Best technique for CNV and mutation analysis

Question

[neuro3030]

A very basic question:

I have 2 specific genes I am interested in. I would like to evaluate these genes for mutation and copy number alteration (specifically, homozygous and hemizygous loss).

What is/are the molecular technique(s) that are optimal for this? I need to analyze both FFPE tissue, frozen tissue, and cell cultures.

SNP? Sanger? MLPA?

Thanks and any help would be appreciated

[btsui]

For accurate CNV counting, my understanding is that you can design probes to target those two genes and then use ddPCR for quantification.

Are you targetting for specific mutations? You might want to read this paper (http://science.sciencemag.org/content/early/2018/01/17/science.aar3247).

[WouterDeCoster]

The scope of Biostars is bioinformatics. I agree that CNV and mutation analysis is bioinformatics, but you are specifically asking about methods, so this might be more appropriate for SeqAnswers.

Anyway,

I would like to evaluate these genes for mutation and copy number alteration

To figure out what the most efficient method is it is important that you mention:

• the size of genes
• the number of individuals in which you want to characterize mutations
• Which technologies you have access to: Illumina sequencers, capillary elektrophoresis, Sanger sequencer,...

Answer 1

[Kevin Blighe]

For copy number aberrations, just do real-time PCR targeting genomic sequence. I was doing this years ago and detecting copy number aberrations in EGFR and ERBB2 / Her2. For calculating relative copy number differences, here are sample formulae: A: How to report and plot qPCR data

For mutation analysis, Sanger is still the Gold standard. NGS is struggling to overcome Sanger due to the uncomfortable amount of error that NGS sequences produce.

Both of these options are 'cheap' but require access to the instruments with which to perform these techniques, of course.

Kevin