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Forum: Illumina/Solexa 09-23-2019, 11:19 AM
Replies: 14
Views: 2,505
Posted By thermophile
our guess is etoh floating to the top of the...

our guess is etoh floating to the top of the flowcell? We had a number of runs with sig more clusters on the bottom than top which started when a new tech started cleaning the pools. I watched her...
Forum: Illumina/Solexa 08-23-2019, 12:46 PM
Replies: 14
Views: 2,505
Posted By thermophile
I agree, that looks low. and your GC isn't going...

I agree, that looks low. and your GC isn't going to cause a problem.

I've been checking the pH of my diluted NaOH with every run-just a drop on pH paper helps me know that my NaOH is ok
Forum: Illumina/Solexa 08-20-2019, 09:19 AM
Replies: 14
Views: 2,505
Posted By thermophile
Have you looked at the thumbnails? does it look...

Have you looked at the thumbnails? does it look like low clustering? What are you sequencing-super high or low GC?
Forum: Illumina/Solexa 05-24-2019, 02:23 PM
Replies: 14
Views: 2,505
Posted By thermophile
is your cluster density the same top and bottom...

is your cluster density the same top and bottom of the flow cell? if you have sig more reads on the bottom you may be carrying over etoh from the ampure cleanup
Forum: Illumina/Solexa 05-24-2019, 02:18 PM
Replies: 2
Views: 1,093
Posted By thermophile
I use a long p200...

I use a long p200 (https://www.usascientific.com/200ul-extra-long-graduated-tip.aspx). Pipet up ~150ul of primer in well, mix with aloquated primer in epitube, add back to well, pipet up and down to...
Forum: Illumina/Solexa 01-08-2019, 02:00 PM
Replies: 1
Views: 1,246
Posted By thermophile
what kind of samples? I'm fighting with difficult...

what kind of samples? I'm fighting with difficult really high gc bacteria that I've truseq pcr free prepped. Ive started heat denaturing after NaOH denaturing. it may be helping a bit? I'm still...
Forum: Illumina/Solexa 01-08-2019, 01:31 PM
Replies: 2
Views: 955
Posted By thermophile
changing between 6 and 8 bp is just a matter of...

changing between 6 and 8 bp is just a matter of putting the index in the sheet. I'm not sure if you can do 25 bp index or if there's a limit either buried in the software. You could either call tech...
Forum: Illumina/Solexa 01-07-2019, 02:58 PM
Replies: 2
Views: 1,185
Posted By thermophile
I tested the new kit on my extreme problem...

I tested the new kit on my extreme problem bacterial genomes (>70% GC). It appears that the cut site bias is basically the same for the flex as it has been for the other flavors of nextera. the...
Forum: Illumina/Solexa 11-07-2018, 01:31 PM
Replies: 3
Views: 1,191
Posted By thermophile
you can contact me as well. User prepped...

you can contact me as well. User prepped libraries are usually sequenced in 1 week. When you are prepping, let us know what kit you need (or you can order the kit from Illumina and have it delivered...
Forum: Metagenomics 04-19-2018, 09:42 AM
Replies: 13
Views: 4,622
Posted By thermophile
good to know, thanks

good to know, thanks
Forum: Metagenomics 04-18-2018, 11:28 AM
Replies: 13
Views: 4,622
Posted By thermophile
have you ever tried ampure cleaning after the...

have you ever tried ampure cleaning after the denaturing step? That should work, right? Carboxyl groups would attract either single or double stranded. But maybe [PEG] would need to be changed?
Forum: Metagenomics 04-17-2018, 01:48 PM
Replies: 13
Views: 4,622
Posted By thermophile
interesting. I've had a few library pools with a...

interesting. I've had a few library pools with a drop off after 50ish bases but couldn't see primer diamers on the tape station (I don't routinely run my amplicon libraries on the tape station, just...
Forum: Metagenomics 04-17-2018, 11:53 AM
Replies: 13
Views: 4,622
Posted By thermophile
Phillip-what conditions have you seen primers...

