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How to do sequencing validation?

Hello,

when I read about validation in sequencing, there it is always claimed to "establish performance characteristics such as accuracy, precision, sensitivity, specificity".

But it is absolutly unclear to me, how I can determine this value. Of course I know things that have influence on it. But in the end I need a value.

So what I have to do to get these values for sanger sequencing or ngs applications?

fin swimmer

sequencing validation
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written by finswimmer finswimmer

For validation of a technique you need to have either a truth set (e.g. SNPs you know that should be there) or you need to perform the same analysis using an orthogonal technique. Since sanger sequencing is often considered the golden standard, you could validate your NGS results using Sanger sequencing and see how much variants were true positive, false positive, false negative, true negative...

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written by WouterDeCoster WouterDeCoster

Hello WouterDeCoster,

well checking ngs results is a possible solution in theory. But who is doing that? If I just have a few target genes - ok. But how can I do it economically justifiable for 50, 100 or more genes?

fin swimmer

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written by finswimmer finswimmer

If you can figure out how to get the Sanger results in a vcf format you can check the concordance with the ngs variant calls.

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written by WouterDeCoster WouterDeCoster

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