SEQanswers

Go Back   SEQanswers > Search Forums


Showing results 1 to 25 of 500
Search took 1.56 seconds.
Search: Posts Made By: pmiguel
Forum: Illumina/Solexa 12-03-2018, 03:49 AM
Replies: 6
Views: 5,740
Posted By pmiguel
Impossible to guess what "much less" means in...

Impossible to guess what "much less" means in this context. The previous poster mentioned 100x less -- that is clearly an issue.

Anyway, you will want to contact Illumina technical support. That...
Forum: Illumina/Solexa 11-30-2018, 04:30 AM
Replies: 3
Views: 464
Posted By pmiguel
No reason why it would not be possible. Since you...

No reason why it would not be possible. Since you don't specify an organism, I presume you mean a mammal. So you want an average of 3 billion bases/sample. So that would be 1000 UDIs. Presuming you...
Forum: Sample Prep / Library Generation 11-26-2018, 07:40 AM
Replies: 4
Views: 333
Posted By pmiguel
Yes, please everyone switch to blue light...

Yes, please everyone switch to blue light illumination for preparative gels! We did so > 20 years ago. I'm just realizing that many labs (and new PI!) are unaware of the problems UV light damage to...
Forum: Illumina/Solexa 11-06-2018, 07:22 AM
Replies: 8
Views: 1,102
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.

--
Phillip
Forum: Illumina/Solexa 11-01-2018, 10:07 AM
Replies: 8
Views: 1,102
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: 438
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.

--
Phillip
Forum: Sample Prep / Library Generation 10-31-2018, 06:56 AM
Replies: 17
Views: 7,626
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: 17
Views: 7,626
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: 17
Views: 7,626
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: 634
Posted By pmiguel
I agree, it will probably work fine for you. ...

I agree, it will probably work fine for you.
--
Phillip
Forum: Sample Prep / Library Generation 08-31-2018, 06:44 AM
Replies: 6
Views: 857
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: 857
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: 857
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: 866
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: 962
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: 962
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, 05:20 AM
Replies: 14
Views: 962
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: 822
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, 08:18 AM
Replies: 3
Views: 1,352
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: 773
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: 666
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: 1,710
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: 1,710
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: 1,710
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: 6
Views: 1,317
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...
Showing results 1 to 25 of 500

 


All times are GMT -8. The time now is 02:57 AM.


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