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Forum: Illumina/Solexa 07-18-2018, 02:39 AM
Replies: 2
Views: 359
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: 703
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: 473
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: 390
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: 1,257
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: 1,257
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: 1,257
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: 643
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: 274
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: 805
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: 1,251
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: 1,505
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: 1,505
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: 1,505
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: 1,505
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: 1,505
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: 624
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: 1,627
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: 1,627
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: 1,627
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: 1,526
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: 1,514
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: 1,514
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: 1,413
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: 648
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...
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