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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
Many thanks, Lisle Gibbs
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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. -
How is such a MID configuration file defined?Comment
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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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I’m not a sequencing expert. I’m a purification scientist who uses NGS to evaluate workflows my group develops. With this perspective, we think about the sample first and the NGS workflow second. The sequencer is an exceptionally honest reporter, but it can only report on what you give it, so whether you get clean, interpretable data from an NGS workflow is largely determined before you begin.
Here are nine questions we think about, in roughly the order they matter, before...06-18-2026, 07:11 AM -
Data variability is still an issue in sequencing technologies despite the advances in reproducibility and accuracy of these platforms. But the problem does not originate in the sequencing itself, but in the previous steps, before the sample reaches the sequencer.
The first step is collection, followed by preservation and sample preparation for analysis. Most scientists overlook those steps, but not being careful might just be skewing the experiment’s results.
...06-02-2026, 10:05 AM
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