Phillip-what conditions have you seen primers annealed to full length amplicons? do you routinely heat denature then snap cool before cleaning amplicons?

OP- I look at %base to see what is...
Forum: Service Providers 04-05-2018, 03:41 PM
Replies: 7
Views: 3,070
Posted By thermophile
Sounds like a fairly straightforward MiSeq run or...

Sounds like a fairly straightforward MiSeq run or single lane of HiSeq. Check out my rates for MiSeq (rates on the page are for genomes run on 2x250 MiSeq)

https://mars.uconn.edu/rates/
Forum: Service Providers 04-05-2018, 01:41 PM
Replies: 7
Views: 3,070
Posted By thermophile
How many genomes do you need to sequence?

How many genomes do you need to sequence?
Forum: Metagenomics 04-02-2018, 08:28 AM
Replies: 2
Views: 3,106
Posted By thermophile
I start with indicator species analysis...

I start with indicator species analysis (indicspecies package in R). you'll need to correct the p-values for multiple comparisons, the package doesn't automatically do that.
Forum: Illumina/Solexa 03-27-2018, 09:49 AM
Replies: 22
Views: 2,674
Posted By thermophile
I run primarily amplicons too so the metrics that...

I run primarily amplicons too so the metrics that I look for are a bit different than the standards that Illumina uses (our runs don't meet the base diversity that makes some of the standard number...
Forum: Metagenomics 03-26-2018, 06:35 AM
Replies: 3
Views: 3,052
Posted By thermophile
I wouldn't move to the longer region on the 2x250...

I wouldn't move to the longer region on the 2x250 kit. Allegedly the 2x300 is better in the past ~year but I haven't tested it out so continue offering amplicons that are <300bp long.
Forum: Illumina/Solexa 03-26-2018, 06:26 AM
Replies: 22
Views: 2,674
Posted By thermophile
have you looked at the thumbnails? I'd guess your...

have you looked at the thumbnails? I'd guess your cluster density is higher than reported.
Forum: Bioinformatics 03-23-2018, 09:48 AM
Replies: 6
Views: 1,500
Posted By thermophile
I don't want to just rsync because I need all the...

I don't want to just rsync because I need all the fastq in a single folder for downstream processing

Here I've echo'd the cp line and added a comma for readability

for f in...
Forum: Bioinformatics 03-22-2018, 03:58 PM
Replies: 6
Views: 1,500
Posted By thermophile
help with basemount copy script

I have a script that I've cobbled together to copy fastq from each sample within a project into one folder on my computer.

for f in...
Forum: Illumina/Solexa 03-21-2018, 12:53 PM
Replies: 7
Views: 2,151
Posted By thermophile
FAE checked over the first miseq. nothing...

FAE checked over the first miseq. nothing horribly out of wack. replaced the syringe, realigned the flowcell clamp (it was at the edge of spec which he thought *could* cause some errors). I showed...
Forum: Illumina/Solexa 03-19-2018, 07:48 AM
Replies: 7
Views: 2,151
Posted By thermophile
thermal issue is certainly on my mind since these...

thermal issue is certainly on my mind since these are custom amplicons (16S v4). I've asked the engineer to bring a thermocouple to test it. 2 years ago the second machine (the one that's currently...
Forum: Illumina/Solexa 03-19-2018, 07:33 AM
Replies: 7
Views: 2,151
Posted By thermophile
The phiX run failed. Engineer is coming tomorrow....

The phiX run failed. Engineer is coming tomorrow. It's possible that it was flow cell related, I used the last of my flowcells from that lot on the phiX run.

I ran another primarily amplicon...
Forum: Illumina/Solexa 03-19-2018, 07:27 AM
Replies: 9
Views: 2,073
Posted By thermophile
Yes I've had runs with 50% phiX fail during...

Yes I've had runs with 50% phiX fail during indexing on the MiSeq-I'd guess something similar would happen if you only use one index (3/4 colors would be dark and the machine would likely throw the...
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