Background

Pyrosequencing techniques allow scientists to perform prokaryotic genome sequencing and achieve draft sequences within a few days. However, the sequencing results always turn out to contain several hundred contigs. A multiplex PCR procedure is then needed to fill all of the gaps and to link the contigs into one full-length genome sequence [[1–10]]. The full-length prokaryotic genome sequence is the gold standard for comparative prokaryotic genome analysis. This study assessed pyrosequencing strategies by using a simulation with 100 prokaryotic genomes.

Results

Our simulation shows the following: first, a single-end 454 Jr Titanium run combined with a paired-end 454 Jr Titanium run may assemble about 90% of 100 genomes into <10 scaffolds and 95% of 100 genomes into <150 contigs; second, the average contig N50 size is more than 331 kb (Table 1); third, the average single base accuracy is >99.99% (Table 1); fourth, the average false gene duplication rate is <0.7% (Table 1); fifth, the average false gene loss rate is <0.4% (Table 1); sixth, the total size of long repeats (both repeat length >300 bp and >700 bp) is significantly correlated to the number of contigs (Table 4); and, seventh, increasing the read length of a pyrosequencing run could improve the assembly quality significantly (Table 1, 2, 3).

Table 1 Main average indices for different sequencing strategies for 100 genomes (400-bp read length)
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Table 2 Main average indices for different sequencing strategies for 100 genomes (100-bp read length)
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Table 3 Main average indices for different sequencing strategies for 100 genomes (200-bp read length)
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Table 4 Linear regression results for 100 genomes, between the genome quality indicators and, for various read lengths, the number of repeats in the genome, the total repeat length of the genome and the percentage of the total repeat length of the genome
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Conclusions

A single-end 454 Jr run combined with a paired-end 454 Jr run is a good strategy for prokaryotic genome sequencing. This strategy provides a solution to producing a high-quality draft genome sequence of almost any prokaryotic organism, selected at random, within days. It could be the first step to achieving the full-length genome sequence. It also makes the subsequent multiplex PCR procedure (for gap filling) much easier, aided by the knowledge of the orders/orientations of most of the contigs. As a result, large-scale full-length prokaryotic genome-sequencing projects could be finished within weeks.