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Forum: Sample Prep / Library Generation 03-12-2014, 03:16 AM
Replies: 4
Views: 3,178
Posted By Palecomic
Thanks Paul! That's an ironic retro GoDaddy style...

Thanks Paul! That's an ironic retro GoDaddy style website ;)
Forum: Sample Prep / Library Generation 03-12-2014, 02:00 AM
Replies: 4
Views: 3,178
Posted By Palecomic
You wouldn't happen to be aware of an alternative...

You wouldn't happen to be aware of an alternative to this method would you? They can be a pain to get hold of, back order dates have slipped repeatedly (popularity?)

I know the binding capacity...
Forum: Bioinformatics 01-15-2014, 01:40 AM
Replies: 8
Views: 2,972
Posted By Palecomic
The best place to ask about this is the bio-linux...

The best place to ask about this is the bio-linux mailing list http://nebc.nerc.ac.uk/about-us/mailing-lists Tim Booth is pretty responsive.

Doesn't Qiime come installed as standard on Bio-linux...
Forum: Bioinformatics 05-06-2013, 11:44 PM
Replies: 2
Views: 1,598
Posted By Palecomic
Hi emartinez, in Mothur you need to use the...

Hi emartinez,

in Mothur you need to use the Get.oturep command: http://www.mothur.org/wiki/Get.oturep which gives you a single sequence per OTU. You might want to check out the weighted parameter...
Forum: Metagenomics 03-14-2013, 05:13 AM
Replies: 6
Views: 3,431
Posted By Palecomic
Biases are endless, and boil down to - keep...

Biases are endless, and boil down to - keep everything the same.

Lots have been covered in the TRFLP and DGGE literature, which were also PCR based, and biases that are detectable using those...
Forum: 454 Pyrosequencing 03-14-2013, 04:52 AM
Replies: 4
Views: 1,730
Posted By Palecomic
We size select twice with Ampure XP for amplicon...

We size select twice with Ampure XP for amplicon sequencing, primer (and/or primer dimer) that is undetectable by bioanalyzer can still lead to a big short read spike. Our amplicon is 676 bp and the...
Forum: 454 Pyrosequencing 03-14-2013, 04:45 AM
Replies: 4
Views: 2,892
Posted By Palecomic
Hi Linking, I'm going to take the opposite...

Hi Linking,

I'm going to take the opposite view and disagree with the reviewer. 16S sequencing and other PCR microbial ecology techniques that have been established for years are considered...
Forum: 454 Pyrosequencing 09-11-2012, 10:15 AM
Replies: 5
Views: 5,163
Posted By Palecomic
Take a look at QIIME http://www.qiime.org and...

Take a look at QIIME http://www.qiime.org and Mothur http://www.mothur.org for the analysis pipeline. BLAST isn't good enough for identifying these sequences, it does local alignments not global,...
Forum: 454 Pyrosequencing 09-11-2012, 09:49 AM
Replies: 9
Views: 13,181
Posted By Palecomic
The guys at Silva (bigger and better aligned 16S...

The guys at Silva (bigger and better aligned 16S database than the other alternatives) have done an exercise checking out all the 16S primers they could get their hands on.

Paper published here...
Forum: 454 Pyrosequencing 07-23-2012, 03:51 AM
Replies: 6
Views: 2,088
Posted By Palecomic
With 16S we've found that you get different...

With 16S we've found that you get different communities of bugs (alterations in relative abundance of each organism) depending on whether you use amplicon or shotgun processing and depending on the...
Forum: 454 Pyrosequencing 07-23-2012, 03:37 AM
Replies: 2
Views: 1,154
Posted By Palecomic
Hi Daniel, The MDA amplification leads to...

Hi Daniel,

The MDA amplification leads to branched DNA formation (there's mention of it here http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003042 and in the subsequent reference...
Forum: 454 Pyrosequencing 09-22-2011, 11:54 AM
Replies: 12
Views: 5,663
Posted By Palecomic
I would guess not, though there might be some...

I would guess not, though there might be some useful aspects of it? I think you might need a friendly local bioinformatician to help get you started.
Forum: 454 Pyrosequencing 09-22-2011, 11:43 AM
Replies: 12
Views: 5,663
Posted By Palecomic
What's your application? Qiime is for 16S rRNA...

