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Forum: Illumina/Solexa 07-09-2014, 04:33 PM
Replies: 5
Views: 2,992
Posted By mcnelson.phd
Your assembly looks pretty bad, N50 of 375 bp and...

Your assembly looks pretty bad, N50 of 375 bp and ~1.24 M contigs for a ~465 Mbp genome.

The problem you're likely having is that your library prep was the wrong choice for your genome. I don't...
Forum: Illumina/Solexa 04-09-2014, 06:21 AM
Replies: 27
Views: 7,214
Posted By mcnelson.phd
Ah, that makes sense now, and that bug doesn't...

Ah, that makes sense now, and that bug doesn't affect us at all either so that's probably why our FAS never mentioned it to me since he's usually pretty good at notifying us of these types of things....
Forum: Illumina/Solexa 04-09-2014, 06:01 AM
Replies: 27
Views: 7,214
Posted By mcnelson.phd
To be somewhat fair to Illumina, back when they...

To be somewhat fair to Illumina, back when they started producing the MiSeq, they weren't producing nearly the same volume of data that they are now. Even three years ago when we got our system, I...
Forum: Illumina/Solexa 04-09-2014, 05:35 AM
Replies: 27
Views: 7,214
Posted By mcnelson.phd
MCS is now at v2.4. If you don't see an upgrade...

MCS is now at v2.4. If you don't see an upgrade button on the MiSeq itself, I'd suggest downloading the installer directly from the Illumina support site and installing the update. It's only been out...
Forum: Illumina/Solexa 04-09-2014, 05:18 AM
Replies: 27
Views: 7,214
Posted By mcnelson.phd
For starters, there's a new update to MCS that...

For starters, there's a new update to MCS that fixes that memory leak issues. I would highly recommend that you install it ASAP.

As for Windows killing itself and thus your run, well, that's what...
Forum: Sample Prep / Library Generation 02-28-2014, 04:34 AM
Replies: 10
Views: 7,533
Posted By mcnelson.phd
We use 0.1N NaOH, not 0.2N as Illumina states in...

We use 0.1N NaOH, not 0.2N as Illumina states in their standard denaturing protocol. I don't think that heat denaturing would work because the strands will likely re-hybridize in the time between...
Forum: Bioinformatics 12-12-2013, 07:32 PM
Replies: 2
Views: 2,683
Posted By mcnelson.phd
You should also look into using...

You should also look into using make_per_library_sff.py. That will take your original sff file and, as the name implies, make sff files for each sample. That way you're uploading your data in the...
Forum: Bioinformatics 12-10-2013, 05:44 AM
Replies: 8
Views: 4,426
Posted By mcnelson.phd
You'll need to get the nucleotide sequence of all...

You'll need to get the nucleotide sequence of all possible ORFs that are found in your contigs. To do that you can use Glimmer, GeneMark, or a few other ORF callers although the first two are...
Forum: Bioinformatics 12-10-2013, 05:41 AM
Replies: 5
Views: 3,337
Posted By mcnelson.phd
I'll echo Tony's statements about assemblies...

I'll echo Tony's statements about assemblies coming straight off the MiSeq as being very hit or miss. For one genome we once got a 2.8Mbp contig that was nearly perfect out of a 3.5Mbp genome, but...
Forum: Illumina/Solexa 12-10-2013, 05:29 AM
Replies: 7
Views: 3,093
Posted By mcnelson.phd
Hi Daniel, You're correct in that the actual...

Hi Daniel,

You're correct in that the actual read length of an Illumina run is always N+1 because the extra base is used for phasing/pre-phasing analysis. Ideally that last base should be trimmed...
Forum: Illumina/Solexa 12-08-2013, 05:42 PM
Replies: 4
Views: 3,996
Posted By mcnelson.phd
Yeah, it looks like you're seeing what I've been...

Yeah, it looks like you're seeing what I've been calling index misassignment. It apparently stems from image alignment and signal bleed issues during index sequencing that causes a cluster to be...
Forum: Bioinformatics 11-29-2013, 10:31 AM
Replies: 6
Views: 2,394
Posted By mcnelson.phd
If your Illumina reads are paired-end, then you...

If your Illumina reads are paired-end, then you might be able to get away with looking at how they map to your contig edges, and from there you can resolve which repeat goes where. This would assume...
Forum: Illumina/Solexa 11-21-2013, 05:34 PM
Replies: 3
Views: 2,152
Posted By mcnelson.phd
I'm half tempted to just reply with RTFM, but...

