Derivation of DNA copy number concentration in the final reaction chamber with and without STA.

Where A = stock DNA concentration (ng/µL); l = length in bp (human and whale genomes ∼3×109 bp); V1 = DNA sample volume for STA (1.25 µL); V2 = STA mixture volume (5 µL); n = Total number of PCR cycles; V3 = Pooled mixture volume, which is derived by multiplying V2 and the number of SNP-GT assays (V3 = V2×SNP-GT assays); D = Dilution factor (5- or 20- fold); V4 = DNA sample volume for genotyping (2.1 µL); V5 = GT sample solution volume (5 µL) and V6 = Reaction chamber volume (6.75 nL)[9].ESX was calculated assuming 100% PCR efficiency.†Use equations 1, 5–6 when estimating copies/reaction chamber without STA.Use equations 1–6 when estimating copies/reaction chamber with STA in simplex.Use equations 1–3, 4♦ and 5–6 when estimating copies/reaction chamber with STA in Multiplex.*The Avogadro number (6.02214179×1023) was taken from Mohr et al[20] (CODATA-2006).•Average molecular weight of DNA base pair used was 615.8771[21].NA – refers to Not Applicable.