Efficient high-throughput resequencing of genomic DNA

Abstract

Targeted resequencing of genomic DNA from organisms such as humans is an important tool enabling experimental access to variation within the species and between similar species. Taking full advantage of the reference genome sequences in designing robust, specific PCR assays and using stringent conditions, resequencing can be done efficiently without purification of the PCR product. By using a 10-fold greater amount of one primer when setting up the PCR initially in a new version of asymmetric PCR, one simply adds the rest of the sequencing reagents at the end of PCR and allows the sequencing reaction to proceed, with the excess PCR primer serving as the sequencing primer. We demonstrated that this streamlined protocol can be used with PCR products up to 1300 bp and had up to a 97% success rate in high-throughput analysis of allele frequencies for >30,000 single-nucleotide polymorphisms (SNPs). SNP primers and characterization results are provided at http://snp.wustl.edu.

Figure 1.

Resequencing flow diagram for SNPs. Databases (e.g., http://www.ncbi.nlm.nih.gov/SNP/; http://snp.cshl.org/) provided the surrounding sequence necessary for primer design (see Methods). With adjustment of input DNA, a similar flow diagram could be used for other resequencing projects, such as for exons.

Figure 2.

Aligned resequencing electropherograms for the region containing SNP rs1040683. From the top, the traces are, respectively, from African Americans, Asians, European Americans, and a reference DNA. The SNP, which maps to the X-chromosome, is highlighted. The reference individual is a C/G heterozygote, and the allele frequencies differ among the populations.