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Whole-genome sequencing and assembly with highthroughput, short read technologies (2007)
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| Venue: | PLoS ONE |
| Citations: | 24 - 1 self |
BibTeX
@ARTICLE{Sundquist07whole-genomesequencing,
author = {Andreas Sundquist and Mostafa Ronaghi and Haixu Tang and Pavel Pevzner and Serafim Batzoglou},
title = {Whole-genome sequencing and assembly with highthroughput, short read technologies},
journal = {PLoS ONE},
year = {2007},
volume = {2},
pages = {484}
}
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Abstract
While recently developed short-read sequencing technologies may dramatically reduce the sequencing cost and eventually achieve the $1000 goal for re-sequencing, their limitations prevent the de novo sequencing of eukaryotic genomes with the standard shotgun sequencing protocol. We present SHRAP (SHort Read Assembly Protocol), a sequencing protocol and assembly methodology that utilizes high-throughput short-read technologies. We describe a variation on hierarchical sequencing with two crucial differences: (1) we select a clone library from the genome randomly rather than as a tiling path and (2) we sample clones from the genome at high coverage and reads from the clones at low coverage. We assume that 200 bp read lengths with a 1 % error rate and inexpensive random fragment cloning on whole mammalian genomes is feasible. Our assembly methodology is based on first ordering the clones and subsequently performing read assembly in three stages: (1) local assemblies of regions significantly smaller than a clone size, (2) clone-sized assemblies of the results of stage 1, and (3) chromosome-sized assemblies. By aggressively localizing the assembly problem during the first stage, our method succeeds in assembling short, unpaired reads sampled from repetitive genomes. We tested our assembler using simulated reads from D. melanogaster and human chromosomes 1, 11, and 21, and produced assemblies with large sets of contiguous sequence and a misassembly rate comparable to other draft assemblies. Tested on D. melanogaster and the entire human genome, our clone-ordering method produces accurate maps, thereby localizing fragment assembly and enabling the parallelization of the subsequent steps of our pipeline. Thus, we have demonstrated that truly inexpensive de novo sequencing of mammalian
Keyphrases
short read technology whole-genome sequencing whole mammalian genome read assembly accurate map short-read sequencing technology high coverage chromosome-sized assembly short read assembly protocol present shrap repetitive genome clone library bp read length unpaired read assembly methodology inexpensive random fragment contiguous sequence fragment assembly sequencing cost large set standard shotgun entire human genome utilizes high-throughput short-read technology simulated read eukaryotic genome clone size assembly problem draft assembly crucial difference human chromosome clone-sized assembly clone-ordering method $1000 goal sequencing protocol first stage low coverage error rate subsequent step local assembly
