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Forum: Illumina/Solexa 11-07-2018, 01:31 PM
Replies: 3
Views: 610
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: 2,131
Posted By thermophile
good to know, thanks

good to know, thanks
Forum: Metagenomics 04-18-2018, 11:28 AM
Replies: 13
Views: 2,131
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: 2,131
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: 2,131
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: 1,188
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: 1,188
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: 1,443
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: 1,767
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: 1,447
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: 1,767
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: 5
Views: 826
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: 5
Views: 826
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: 1,675
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: 1,675
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: 1,675
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: 1,698
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...
Forum: Illumina/Solexa 03-16-2018, 10:27 AM
Replies: 9
Views: 1,698
Posted By thermophile
Only one index could cause the run to fail during...

Only one index could cause the run to fail during the index sequencing because there isn't any diversity in each cycle. I'd just set up the sample sheet with 0 index
Forum: Illumina/Solexa 03-16-2018, 09:27 AM
Replies: 11
Views: 2,119
Posted By thermophile
you reverse complimented your i7 barcodes right?

you reverse complimented your i7 barcodes right?
Forum: Illumina/Solexa 03-16-2018, 08:30 AM
Replies: 7
Views: 1,675
Posted By thermophile
The error that halted the first run was (can't...

The error that halted the first run was (can't remember exact wording) can't find focus without boosting signal beyond threshold. Looking at the thumbnails, the actual focus was fine but there...
Forum: Illumina/Solexa 03-15-2018, 01:05 PM
Replies: 11
Views: 2,119
Posted By thermophile
Here's how my header looks for custom amplicon...

Here's how my header looks for custom amplicon sequencing.

Workflow GenerateFASTQ
Application FASTQ Only
Assay Nextera
Description
Chemistry Amplicon
Forum: Illumina/Solexa 03-13-2018, 12:22 PM
Replies: 7
Views: 1,675
Posted By thermophile
miseq clustering higher bottom than top of flow cell

What can cause different clustering levels on the top vs bottom of the flow cell. I run amplicons, so undercluster generally aim for ~750k clusters. I have a library that I've run twice, both times...
Forum: Illumina/Solexa 02-23-2018, 01:38 PM
Replies: 12
Views: 2,142
Posted By thermophile
Both of those look very underclustered. No where...

Both of those look very underclustered. No where near 500k. One tiny thing that can ruin your cluster density is old NaOH. When was the last time you've made 2N from 10N stock? You're diluting the 2N...
Forum: Illumina/Solexa 02-16-2018, 06:59 AM
Replies: 11
Views: 2,119
Posted By thermophile
Look for WorkflowLog.txt (I demultiplex in...

Look for WorkflowLog.txt (I demultiplex in basespace where it's in fastqc/files), scroll to the bottom and it will tell you exactly what has caused the sample sheet failure.
Forum: Illumina/Solexa 02-14-2018, 02:11 PM
Replies: 2
Views: 704
Posted By thermophile
I don't use the EMP ITS, I use the original ITS3...

I don't use the EMP ITS, I use the original ITS3 and ITS4 from White (same reverse, different forward). you have to adjust the pad to increase the melting temp because ITS3/ITS4 have a theoretical TM...
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