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Forum: Ion Torrent 01-16-2018, 08:52 AM
Replies: 3
Views: 140
Posted By thermophile
Why IonTorrent? MiSeq is cheaper and much better...

Why IonTorrent? MiSeq is cheaper and much better sequence quality for amplicons? Besides dada2 and deblur are both for illumina sequencing, applying them to IonTorrent would require quite a bit of...
Forum: Illumina/Solexa 01-16-2018, 08:47 AM
Replies: 29
Views: 4,369
Posted By thermophile
did tech support check your lot numbers to be...

did tech support check your lot numbers to be sure that you were using the "new and improved" chemistry? Though I haven't been confident enough to try to convince a user to try the new v3 yet
Forum: 454 Pyrosequencing 01-11-2018, 06:59 AM
Replies: 2
Views: 207
Posted By thermophile
I don't know qiime well (only used enough to know...

I don't know qiime well (only used enough to know that I strongly prefer mothur). So can't tell you exactly where to combine but you can't combine OTU tables if you do denovo clustering. In mothur, I...
Forum: Illumina/Solexa 12-18-2017, 04:09 PM
Replies: 2
Views: 506
Posted By thermophile
if you have a lot of the shorter amplicons,...

if you have a lot of the shorter amplicons, there's a chance that the software will just kill the run because of difficulty focusing when a lot of the clusters are dark. I've run up to 25% or so...
Forum: Illumina/Solexa 11-07-2017, 08:29 AM
Replies: 13
Views: 991
Posted By thermophile
your %base also suggest short fragments and...

your %base also suggest short fragments and reading into the flowcell.
Forum: Sample Prep / Library Generation 11-03-2017, 09:24 AM
Replies: 2
Views: 578
Posted By thermophile
https://www.alpaqua.com/Products/Magnet-Plates/Mag...

https://www.alpaqua.com/Products/Magnet-Plates/Magnum-FLX

by far the best plate mag I've tried. it's expensive but if you are doing lots of cleanups and want speed and small volumes it's probably...
Forum: Illumina/Solexa 10-31-2017, 08:59 AM
Replies: 13
Views: 991
Posted By thermophile
Are you sure your template is more than 80bp?

Are you sure your template is more than 80bp?
Forum: Illumina/Solexa 10-26-2017, 12:53 PM
Replies: 13
Views: 836
Posted By thermophile
The poor pf and wrong indices are 2 different...

The poor pf and wrong indices are 2 different issues. Bad libraries shouldn't lead to poor pf unless you were loosing all of a certain type of libraries and ended up with just low diversity? Might...
Forum: Illumina/Solexa 10-19-2017, 10:49 AM
Replies: 2
Views: 485
Posted By thermophile
The only concern is index diversity, Check your...

The only concern is index diversity, Check your combinations to make sure that all channels are roughly equally represented across the indexes given that one index will be ~40% of the reads.
Forum: Sample Prep / Library Generation 10-12-2017, 02:02 PM
Replies: 1
Views: 461
Posted By thermophile
My Qiagen rep was just here talking about this....

My Qiagen rep was just here talking about this. He claimed that it isn't sequence specific cleaving at all but beyond that I couldn't get much info out of him. Showed some data comparing sequencing...
Forum: Sample Prep / Library Generation 09-14-2017, 01:14 PM
Replies: 2
Views: 442
Posted By thermophile
I'd prefer not to use phenol - there's no way to...

I'd prefer not to use phenol - there's no way to set up the robot in a hood for one.

I'll check out the zymo kit, thanks
Forum: Sample Prep / Library Generation 09-07-2017, 10:11 AM
Replies: 2
Views: 442
Posted By thermophile
HTP RNA extraction from plants?

