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Forum: Illumina/Solexa 03-03-2016, 08:04 AM
Replies: 8
Views: 1,407
Posted By Egansbay
Thanks Thermophile - I think the seq co must have...

Thanks Thermophile - I think the seq co must have loaded the max, as 24 samples yielded 22m raw reads.

Not sure if the seq was too deep though, as when I run rarefaction down to 100,000...
Forum: Illumina/Solexa 02-25-2016, 11:13 AM
Replies: 8
Views: 1,407
Posted By Egansbay
Thank you - I have run 2 x 300bp, but have to...

Thank you - I have run 2 x 300bp, but have to trim as the quality of the reverse read is not that good. Assume this is what you're referring to
Forum: Illumina/Solexa 02-25-2016, 11:05 AM
Replies: 8
Views: 1,407
Posted By Egansbay
I am just referring to the statement in the MiSeq...

I am just referring to the statement in the MiSeq wet lab protocol where it reads "The MiSeq run output is >20m reads, and assuming 96 indexed samples, can generate >100,000 reads per sample,...
Forum: Illumina/Solexa 02-25-2016, 10:52 AM
Replies: 8
Views: 1,407
Posted By Egansbay
Thank you for your reply GW_OK So from the...

Thank you for your reply GW_OK

So from the number of ~20m reads, Illumina states that you should get ~100,000 seqs per sample, assuming 96 samples were run.

Do you think that this number refers...
Forum: Illumina/Solexa 02-25-2016, 10:12 AM
Replies: 8
Views: 1,407
Posted By Egansbay
Total reads from MiSeq 16S V4 PE run

Hi everyone

Probably a stupid question, but i'm finding it hard to find meaningful info on sequencing depth and read numbers on the MiSeq.

Illumina states that a 16S V4 run should deliver ~20m...
Forum: Illumina/Solexa 12-04-2015, 12:16 PM
Replies: 16
Views: 2,448
Posted By Egansbay
Triplicate pcr's - a lot of studies pubkished...

Triplicate pcr's - a lot of studies pubkished recently on fish gut microbiome are finding mycoplasma, so I'm not sure if it's contamination. Plus we tested samples of the reagents as neg controls too...
Forum: Illumina/Solexa 12-04-2015, 01:51 AM
Replies: 16
Views: 2,448
Posted By Egansbay
Ok I see - we usually get approx. 900,000 reads...

Ok I see - we usually get approx. 900,000 reads per sample after the sequencing run of 24 samples, but after quality filtering this drops by anywhere from 30-60%.

How many sequences do you then...
Forum: Illumina/Solexa 12-04-2015, 01:34 AM
Replies: 16
Views: 2,448
Posted By Egansbay
Hi Alex - sorry to be a pain, but just want to...

Hi Alex - sorry to be a pain, but just want to clarify.

Do you mean that you have a minimum of 100,000 sequences per sample left over after all quality filtering steps? Or is this your starting...
Forum: Illumina/Solexa 12-02-2015, 05:44 AM
Replies: 16
Views: 2,448
Posted By Egansbay
Ok yes I have also used 2 x 250bp before too ...

Ok yes I have also used 2 x 250bp before too

Yes I am getting a diverse community besides the primary OTU, and anywhere from 100,000-600,000 seqs lefts over after quality filtering

So after...
Forum: Illumina/Solexa 12-02-2015, 03:55 AM
Replies: 16
Views: 2,448
Posted By Egansbay
Ok I see - we tested an extraction from the...

Ok I see - we tested an extraction from the reagents we are using but the otu was not present. We also tested an extraction from the diets used to feed our fish but again negative.

Are you using...
Forum: Illumina/Solexa 12-02-2015, 02:38 AM
Replies: 16
Views: 2,448
Posted By Egansbay
Hi Alex, appears to be a Mycoplasma. Appearing a...

Hi Alex, appears to be a Mycoplasma. Appearing a lot in the fish gut microbiome literature but also a contaminant of cell culture. Same otu?
Forum: Illumina/Solexa 12-02-2015, 01:33 AM
Replies: 16
Views: 2,448
Posted By Egansbay
Hi Cliff - yes I have found this to indeed be the...

