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2013 ; 14
(1
): 711
Nephropedia Template TP
gab.com Text
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English Wikipedia
Pseudo-Sanger sequencing: massively parallel production of long and near
error-free reads using NGS technology
#MMPMID24134808
Ruan J
; Jiang L
; Chong Z
; Gong Q
; Li H
; Li C
; Tao Y
; Zheng C
; Zhai W
; Turissini D
; Cannon CH
; Lu X
; Wu CI
BMC Genomics
2013[Oct]; 14
(1
): 711
PMID24134808
show ga
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.