Forum: 454 Pyrosequencing
10-04-2012, 06:46 AM
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Replies: 3
Views: 740
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Forum: 454 Pyrosequencing
07-25-2012, 08:23 AM
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Replies: 6
Views: 966
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Forum: 454 Pyrosequencing
04-30-2012, 08:54 AM
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Replies: 6
Views: 966
We utilize the sfffile utility (a command line...
We utilize the sfffile utility (a command line tool included with the Roche software) to split the original sff file first by MID, and then by primer sequence.
The first step is to do the primary...
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Forum: 454 Pyrosequencing
04-04-2012, 12:55 PM
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Replies: 6
Views: 966
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Forum: 454 Pyrosequencing
03-12-2012, 01:49 PM
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Replies: 10
Views: 1,390
I don't buy the tired excuse of small fragment...
I don't buy the tired excuse of small fragment problems leading to bad amplicon sequencing. No matter how many times we SPRI purify and no matter how clean our traces look, we have had many months of...
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Forum: 454 Pyrosequencing
02-03-2012, 09:37 AM
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Replies: 17
Views: 2,549
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Forum: 454 Pyrosequencing
01-31-2012, 12:31 PM
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Replies: 3
Views: 1,214
We've seen the same problem: when mixing...
We've seen the same problem: when mixing amplicons, particularly of different sizes, FLX+ has significant issues. I would note that we routinely pool different sized amplicons on a GS-Jr and don't...
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Forum: 454 Pyrosequencing
01-31-2012, 12:28 PM
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Replies: 7
Views: 1,205
Mis-priming of amplicons during emPCR
Hi guys.
I've been having problems recently with the Roche/454 amplification primers mis-priming my amplicon templates in regions with some weak complimentarity. This is really messing up my data...
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Forum: 454 Pyrosequencing
01-31-2012, 12:24 PM
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Replies: 3
Views: 740
I'm a little biased, since I do home-brew HLA...
I'm a little biased, since I do home-brew HLA typing with Roche/454 (not using their kits) but I don't think it's possible to add anything to their kits since they come with the primers already added...
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Forum: 454 Pyrosequencing
11-01-2011, 10:17 AM
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Replies: 6
Views: 1,362
Indeed my previous thread was similar, but in...
Indeed my previous thread was similar, but in that case I was eventually able to turn off most of the filters and find my missing A reads (they were chimeric products generated with one of the Roche...
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Forum: 454 Pyrosequencing
10-31-2011, 10:59 AM
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Replies: 6
Views: 1,362
View all Raw reads?
I have experience with re-processing 454 data using the custom xml file, but I'm wondering if there's any relatively easy way to pass ALL of the raw well reads into an sfffile without any filtering...
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Forum: 454 Pyrosequencing
10-31-2011, 10:26 AM
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Replies: 1
Views: 775
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Forum: 454 Pyrosequencing
08-30-2011, 09:45 AM
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Replies: 4
Views: 1,243
I second the idea of reprocessing with a...
I second the idea of reprocessing with a non-amplicon pipeline and turning many of the quality filters off. We have successfully uncovered problems (such as incompatibilities between our amplicon and...
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Forum: 454 Pyrosequencing
08-29-2011, 09:57 AM
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Replies: 4
Views: 1,243
We have not been able to figure out what the...
We have not been able to figure out what the problem is yet. For us, bad runs seem to come in batches so we figured it was a problem with one of our lots.
Some things to consider, based on our...
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Forum: 454 Pyrosequencing
08-15-2011, 07:46 AM
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Replies: 9
Views: 1,612
I've done 454 FLX and Jr sequencing on 1,000 bp...
I've done 454 FLX and Jr sequencing on 1,000 bp amplicons without modifying the protocol at all and not had problems. Have you checked that there's not some other problem going on with your template...
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Forum: 454 Pyrosequencing
08-11-2011, 08:11 AM
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Replies: 4
Views: 1,243
Anyone else having Lib-A GS Jr problems recently?
Recently we've been having problems related to the quality of our Lib-A sequencing on the Jr. Very few passed filter reads, mixed-like flowgrams when our enrichment is correct, or problems with the...
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Forum: Bioinformatics
05-25-2011, 01:19 PM
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Replies: 26
Views: 2,865
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Forum: Bioinformatics
02-22-2011, 08:10 AM
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Replies: 2
Views: 998
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Forum: Bioinformatics
02-21-2011, 12:49 PM
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Replies: 2
Views: 998
Recovering all keypass 454 reads
I've done several Lib-A experiments where a significant percentage of my "A" reads are getting lost somewhere.
I know I have amplified template, since I get the corresponding "B" reads that...
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Forum: 454 Pyrosequencing
08-01-2010, 10:04 AM
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Replies: 21
Views: 6,738
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Forum: Bioinformatics
11-12-2009, 10:04 AM
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Replies: 3
Views: 3,425
We do highly multiplexed amplicon-based...
We do highly multiplexed amplicon-based genotyping with 454 and have found AVA inadequate.
Here's how we deconvolute and analyze the tagged sequence data. The key is to split apart the .sff files...
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