freqpcr: Estimation of population allele frequency using qPCR ΔΔCq measures from bulk samples

Abstract

Estimating the frequency of specific alleles in populations is a technique ubiquitous in population genetics, molecular ecology, evolutionary biology and their areas of application. Indices of genetic differentiation between populations are defined based on allele frequency measurement for one or more loci, on which phylogenetic analyses have been established (Takezaki & Nei, 1996;Wright, 1965). Allele frequencies fluctuate between generations due to adaptation or genetic drift. In evolutionary genetics, multilocus and/or time-series data of single nucleotide polymorphisms (SNPs) are used to detect natural selection (Nielsen, 2005), adaptive introgression (Hedrick, 2013) and historical events such as population bottlenecks

The complex II resistance mutation H258Y in succinate dehydrogenase subunit B causes fitness penalties associated with mitochondrial respiratory deficiency

“…This variation can be limited leading to a higher achieved precision by increasing the number of replicates per bulk sample. Unfortunately, this also increases the cost of sample processing and sequencing 66 . The lack of a perfect correlation between allele frequency values obtained using ddPCR and proportional Sanger sequencing can be attributed to a number of reasons.…”

“…The Sanger sequencing method has a relatively low sensitivity and suffers from the fact that different sequencing chromatographs, and thus relative abundance of peak residues, are obtained when using the forward and reverse primers. 66 By adopting the highly sensitive ddPCR technique developed by Mavridis et al 57 with overall detection limit of 1%, much lower allele frequencies can be detected in the pooled DNA samples. This can be very useful in the field to detect resistance mutations in the early phase of resistance development when resistant allele frequencies are usually very low.…”

“…The lack of a perfect correlation between allele frequency values obtained using ddPCR and proportional sanger sequencing can be attributed to a number of reasons. The sanger sequencing method has a relatively low sensitivity and suffers from the fact that different sequencing chromatographs, and thus relative abundance of peak residues, are obtained when using the forward and reverse primers 66 . By adopting the highly sensitive ddPCR technique developed by Mavridis et al 57 , with overall detection limit of 1%, much lower allele frequencies can be detected in the pooled DNA samples.…”

A mutation in chitin synthase I associated with etoxazole resistance in the citrus red mite Panonychus citri (Acari: Tetranychidae) and its uneven geographical distribution in Japan

“…37 This package was developed for maximum-likelihood estimation with interval estimation of the population allele frequency by qPCR based on the RED-ΔΔCt method. 38 The experimental parameters required for 'freqpcr' were determined using the 'knownqpcr_unpaired' module included in the package, using a triplicate qPCR data set obtained from DNA solutions with nine mixing ratios of X R /(X R + X S ) = {0.001, 0.005, 0.01, 0.05, 0.1, 0.25, 0.5, 0.75, 1}. X R and X S are the DNA solution (1 ng μL −1 ) volumes extracted as above from 40-70 females of FoOm (I1017F) and KgOi (I1017), respectively (Fig.…”

“…This approximation of Cq exp is obtained by substituting 0 for the amount of DNA extracted from the resistant individuals in eqn (11) of Sudo and Osakabe. 38 The justification is explained in the package vignette: https://cran.r-project. org/web/packages/freqpcr/vignettes/freqpcr-intro.html (the section 'To circumvent frequent missing values of target1').…”