I'm half tempted to just reply with RTFM, but I'll be kinder and say that if you're unsure then look on Illumina's support site for their new denaturing guidelines for using the V3 MiSeq kits.
Forum: Illumina/Solexa 11-13-2013, 06:41 PM
Replies: 6
Views: 3,329
Posted By mcnelson.phd
I'm really not sure I follow what you're talking...

I'm really not sure I follow what you're talking about. If you're saying that your post-PCR cleanup library is 0.2ng/ul with an avg. size of 800bp, then you DO NOT have a library that would meet the...
Forum: Illumina/Solexa 11-13-2013, 06:32 PM
Replies: 2
Views: 5,235
Posted By mcnelson.phd
The simple answer is that yes, you can use...

The simple answer is that yes, you can use Nextera/XT on your cDNA to make a pretty decent and quick RNASeq library.

However, you will lose the 5' and 3' ends, which can bias some analyses. There...
Forum: Illumina/Solexa 11-08-2013, 12:50 PM
Replies: 1
Views: 2,169
Posted By mcnelson.phd
Even though Illumina says that the phix is...

Even though Illumina says that the phix is supplied at 10nM, I've found that the actual value does indeed seem to jump around a little bit.

I asked my FAS about that once and they confirmed that...
Forum: Bioinformatics 11-07-2013, 02:42 PM
Replies: 4
Views: 3,733
Posted By mcnelson.phd
We run Trinity on our MacPro without problems,...

We run Trinity on our MacPro without problems, although I'll admit that it hasn't been updated for a while so it's not the current version (we're still using version 2012-06-08 since we don't run...
Forum: Bioinformatics 11-05-2013, 08:08 AM
Replies: 2
Views: 1,467
Posted By mcnelson.phd
You're reads were most likely NOT adapter...

You're reads were most likely NOT adapter trimmed. This has caused a number of people problems with assembly of Nextera data.

Do that before assembling and you should get normal looking N50 values.
Forum: Sample Prep / Library Generation 11-02-2013, 07:26 AM
Replies: 4
Views: 9,374
Posted By mcnelson.phd
The concentration values that are shown on the...

The concentration values that are shown on the Qubit are the concentration of DNA in the tube that you are reading, and not of the original sample. This means that you need to take the value that it...
Forum: Bioinformatics 11-01-2013, 11:53 AM
Replies: 17
Views: 4,558
Posted By mcnelson.phd
We're currently OK with RAM usage and capacity,...

We're currently OK with RAM usage and capacity, and my intention wasn't that compressed memory would allow us to analyze a larger dataset than the system is capable of handling.

My main line of...
Forum: Illumina/Solexa 10-28-2013, 05:10 PM
Replies: 11
Views: 8,939
Posted By mcnelson.phd
For cleanup, we're using the full 50ul volume...

For cleanup, we're using the full 50ul volume from the PCR step. That requires only 30ul of AMPure, so it's not a lot (esp. compared to what TruSeq required). It is completely ridiculous, however,...
Forum: Illumina/Solexa 10-28-2013, 03:14 PM
Replies: 9
Views: 5,747
Posted By mcnelson.phd
As I said before, this is probably Illumina's...

As I said before, this is probably Illumina's biggest failing, making their workflows/kits/systems seem overly simple.

Cluster density is probably the single most important factor affecting run...
Forum: Illumina/Solexa 10-28-2013, 01:14 PM
Replies: 9
Views: 5,747
Posted By mcnelson.phd
260K is low, and could be due to the bead...

260K is low, and could be due to the bead normalization and denaturing protocol or simply that some of your libraries weren't that good and didn't form viable clusters. You'll get some idea after the...
Forum: Illumina/Solexa 10-28-2013, 11:38 AM
Replies: 9
Views: 5,747
Posted By mcnelson.phd
If this is your first run, then I'd recommend...

If this is your first run, then I'd recommend adding phiX. This will help with troubleshooting if anything goes wrong. For example, if the run fails immediately because it can't find clusters, then...
Forum: Bioinformatics 10-28-2013, 05:05 AM
Replies: 11
Views: 2,621
Posted By mcnelson.phd
PCA will let your group samples that are...

PCA will let your group samples that are "similar" but it won't tell you if there's a correlation between the expression patterns. Correlations are good for telling you if there's a relationship and...
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