What's your application? Qiime is for 16S rRNA amplicon sequencing or other microbial ecology stuff.
Forum: 454 Pyrosequencing 09-22-2011, 11:34 AM
Replies: 4
Views: 3,427
Posted By Palecomic
I expect that's why the Jr is just Jr and not FLX...

I expect that's why the Jr is just Jr and not FLX Jr. Not that I'm bitter, just hoping that Jr hits puberty and becomes Jr+ ;)
Forum: 454 Pyrosequencing 09-22-2011, 11:09 AM
Replies: 12
Views: 5,663
Posted By Palecomic
I'm fairly sure the virtual box root password...

I'm fairly sure the virtual box root password used to be 'qiime', but that may not be the case in the most recent version.

If you're using qiime on a mac, save yourself effort by installing...
Forum: 454 Pyrosequencing 08-05-2011, 02:50 AM
Replies: 9
Views: 4,633
Posted By Palecomic
Have commented on Nick's blog and updated here:...

Have commented on Nick's blog and updated here: http://seqanswers.com/forums/showpost.php?p=48280&postcount=27 we're using the V3-V5 primers and you can shift some of those medium length reads...
Forum: 454 Pyrosequencing 08-05-2011, 02:45 AM
Replies: 2
Views: 1,965
Posted By Palecomic
QIIME should help you to do some aspects of what...

QIIME should help you to do some aspects of what you want to do.

You can cluster sequences by identity using uclust (and other methods), pick representative sequences from the clusters and build...
Forum: 454 Pyrosequencing 08-05-2011, 02:42 AM
Replies: 27
Views: 11,054
Posted By Palecomic
Dredging this thread from the depths as have some...

Dredging this thread from the depths as have some updates that might be useful:

We've been having problems with really random runs (16S amplicons) on our Junior and have solved a couple of...
Forum: 454 Pyrosequencing 08-05-2011, 02:35 AM
Replies: 3
Views: 1,597
Posted By Palecomic
I've seen this once on our Junior, and it was...

I've seen this once on our Junior, and it was also about the top third that worked and the bottom third that failed. This turned out to be due to the centrifuge we were using, the Eppendorf 5450 (I...
Forum: 454 Pyrosequencing 04-19-2011, 01:30 AM
Replies: 27
Views: 11,054
Posted By Palecomic
OK - to update, we have the same problem again. ...

OK - to update, we have the same problem again. A very small volume of clearing at the bottom of the emPCR, no classic banding of the emulsion and this time it is possible to see a small pellet of...
Forum: 454 Pyrosequencing 04-18-2011, 01:54 AM
Replies: 9
Views: 4,666
Posted By Palecomic
We have lots of primer dimer, and it is tricky to...

We have lots of primer dimer, and it is tricky to remove, but have had success with an initial ethanol/sodium acetate precipitation (to pool replicate PCRs together) followed by two rounds of Ampure...
Forum: 454 Pyrosequencing 04-18-2011, 01:42 AM
Replies: 27
Views: 11,054
Posted By Palecomic
Hi Yaximik, With your emulsions, do you see...

Hi Yaximik,

With your emulsions, do you see a small clearing at the very bottom of the tubes?

We're doing amplicons and have carefully optimised our cpb ratio so that we get 5 % enrichment...
Forum: Bioinformatics 04-11-2011, 07:20 AM
Replies: 16
Views: 4,383
Posted By Palecomic
Sorry, just spotted your update. MEGAN would do...

Sorry, just spotted your update. MEGAN would do the job, but check out that PhylOTU paper for a brief discussion of its limitations.
Forum: Bioinformatics 04-11-2011, 07:16 AM
Replies: 16
Views: 4,383
Posted By Palecomic
I'd also check out Silva http://www.arb-silva.de/...

I'd also check out Silva http://www.arb-silva.de/ it's from the guys who made Arb. The plus points are that the alignment is better than greengenes (which uses an old alignment from the Silva group)...
Forum: Sample Prep / Library Generation 01-20-2011, 01:50 AM
Replies: 0
Views: 2,836
Posted By Palecomic
PCR product normalization

We're multiplexing quite variable PCR products from environmental samples in single runs on a 454 Junior. Increasing the multiplex level to the most cost effective (we've got lots of samples!) means...
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