I need to extract RNA from ~1200 leaf and stem tissues. So far I've found a protocol using the qiagen 96well RNA easy kit for plants. Is that the only high throughput RNA kit that has been used for...
Forum: Illumina/Solexa 08-30-2017, 12:30 PM
Replies: 22
Views: 2,059
Posted By thermophile
lower than expected phiX indicates that your...

lower than expected phiX indicates that your quant was off. If you loaded your library to get 30% and only could align 15% phiX, that means that your 16S was approximately twice as concentrated as...
Forum: Bioinformatics 07-25-2017, 12:51 PM
Replies: 2
Views: 435
Posted By thermophile
do you only have the 3 samples? I'd look at a...

do you only have the 3 samples? I'd look at a dissimilarity measure (bray-curtis is a good one) and see how similar they are. if you have triplicates of many samples then plot the dissimilarities in...
Forum: Illumina/Solexa 07-21-2017, 07:41 AM
Replies: 22
Views: 2,059
Posted By thermophile
I run mostly 16s libraries as well. 15% 8pM phiX...

I run mostly 16s libraries as well. 15% 8pM phiX should be enough. I only quibit my libraries so not as accurate as kapa, but I load 8pm library. Illumina tech support has said to try not to go below...
Forum: Illumina/Solexa 07-11-2017, 06:47 AM
Replies: 6
Views: 692
Posted By thermophile
My primary type is 16s v4 (~300bp). I've mixed up...

My primary type is 16s v4 (~300bp). I've mixed up to 30% of a run with ITS2 (~250-400bp) or custom ~150bp amplicons with no issues and no noticeable decrease in reads for the longer amplicons on the...
Forum: Illumina/Solexa 07-06-2017, 11:09 AM
Replies: 6
Views: 692
Posted By thermophile
length bias still exists on illumina flowcells...

length bias still exists on illumina flowcells but it's not anywhere close to as strong as it was with 454. You may need to tweak your load ratios a small amount but not much. 5-10% as a complete...
Forum: Bioinformatics 06-27-2017, 09:11 AM
Replies: 3
Views: 572
Posted By thermophile
Is the Atlantic ocean the best ocean? I don't...

Is the Atlantic ocean the best ocean? I don't think best exists.


I prefer mothur because it is very transparent with methods used and simple to install on any OS (there are a few scientific...
Forum: Metagenomics 06-23-2017, 11:47 AM
Replies: 3
Views: 951
Posted By thermophile
The 515F/806R primers that are out there...

The 515F/806R primers that are out there (Caparosso or Kozich primers) are designed to be biased against chloroplast and mitochondria. My tricks for tissues are use a ton of DNA because most of what...
Forum: Bioinformatics 06-23-2017, 11:41 AM
Replies: 3
Views: 572
Posted By thermophile
I've only used the Illumina pipeline once (for...

I've only used the Illumina pipeline once (for that matter I prefer mothur to qiime). The Illumina pipeline is a total black box, do you trust every decision they made for you but didn't document?...
Forum: Metagenomics 06-13-2017, 07:28 AM
Replies: 3
Views: 977
Posted By thermophile
Use mothur with the silva alignment. you may need...

Use mothur with the silva alignment. you may need to format the LSU alignment for mothur but there are instructions on the mothur website. Also the mothur forum is fairly active if you have any...
Forum: Illumina/Solexa 06-13-2017, 07:23 AM
Replies: 4
Views: 440
Posted By thermophile
As long as they are roughly the same sheared...

As long as they are roughly the same sheared size, this will work well. Total size of the genome doesn't effect the run quality, you just need to account for it when deciding how much of each genome...
Forum: Metagenomics 06-13-2017, 07:18 AM
Replies: 3
Views: 977
Posted By thermophile
what kind of metabarcode target? Can your target...

what kind of metabarcode target? Can your target be aligned? If so, mothur. If not, doesn't matter as much. I like mothur because of many reasons including it's easy to install natively (my datasets...
Forum: Sample Prep / Library Generation 06-12-2017, 07:41 AM
Replies: 3
Views: 834
Posted By thermophile
Remember quibit is a single point calibration...

Remember quibit is a single point calibration (with blank-s1 0ng, s2 10ng). So the further you are from 10ng/ul the worse the quantification will be. Are you doing replicates with your picogreen?...
Forum: Illumina/Solexa 05-30-2017, 08:04 AM
Replies: 22
Views: 5,161
Posted By thermophile
That makes sense to me and is a good idea but I...

That makes sense to me and is a good idea but I thought that basemount was supposed to enable uploading projects as well as downloading (i.e. if you don't want to use standard demultiplexing).

My...
Showing results 1 to 25 of 205

 


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