Heejung Bang, Marie Davidian
Experimental statistics for biological sciences
Methods in molecular biology (Clifton, N.J.)
detection validation subsequent interpretation potentially mosaic singlenucleotide variants snv within nextgeneration sequencing data remains challenge research clinical laboratory settings ability identify mosaic variants high genome coverage sequencing data levels 1 underscores necessity developing guidelines best practices verify variants orthogonally droplet digital pcr ddpcr proven powerful precise method allows determination lowlevel variant fractions within given sample herein describe two precise ddpcr methods using either fluorescent taqman hydrolysis probe approach evagreen fluorescent dye protocol taqman approach relies two different fluorescent probes fam hexvic designed amplify selectively presence single nucleotide change denoting variant reference position fractional abundance calculated determine relative quantities alleles final sample evagreen protocol relies two independent reactions oligonucleotide primers designed single nucleotide change denoting variant penultimate position primer relative amplification efficiency primer sets reference variant compared determine mosaic level given variant cost highcoverage sequencing continues decrease identification potentially mosaic variants also increase approaches outlined allow clinicians researchers precise determination true mosaic level given variant allowing better assess potential pathogenicity also possible recurrence risk offering genetic counseling families 2024 wiley periodicals llc basic protocol droplet digital pcr ddpcr taqman hydrolysis probes alternate protocol evagreen oligonucleotidespecific ddpcr