Hi Cliff - yes I have found this to indeed be the case when I have sequenced further sample sets.

How many v4 reads per sample are you generally using for classification, after quality filtering?...
Forum: Illumina/Solexa 03-18-2015, 02:30 PM
Replies: 16
Views: 2,448
Posted By Egansbay
I set the cutoff=80 on the classify.seqs command....

I set the cutoff=80 on the classify.seqs command. About 500-800 otu's total. Not sure if I've done something wrong or whether this is a true representation
Forum: Illumina/Solexa 03-18-2015, 11:31 AM
Replies: 16
Views: 2,448
Posted By Egansbay
MiSeq V4 16S run low OTU evenness

Hi everyone

Recently received my sequences back from an Illumina MiSeq 16S V4 300bp PE run, performed by a biotech company. The DNA was extracted from the gut of fish.

Although I received many...
Forum: Illumina/Solexa 01-21-2015, 01:51 AM
Replies: 1
Views: 1,273
Posted By Egansbay
Smile Is there a manual way of producing Rarefaction curves?

Hi everyone

Just wondering if there's an easy way to produce rarefaction curves from simple 16S V4 amplicon read abundance data from MiSeq? Or does the data have to be passed back through a...
Forum: Illumina/Solexa 01-07-2015, 12:26 PM
Replies: 13
Views: 2,600
Posted By Egansbay
Thanks microgirl, I'll try it, just have to trust...

Thanks microgirl, I'll try it, just have to trust the bands are pure microbial dna and hopefully everything will work out!
Forum: Illumina/Solexa 01-07-2015, 12:16 PM
Replies: 13
Views: 2,600
Posted By Egansbay
Yes measuring with qubit and also agilent...

Yes measuring with qubit and also agilent bioanalyzer. The conc recorded from the agilent is slightly lower but qubit quite reliable as measures dsDNA only?
Forum: Illumina/Solexa 01-07-2015, 12:12 PM
Replies: 13
Views: 2,600
Posted By Egansbay
My only worry is that the DNA conc might be a bit...

My only worry is that the DNA conc might be a bit low, but then it's hard to know how much is enough. I'm only adding an average of 10ng per sample into the index PCR. I'm hoping this will be enough?
Forum: Illumina/Solexa 01-07-2015, 11:58 AM
Replies: 13
Views: 2,600
Posted By Egansbay
Hi microgirl, thanks for your feedback, I am...

Hi microgirl, thanks for your feedback, I am loading a total of 2.5ul of the first pc product? Is that similar to your volume for the index PCR? Hopefully the run will go well, only one way to find...
Forum: Illumina/Solexa 01-07-2015, 08:51 AM
Replies: 13
Views: 2,600
Posted By Egansbay
Thanks Microgirl, by sample fall out, what...

Thanks Microgirl, by sample fall out, what happens exactly? Are there any reliable indicators of potential sample performance on the MiSeq?
Forum: Illumina/Solexa 01-07-2015, 07:57 AM
Replies: 13
Views: 2,600
Posted By Egansbay
16S V4 Indexing PCR - Thoughts on this gel?

Hi all

I'm running a microbiome analysis of fish gut microdlora using Illumina MiSeq 16s pipeline. I'd like to get your thoughts on my indexing PCR gel - some of the bands seem a little faint. Can...
Forum: Illumina/Solexa 01-05-2015, 08:56 AM
Replies: 3
Views: 1,286
Posted By Egansbay
Thank you microgirl. So in your experience are...

Thank you microgirl. So in your experience are my concentrations of product too low to proceed with second round pcr?
Forum: Illumina/Solexa 01-05-2015, 07:28 AM
Replies: 3
Views: 1,286
Posted By Egansbay
Smile 16S V4 amplicon concentration after first PCR step

Dear all

I am analyzing gut samples from fish using illumina's 16S protocol, amplifying the v4 hypervariable region. I am getting ok bands after the first PCR step, and a nice peak on the agilent...
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