A simple method using PyrosequencingTM to identify de novo SNPs in pooled DNA samples

Abstract

A practical way to reduce the cost of surveying single-nucleotide polymorphism (SNP) in a large number of individuals is to measure the allele frequencies in pooled DNA samples. Pyrosequencing(TM) has been frequently used for this application because signals generated by this approach are proportional to the amount of DNA templates. The Pyrosequencing(TM) pyrogram is determined by the dispensing order of dNTPs, which is usually designed based on the known SNPs to avoid asynchronistic extensions of heterozygous sequences. Therefore, utilizing the pyrogram signals to identify de novo SNPs in DNA pools has never been undertook. Here, in this study we developed an algorithm to address this issue. With the sequence and pyrogram of the wild-type allele known in advance, we could use the pyrogram obtained from the pooled DNA sample to predict the sequence of the unknown mutant allele (de novo SNP) and estimate its allele frequency. Both computational simulation and experimental Pyrosequencing(TM) test results suggested that our method performs well. The web interface of our method is available at http://life.nctu.edu.tw/∼yslin/PSM/.

Figure 1.

The flowchart of the algorithm developed in this study.

Figure 2.

(A) The hypothetical pyrogram profile, W, for the wild-type DNA fragment, GATCGGTTCACGTC; (B) the hypothetical pyrogram profile, M, for the mutant allele, GAGCGGTTCACGTC; (C) the expected pyrogram profile, S, for the pooled DNA sample with 95% wild-type allele and 5% mutant allele (95% black bars + 5% white bars). All the three pyrogram profiles were simulated under the same Pyrosequencing^TM dispensing order of dNTPs, GATCGTCACGTC, with CV = 0.5%.

Figure 3.

(A) The blue bars represent the pyrogram, S^blue, of a pooled DNA sample composed of 95% wild-type allele and 5% mutant allele as in Figure 2C. The red bars represent the pyrogram, S^red, of a DNA sample composed of 100% wild-type allele. The two pyrogram profiles were simulated with CV = 0.5%. (B) The ratio profiles R^blue and R^red. (C) The relative cumulative frequencies of profiles Q^blue (blue circles) and Q^red (red triangles). The blue crosses represent the expected cumulative normal distribution, E^blue, which has the same mean and standard deviation as Q^blue. See the main text for the details.

Figure 4.

The alignment between (A) the profile T^blue, which is basically proportional to the unknown profile M, and (B) the profile W. See the main text for the details.

Figure 5.

The real Pyrosequencing^TM examination of the mitochondrial cytochrome b gene of P. parva: (A) the pyrogram of the wild-type DNA fragment, W; (B) the pyrogram of a pooled DNA sample containing 10% mutant DNA, S; (C) the profile R; (D) the profile T; (E) the profile W which is aligned to profile T. See the main text for the details.