Whole-genome sequencing and assembly with high-throughput, short-read technologies.

@article{citeulike:1543434, 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 genomes will soon be possible with high-throughput, short-read technologies using our methodology.}, address = {Department of Computer Science, Stanford University, Stanford, California, United States of America. asundqui@cs.stanford.edu}, author = {Sundquist, A. and Ronaghi, M. and Tang, H. and Pevzner, P. and Batzoglou, S. }, citeulike-article-id = {1543434}, doi = {10.1371/journal.pone.0000484}, issn = {1932-6203}, journal = {PLoS ONE}, keywords = {genome-assembly, sequencing-by-hybridization-graph, short-read-sequencing}, posted-at = {2007-08-08 15:24:22}, priority = {2}, title = {Whole-genome sequencing and assembly with high-throughput, short-read technologies.}, url = {http://dx.doi.org/10.1371/journal.pone.0000484}, volume = {2}, year = {2007} }

TY - JOUR ID - citeulike:1543434 L3 - citeulike-article-id:1543434 TI - Whole-genome sequencing and assembly with high-throughput, short-read technologies. JF - PLoS ONE VL - 2 AD - Department of Computer Science, Stanford University, Stanford, California, United States of America. asundqui@cs.stanford.edu SN - 1932-6203 N2 - 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 genomes will soon be possible with high-throughput, short-read technologies using our methodology. KW - genome-assembly KW - sequencing-by-hybridization-graph KW - short-read-sequencing AU - Sundquist, A AU - Ronaghi, M AU - Tang, H AU - Pevzner, P AU - Batzoglou, S PY - 2007/// UR - http://dx.doi.org/10.1371/journal.pone.0000484 ER -