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Forum: Bioinformatics 09-20-2017, 09:22 PM
Replies: 21
Views: 13,864
Posted By koadman
Hi Coline, Mauve is unable to take gff files as...

Hi Coline, Mauve is unable to take gff files as input, it can take .fasta and Genbank flat files, with the .gbk suffix.
Forum: Illumina/Solexa 10-26-2014, 07:34 PM
Replies: 4
Views: 1,254
Posted By koadman
Hi ramarquezo, kcchan, yes that is indeed what we...

Hi ramarquezo, kcchan, yes that is indeed what we typically do here at UTS. Prep lots of libraries, QC them by generating a little bit of data on a nano flowcell, then balance them precisely for a...
Forum: Illumina/Solexa 02-20-2014, 11:59 AM
Replies: 50
Views: 36,459
Posted By koadman
Well done creeves on pushing the multiplexing so...

Well done creeves on pushing the multiplexing so far. What was the edit distance between your barcodes? The default parameters for demultiplexing tolerate 1 mismatch in each barcode I believe. If...
Forum: Illumina/Solexa 10-24-2013, 06:16 AM
Replies: 12
Views: 6,372
Posted By koadman
Hi James, Yes, I've fallen victim to this...

Hi James,
Yes, I've fallen victim to this problem previously, see the post from February this year:

http://seqanswers.com/forums/showpost.php?p=96466&postcount=14

I've been working mostly with...
Forum: Academic/Non-Profit Jobs 05-22-2013, 10:18 PM
Replies: 1
Views: 3,847
Posted By koadman
Postdoc: scalable Bayesian methods to infer genome evolution, Sydney, Australia

The Darling lab at the University of Technology Sydney, in collaboration with Dr. Erick Matsen's group at Fred Hutchison Cancer Research Center, is exploring statistical inference algorithms for the...
Forum: De novo discovery 03-24-2013, 01:32 AM
Replies: 9
Views: 2,640
Posted By koadman
I'm surprised no secret operatives from OpGen or...

I'm surprised no secret operatives from OpGen or BioNano have chimed in here. Seems like a perfect use-case for their tech. Not much in the way of 3rd party assembly tools for these data types...
Forum: Illumina/Solexa 03-15-2013, 01:15 PM
Replies: 4
Views: 2,031
Posted By koadman
Well of course I was hoping not to be the...

Well of course I was hoping not to be the sacrificial lamb, but if nobody else has tried this and able to share data I guess I will take one for the team! Now just need to find a willing pair of lab...
Forum: Illumina/Solexa 03-13-2013, 03:52 AM
Replies: 4
Views: 2,031
Posted By koadman
1x500 miseq reads instead of 2x250?

A while back ECO posted some great info on a 1x300 miseq run. Has anybody tried a similar run with an upgraded instrument to achieve a 500 nt read? Will the software permit this? How do the base...
Forum: Illumina/Solexa 02-15-2013, 11:06 AM
Replies: 18
Views: 13,980
Posted By koadman
To be honest very little of it ever made sense to...

To be honest very little of it ever made sense to me. I don't run the instruments, the core facility does, and I don't communicate with Illumina tech support, the core facility does. I'm an...
Forum: Illumina/Solexa 02-15-2013, 12:25 AM
Replies: 18
Views: 13,980
Posted By koadman
Following up on my earlier posting I want to warn...

Following up on my earlier posting I want to warn others of some problems we encountered with dual-index sequencing on the hiseq and how we resolved them to ultimately arrive at a good result. Our...
Forum: Illumina/Solexa 01-25-2013, 01:15 AM
Replies: 11
Views: 4,048
Posted By koadman
Hi microgirl123, One way to reduce the primer...

Hi microgirl123,
One way to reduce the primer cost is to use the dual-barcode capabilities of the miseq. You can modify the Caporaso et al 2012 oligos to have a barcode at both ends. I have had...
Forum: Illumina/Solexa 12-08-2012, 11:11 AM
Replies: 13
Views: 10,124
Posted By koadman
We have been using lower input quantities with...

We have been using lower input quantities with the Nextera XT kits, although it requires some fine-tuning to get the normalization step right. As discussed in an earlier thread in this forum, it...
Forum: Illumina/Solexa 10-29-2012, 10:34 PM
Replies: 2
Views: 1,641
Posted By koadman
The bead-based approach bundled in the Nextera XT...

