BACKGROUND: Pyrosequencing techniques allow scientists to perform prokaryotic genome sequencing to achieve the draft genomic sequences within a few days. However, the assemblies with shotgun sequencing are usually composed of hundreds of contigs. A further multiplex PCR procedure is needed to fill all the gaps and link contigs into complete chromosomal sequence, which is the basis for prokaryotic comparative genomic studies. In this article, we study various pyrosequencing strategies by simulated assembling from 100 prokaryotic genomes. FINDINGS: Simulation study shows that a single end 454 Jr. run combined with a paired end 454 Jr. run (8 kb library) can produce: 1) ~90% of 100 assemblies with < 10 scaffolds and ~95% of 100 assemblies with < 150 contigs; 2) average contig N50 size is over 331 kb; 3) average single base accuracy is > 99.99%; 4) average false gene duplication rate is < 0.7%; 5) average false gene loss rate is < 0.4%. CONCLUSIONS: A single end 454 Jr. run combined with a paired end 454 Jr. run (8 kb library) is a cost-effective way for prokaryotic whole genome sequencing. This strategy provides solution to produce high quality draft assemblies for most of prokaryotic organisms within days. Due to the small number of assembled scaffolds, the following multiplex PCR procedure (for gap filling) would be easy. As a result, large scale prokaryotic whole genome sequencing projects may be finished within weeks.
BACKGROUND: Pyrosequencing techniques allow scientists to perform prokaryotic genome sequencing to achieve the draft genomic sequences within a few days. However, the assemblies with shotgun sequencing are usually composed of hundreds of contigs. A further multiplex PCR procedure is needed to fill all the gaps and link contigs into complete chromosomal sequence, which is the basis for prokaryotic comparative genomic studies. In this article, we study various pyrosequencing strategies by simulated assembling from 100 prokaryotic genomes. FINDINGS: Simulation study shows that a single end 454 Jr. run combined with a paired end 454 Jr. run (8 kb library) can produce: 1) ~90% of 100 assemblies with < 10 scaffolds and ~95% of 100 assemblies with < 150 contigs; 2) average contig N50 size is over 331 kb; 3) average single base accuracy is > 99.99%; 4) average false gene duplication rate is < 0.7%; 5) average false gene loss rate is < 0.4%. CONCLUSIONS: A single end 454 Jr. run combined with a paired end 454 Jr. run (8 kb library) is a cost-effective way for prokaryotic whole genome sequencing. This strategy provides solution to produce high quality draft assemblies for most of prokaryotic organisms within days. Due to the small number of assembled scaffolds, the following multiplex PCR procedure (for gap filling) would be easy. As a result, large scale prokaryotic whole genome sequencing projects may be finished within weeks.
Authors: Irina A Kataeva; Sung-Jae Yang; Phuongan Dam; Farris L Poole; Yanbin Yin; Fengfeng Zhou; Wen-chi Chou; Ying Xu; Lynne Goodwin; David R Sims; John C Detter; Loren J Hauser; Janet Westpheling; Michael W W Adams Journal: J Bacteriol Date: 2009-04-03 Impact factor: 3.490
Authors: Daniel Wibberg; Jochen Blom; Sebastian Jaenicke; Florian Kollin; Oliver Rupp; Birgit Scharf; Susanne Schneiker-Bekel; Rafael Sczcepanowski; Alexander Goesmann; Joao Carlos Setubal; Rüdiger Schmitt; Alfred Pühler; Andreas Schlüter Journal: J Biotechnol Date: 2011-02-15 Impact factor: 3.307
Authors: David A Baltrus; Manuel R Amieva; Antonello Covacci; Todd M Lowe; D Scott Merrell; Karen M Ottemann; Markus Stein; Nina R Salama; Karen Guillemin Journal: J Bacteriol Date: 2008-10-24 Impact factor: 3.490
Authors: Maria Pia Di Bonaventura; Rob DeSalle; Mihai Pop; Niranjan Nagarajan; David H Figurski; Daniel H Fine; Jeffrey B Kaplan; Paul J Planet Journal: J Bacteriol Date: 2009-05-15 Impact factor: 3.490