6-10× pyrosequencing is a practical approach for whole prokaryote genome studies.

Next generation 454 pyrosequencing technology for whole bacterial genome sequencing involves a deep sequencing strategy with at least 15-20× in depth proposed by official protocols but usually done with over 20× in practices. In this study, we carried out a comprehensive evaluation of quality of the de novo assemblies based on realistic pyrosequencing simulated data from 1480 prokaryote genomes and 7 runs of machine-generated data. Our results demonstrated that for most of the prokaryote genomes, 6-10× sequencing in qualified runs with 400 bp reads could produce high quality draft assembly (>98% genome coverage, <100 contigs with N50 size >100 kb, single base accuracy >99.99, indel error rate <0.01%, false gene loss/duplication rate <0.5%). Our study proves the power of low depth pyrosequencing strategy, which provides a cost-effective way for sequencing whole prokaryote genomes in a short time and enables further studies in microbial population diversity and comparative genomics.