title: Pseudo-Sanger sequencing: massively parallel production of long and near error-free reads using NGS technology.
abstract: BACKGROUND
  Usually, next generation sequencing (NGS) technology has the property of ultra-high throughput but the read length is remarkably short compared to conventional Sanger sequencing. Paired-end NGS could computationally extend the read length but with a lot of practical inconvenience because of the inherent gaps. Now that Illumina paired-end sequencing has the ability of read both ends from 600 bp or even 800 bp DNA fragments, how to fill in the gaps between paired ends to produce accurate long reads is intriguing but challenging.
  
  
  RESULTS
  We have developed a new technology, referred to as pseudo-Sanger (PS) sequencing. It tries to fill in the gaps between paired ends and could generate near error-free sequences equivalent to the conventional Sanger reads in length but with the high throughput of the Next Generation Sequencing. The major novelty of PS method lies on that the gap filling is based on local assembly of paired-end reads which have overlaps with at either end. Thus, we are able to fill in the gaps in repetitive genomic region correctly. The PS sequencing starts with short reads from NGS platforms, using a series of paired-end libraries of stepwise decreasing insert sizes. A computational method is introduced to transform these special paired-end reads into long and near error-free PS sequences, which correspond in length to those with the largest insert sizes. The PS construction has 3 advantages over untransformed reads: gap filling, error correction and heterozygote tolerance. Among the many applications of the PS construction is de novo genome assembly, which we tested in this study. Assembly of PS reads from a non-isogenic strain of Drosophila melanogaster yields an N50 contig of 190 kb, a 5 fold improvement over the existing de novo assembly methods and a 3 fold advantage over the assembly of long reads from 454 sequencing.
  
  
  CONCLUSIONS
  Our method generated near error-free long reads from NGS paired-end sequencing. We demonstrated that de novo assembly could benefit a lot from these Sanger-like reads. Besides, the characteristic of the long reads could be applied to such applications as structural variations detection and metagenomics.
authors.forename: Jue
authors.initials: J
authors.lastname: Ruan
authors.forename: Lan
authors.initials: L
authors.lastname: Jiang
authors.forename: Zechen
authors.initials: Z
authors.lastname: Chong
authors.forename: Qiang
authors.initials: Q
authors.lastname: Gong
authors.forename: Heng
authors.initials: H
authors.lastname: Li
authors.forename: Chunyan
authors.initials: C
authors.lastname: Li
authors.forename: Yong
authors.initials: Y
authors.lastname: Tao
authors.forename: Caihong
authors.initials: C
authors.lastname: Zheng
authors.forename: Weiwei
authors.initials: W
authors.lastname: Zhai
authors.forename: David
authors.initials: D
authors.lastname: Turissini
authors.forename: Charles H
authors.initials: CH
authors.lastname: Cannon
authors.affiliation: Laboratory of Disease Genomics and Individualized Medicine, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing 100101, People's Republic of China. luxm@big.ac.cn.
authors.forename: Xuemei
authors.initials: X
authors.lastname: Lu
authors.forename: Chung-I
authors.initials: CI
authors.lastname: Wu
mesh_terms: D000465:Algorithms; D000818:Animals; D004247:DNA / Q000032:analysis*; D004331:Drosophila melanogaster / Q000235:genetics; D016678:Genome; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D017422:Sequence Analysis, DNA / Q000379:methods*
chemical_list: D004247:DNA