The bead-based approach bundled in the Nextera XT kit seems to work fairly well for final library pooling. We've had some luck with picogreen assays on a tecan plate reader but it's not nearly as...
Forum: Illumina/Solexa 10-24-2012, 08:06 PM
Replies: 8
Views: 3,917
Posted By koadman
If you really want to use this kit you could try...

If you really want to use this kit you could try using only 1-2ng input to see if the fragment size distribution gets in the 200-300 range.
Forum: Illumina/Solexa 10-16-2012, 11:06 AM
Replies: 3
Views: 2,529
Posted By koadman
Hi JPC, in our experience when using the...

Hi JPC, in our experience when using the protocol's suggested dilution into hybridization buffer at the last step, the number of reads varies widely by sample type. I'm not sure whether this is due...
Forum: Illumina/Solexa 10-02-2012, 04:32 PM
Replies: 7
Views: 6,073
Posted By koadman
Hi all, following up to give this thread some...

Hi all, following up to give this thread some closure. We remade libraries for the 16 samples from the xy-plot above (all same sample type, DNA extraction, quantitation) using 0.5ng input and 14...
Forum: Illumina/Solexa 10-02-2012, 04:19 PM
Replies: 1
Views: 2,794
Posted By koadman
We have accidentally created nextera (though not...

We have accidentally created nextera (though not nextera XT) libraries with small median insert sizes, ~120nt. The Adey et al 2010 paper claims stearic hindrance prevents generating inserts much...
Forum: Illumina/Solexa 10-02-2012, 04:00 PM
Replies: 18
Views: 13,980
Posted By koadman
Hi all, I've been looking at this as well....

Hi all, I've been looking at this as well. Reading through the Illumina protocol pdf, I noticed the following:



Which suggests that one might be able to play with the relative amounts of...
Forum: Illumina/Solexa 09-25-2012, 07:28 PM
Replies: 7
Views: 6,073
Posted By koadman
That's a good point, we've noticed similar issues...

That's a good point, we've noticed similar issues in the past too and the samples in the histogram I posted above are indeed from at least 4 different sample types, at least 4 different DNA...
Forum: Illumina/Solexa 09-25-2012, 03:42 PM
Replies: 7
Views: 6,073
Posted By koadman
Thanks for the reply Scott. Based on your...

Thanks for the reply Scott. Based on your suggestion I decided to look at the relationship between median insert size in mapped reads and the number of clusters per sample. See the attached plot....
Forum: Illumina/Solexa 09-19-2012, 05:53 AM
Replies: 7
Views: 6,073
Posted By koadman
Thanks for the reply MrGuy, it hadn't occurred to...

Thanks for the reply MrGuy, it hadn't occurred to me that we might be underloading some samples. The Nextera XT protocol had us tagment 1ng material per sample, then amplify that material with 12...
Forum: Illumina/Solexa 09-18-2012, 01:47 PM
Replies: 7
Views: 6,073
Posted By koadman
Nextera XT multiplex sample normalization

Hi all,
Our lab just did our first Nextera XT run on a MiSeq. I'm just the informatics guy and didn't actually prep the libraries myself, but the person who did says the protocol is nicely...
Forum: Illumina/Solexa 08-16-2012, 07:01 PM
Replies: 6
Views: 4,052
Posted By koadman
very interesting! this seems to suggest that...

very interesting! this seems to suggest that signal phasing and basecalling may be contributing more to the reduction in quality near the end of reads than the chemistry/imaging. if that's true then...
Forum: Illumina/Solexa 08-14-2012, 08:35 PM
Replies: 18
Views: 6,953
Posted By koadman
@ScottC, did you sequence anything from the lower...

@ScottC, did you sequence anything from the lower input preps, e.g. 0.2-0.5ng or just bioanalyze it? If sequenced how did the library complexity look? Did you have many PCR duplicates? How many PCR...
Forum: Illumina/Solexa 08-08-2012, 08:36 PM
Replies: 10
Views: 9,316
Posted By koadman
I wonder if the relatively minor increase in FHWM...

I wonder if the relatively minor increase in FHWM with larger fragments is because the image processing software doesn't recognize larger clusters, possibly because the signal is less intense moving...
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