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Forum: Illumina/Solexa 11-06-2018, 07:22 AM
Replies: 6
Views: 784
Posted By pmiguel
SAV 2.4.5 includes an "occupied count" and "%...

SAV 2.4.5 includes an "occupied count" and "% occupied" metric in the drop down list of the Flow Cell Chart pane of the Analysis tab.

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Phillip
Forum: Illumina/Solexa 11-01-2018, 10:07 AM
Replies: 6
Views: 784
Posted By pmiguel
The new version of SAV has a metric "% occupancy"...

The new version of SAV has a metric "% occupancy" for each tile. You should check that.
It looks like you either had very low occupancy (very under-clustered) or very massive over-clustering....
Forum: Sample Prep / Library Generation 10-31-2018, 12:10 PM
Replies: 1
Views: 255
Posted By pmiguel
No, but if you post your spectrum here, one of ...

No, but if you post your spectrum here, one of us might recognize it.

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Phillip
Forum: Sample Prep / Library Generation 10-31-2018, 06:56 AM
Replies: 16
Views: 7,254
Posted By pmiguel
You would probably need to sequence it to find...

You would probably need to sequence it to find out what it is. But my guess would be lots of tRNA, or some other small RNA. Or something unexpected.

Since the rRNA peaks appear to be intact, it...
Forum: Sample Prep / Library Generation 10-31-2018, 04:08 AM
Replies: 16
Views: 7,254
Posted By pmiguel
Most likely it is a mixture of small RNAs -- 5S,...

Most likely it is a mixture of small RNAs -- 5S, tRNA, etc. "TRIfast" I have not heard of, but I presume it is another version of "Trizol", the commercial acid phenol reagents. For whatever reason,...
Forum: Sample Prep / Library Generation 10-31-2018, 04:01 AM
Replies: 16
Views: 7,254
Posted By pmiguel
Just looking at this ancient thread that...

Just looking at this ancient thread that mcheckula revived. The answer I gave in 2013 misses the actual issue here. The "signal in the fast region" is the actual 25 nt standard peak. The software has...
Forum: Illumina/Solexa 10-16-2018, 01:04 PM
Replies: 2
Views: 470
Posted By pmiguel
I agree, it will probably work fine for you. ...

I agree, it will probably work fine for you.
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Phillip
Forum: Sample Prep / Library Generation 08-31-2018, 06:44 AM
Replies: 6
Views: 779
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, 04:03 AM
Replies: 6
Views: 779
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, 09:38 AM
Replies: 6
Views: 779
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, 09:18 AM
Replies: 2
Views: 798
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, 09:40 AM
Replies: 14
Views: 884
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, 07:02 AM
Replies: 14
Views: 884
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?

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Phillip
Forum: Illumina/Solexa 07-25-2018, 05:20 AM
Replies: 14
Views: 884
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, 03:39 AM
Replies: 2
Views: 781
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.

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Phillip
Forum: Sample Prep / Library Generation 06-25-2018, 08:18 AM
Replies: 3
Views: 1,276
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, 01:56 PM
Replies: 3
Views: 744
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, 09:37 AM
Replies: 4
Views: 638
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, 05:11 AM
Replies: 10
Views: 1,650
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, 12:07 PM
Replies: 10
Views: 1,650
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, 08:58 AM
Replies: 10
Views: 1,650
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, 12:00 PM
Replies: 5
Views: 1,151
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, 07:14 AM
Replies: 1
Views: 337
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, 09:48 AM
Replies: 6
Views: 1,162
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, 07:40 AM
Replies: 7
Views: 1,476
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...
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