Roche 454 Amplicon Variant Analysis software

Lisle · 09-18-2009, 11:22 AM

Any suggestions for software alternatives to the Roche AVA module for analyzing amplicon variation in 454 data where individuals are identified with tagged primers?

Many thanks, Lisle Gibbs

proteasome · 11-12-2009, 10:04 AM

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 from the sequencer using Roche's command-line tool called "sfffile" which is available as part of the Roche-454 off-instrument software package. By using the correct arguments and MID-configuration file this will break your data into MID specific .sff files.

Then, we analyze each patient specific sequence data individually. A scriptable assembly software package will allow you to do this in batch. In our case we use Seqman, from DNAstar, to assemble the reads at high identity (99.5 - 100%) and then blast them against a genotyping database.

maasha · 02-25-2010, 01:25 AM

How is such a MID configuration file defined?

westerman · 02-25-2010, 10:26 AM

In your 454 software's config directory there will be an MIDConfig.parse file that contains the information in the form:

GSMIDs
{
mid = "MID1", "ACGAGTGCGT", 2;
mid = "MID2", "ACGCTCGACA", 2;
...

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09-18-2009, 11:22 AM	#1
Lisle Junior Member Location: USA Join Date: Sep 2009 Posts: 1	Roche 454 Amplicon Variant Analysis software Any suggestions for software alternatives to the Roche AVA module for analyzing amplicon variation in 454 data where individuals are identified with tagged primers? Many thanks, Lisle Gibbs

11-12-2009, 10:04 AM	#2
proteasome Member Location: Wisconsin Join Date: Jul 2009 Posts: 21	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 from the sequencer using Roche's command-line tool called "sfffile" which is available as part of the Roche-454 off-instrument software package. By using the correct arguments and MID-configuration file this will break your data into MID specific .sff files. Then, we analyze each patient specific sequence data individually. A scriptable assembly software package will allow you to do this in batch. In our case we use Seqman, from DNAstar, to assemble the reads at high identity (99.5 - 100%) and then blast them against a genotyping database.

02-25-2010, 01:25 AM	#3
maasha Senior Member Location: Denmark Join Date: Apr 2009 Posts: 137	How is such a MID configuration file defined?

02-25-2010, 10:26 AM	#4
westerman Rick Westerman Location: Purdue University, Indiana, USA Join Date: Jun 2008 Posts: 685	In your 454 software's config directory there will be an MIDConfig.parse file that contains the information in the form: GSMIDs { mid = "MID1", "ACGAGTGCGT", 2; mid = "MID2", "ACGCTCGACA", 2; ...