HTS-PEG: a method for high throughput sequencing of the paired-ends of genomic libraries

PLoS One. 2012;7(12):e52257. doi: 10.1371/journal.pone.0052257. Epub 2012 Dec 20.

Abstract

Second generation sequencing has been widely used to sequence whole genomes. Though various paired-end sequencing methods have been developed to construct the long scaffold from contigs derived from shotgun sequencing, the classical paired-end sequencing of the Bacteria Artificial Chromosome (BAC) or fosmid libraries by the Sanger method still plays an important role in genome assembly. However, sequencing libraries with the Sanger method is expensive and time-consuming. Here we report a new strategy to sequence the paired-ends of genomic libraries with parallel pyrosequencing, using a Chinese amphioxus (Branchiostoma belcheri) BAC library as an example. In total, approximately 12,670 non-redundant paired-end sequences were generated. Mapping them to the primary scaffolds of Chinese amphioxus, we obtained 413 ultra-scaffolds from 1,182 primary scaffolds, and the N50 scaffold length was increased approximately 55 kb, which is about a 10% improvement. We provide a universal and cost-effective method for sequencing the ultra-long paired-ends of genomic libraries. This method can be very easily implemented in other second generation sequencing platforms.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Chromosomes, Artificial, Bacterial*
  • Contig Mapping
  • Gene Library

Grants and funding

This work was supported by the key project (No. 30730089) of the National Natural Science Foundation of China (to AX), the National Basic Research Program of China (973 Program; No. 2011CB946101 and 2007CB815800) (to AX), Fundamental Research Funds for the Central Universities (to YF), the National Natural Science Foundation of China (No. 30801012) (to YF), the State High-Tech Development Project (863 Project 2008AA092601) (to SH) and the National Natural Science Foundation of China (No. 31171193) (to SH). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.