Use of Next Generation Sequencing (NGS) Platforms for CE Sequencing on Thousands of Samples

Although there are limitations (throughput, scalability, speed, labor intensiveness, and cost), sequence information for specific targeted regions of interest, has traditionally been generated by capillary electrophoresis (CE)-based Sanger sequencing. Next-Generation Sequencing (NGS) enables inexpensive (per base), massive, rapid, parallel sequencing of entire eukaryotic genomes. However, these attributes do not translate to single targets, where the cost of NGS lies in the sample preparation more than the per base cost of data generation. We have developed a method that inexpensively and in high throughput creates libraries of single (or up to 1000 plex) targets over thousands of samples for NGS. This method, HELSR, (hybridization extension ligation sequencing reaction) allows sequencing resources to be focused on single or multiple informative loci. Custom HELSR probes can be designed to generate overlapping amplicons to provide complete coverage of entire targeted single or multiple regions. With the use of Eureka Genomics' EasySeq indices, all the desired targets from any sample are assigned a unique sample ID sequence (index). This can simultaneously occur in thousands of samples. These indexed samples are pooled into a single sequencing library and NGS data is generated. Based on the sample ID index, the reads are sorted by sample and provided to the biologist. This method (with Eureka Genomics' EasySeq indices) has been used in a Mycobacterium tuberculosis (MTB) multi-drug resistant screening assay (Investigen) to assess 70 target regions, over 16 genes and containing 317 known mutations. The method is being implemented in Eureka Genomics' assays for human colorectal (261 targets, 7 genes spanning 21337 bp of coding regions), prostate cancer (129 loci), and in collaboration with Columbia University for human respiratory viruses (98 targets, 37 viruses).