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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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Love this - good data definitely starts from good input, and poor input can only give relatively poor data. I particularly like the mention of Nanodrop/absorbance based methods for quantification. It's such a toss up if you'll get an accurate reading or what amounts to a randomly generated number, and a lot of library/sequencing related issues can be traced back to poor quant.Yesterday, 11:43 AM
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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...
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