:14 AM
Replies: 2
Views: 1,113
Posted By pmiguel
Illumina reagent sets include primers mixes that...

Illumina reagent sets include primers mixes that work for various adapters. For instrument/reagent sets that pre-date the acquisition of Epicentre by Illumina, it is necessary to use custom primers...
Forum: Sample Prep / Library Generation 03-14-2018, 10:55 AM
Replies: 1
Views: 432
Posted By pmiguel
We've sequence 2x150 ATAC-seq libraries on a...

We've sequence 2x150 ATAC-seq libraries on a NovaSeq 6000. If the insert is too short then, the read will continue into the adapter on the other side and after that you will just get very low quality...
Forum: Illumina/Solexa 03-14-2018, 10:49 AM
Replies: 7
Views: 1,321
Posted By pmiguel
Up to this point I'm just thinking "fluidics...

Up to this point I'm just thinking "fluidics issue". But you go on to write:



What?
I mean even running the same pool on two MiSeqs could just mean both MiSeq have the same issue -- like a...
Forum: Core Facilities 03-14-2018, 10:13 AM
Replies: 7
Views: 993
Posted By pmiguel
(1) Yes, we accept libraries from customers and...

(1) Yes, we accept libraries from customers and pool them with other libraries. But the libraries have to be unique dual indexed and we do some QC to make sure they would not be a major source of...
Forum: Sample Prep / Library Generation 03-13-2018, 12:51 PM
Replies: 2
Views: 915
Posted By pmiguel
I agree with jwfoley. Unless you were going to...

I agree with jwfoley.
Unless you were going to have them sequenced on a patterned flowcell instrument (HiSeq 3000/4000/X, NovaSeq) -- then you want another ampure because extra adapter oligos are...
Forum: Metagenomics 03-13-2018, 11:24 AM
Replies: 3
Views: 686
Posted By pmiguel
I think the critical factor is that at a cluster...

I think the critical factor is that at a cluster density where you produce the most sequence (even the most Q30 bases), the quality of single-plex amplicon runs deteriorates badly over the last 50...
Forum: Sample Prep / Library Generation 03-08-2018, 08:35 AM
Replies: 3
Views: 694
Posted By pmiguel
I hate Sanger sequencing and wish it were no...

I hate Sanger sequencing and wish it were no longer needed and could be kicked to the curb! That said, with current technology, this is a job for Sanger sequencing. Clone your PCR fragment. Sequence...
Forum: Illumina/Solexa 02-15-2018, 10:27 AM
Replies: 11
Views: 1,650
Posted By pmiguel
Well, one thing. You mentioned you changed the...

Well, one thing. You mentioned you changed the sample sheet after the run. What sample sheet was used by the MiSeq during the run?

The length of the i7 index read is determined by the length of...
Forum: Illumina/Solexa 02-15-2018, 10:22 AM
Replies: 11
Views: 1,650
Posted By pmiguel
Nothing leaps out at me as being wrong with that...

Nothing leaps out at me as being wrong with that sample sheet. You will probably need to take this up with Illumina.

--
Phillip
Forum: Illumina/Solexa 02-15-2018, 03:43 AM
Replies: 11
Views: 1,650
Posted By pmiguel
What was the error? -- Phillip

What was the error?

--
Phillip
Forum: Bioinformatics 02-13-2018, 11:10 AM
Replies: 1
Views: 627
Posted By pmiguel
"something" being "command". That is, "command...

"something" being "command". That is, "command not found". Could we see the result of:

ls ../cap3

If that exists, then try:

chmod +x ../cap3

prior to rerunning your command.
--
Forum: Sample Prep / Library Generation 02-13-2018, 08:32 AM
Replies: 69
Views: 40,962
Posted By pmiguel
The NexteraXT index kit could work--however the...

The NexteraXT index kit could work--however the standard ATACseq protocol expects the primer concentrations to be ~5X higher than they are supplied in the NexteraXT index kits. To find this out we...
Forum: Bioinformatics 01-26-2018, 03:11 AM
Replies: 2
Views: 403
Posted By pmiguel
I've never understood why people think that...

I've never understood why people think that pair-merging prior to de novo assembly is a good idea. Seems like were that the case, assembly engines would just do pair-merging themselves prior to other...
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