Efficient approach to unique single-nucleotide polymorphism discovery

Abstract

Single-nucleotide polymorphisms (SNPs) are the most frequently found DNA sequence variations in the human genome. It has been argued that a dense set of SNP markers can be used to identify genetic factors associated with complex disease traits. Because all high-throughput genotyping methods require precise sequence knowledge of the SNPs, any SNP discovery approach must involve both the determination of DNA sequence and allele frequencies. Furthermore, high-throughput genotyping also requires a genomic DNA amplification step, making it necessary to develop sequence-tagged sites (STSs) that amplify only the DNA fragment containing the SNP and nothing else from the rest of the genome. In this report, we demonstrate the utility of a SNP-screening approach that yields the DNA sequence and allele frequency information while screening out duplications with minimal cost and effort. Our approach is based on the use of a homozygous complete hydatidiform mole (CHM) as the reference. With this homozygous reference, one can identify and estimate the allele frequencies of common SNPs with a pooled DNA-sequencing approach (rather than having to sequence numerous individuals as is commonly done). More importantly, the CHM reference is preferable to a single individual reference because it reveals readily any duplicated regions of the genome amplified by the PCR assay before the duplicated sequences are found in GenBank. This approach reduces the cost of SNP discovery by 60% and eliminates the costly development of SNP markers that cannot be amplified uniquely from the genome.

Figure 1

The results of scanning sWXD3868 for SNPs by method 1 is shown in A. CEPH parents are 1, 2, 3, 4, and the CEPH population pool is 5 (additional detail about DNAs used are included in Methods). Sequencing was done with the dRhodamine terminators in A. The results for method 2 are shown in B. The CHM1 is sample 6 and the CEPH population pool is sample 5. Sequencing was done with the BigDye terminators in B. (↓) SNP locations. The small blue underhand for the T peak (↓) in B, sample 6 is a common sequencing artifact and was seen in a number of T peaks in this sequencing trace.

Figure 2

The results of scanning sWXD3654 for SNPs by methods 1 and 2 are shown. (Method 1) CEPH parents are 1, 2, 3, and 4 and the CEPH population pool is 5 (additional detail about DNAs used is included in Methods); (method 2) CHM1 is sample 6, and the CEPH population pool is sample 5. The CEPH population pool is shown only once. Sequencing was done with the BigDye terminators. (↓) SNP locations.

Figure 3

Results of testing STSs sWXD3555 (A) and sWXD3857 (B) against the chromosome panel pools (1–8), human genomic DNA (9), hamster genomic DNA (10), and mouse genomic DNA (11). sWXD3555 amplified only pools 2 and 8 and the human control, indicating that it was found on the X chromosome. sWXD3857 amplified all eight pools and the human control, indicating that it was found on multiple chromosomes. (Shaded arrows) The 700- and 1000-bp molecular weight standards (50–2000 bp, Bio-Rad, Hercules, CA); (black arrows) the specific PCR product. Detail descriptions of the chromosome panel pools are included in Methods.