Next generation sequencing (NGS) technologies can rapidly and economically produce a draft genome of an organism de novo. However, the quality of the draft data is seldom more than 80% complete with >10e5 contigs for large genomes, which is insufficient for many applications. Most contigs begin and end with a repeat with existing library construction technologies. Sequence data that is closer to 95% finished with the unambiguous order and placement of genes would have the greatest utility for scientific and commercial research. New molecular tools that bridge the gaps between massively parallel short read sequencing technologies (35-500 bases) and the need for large scaffolds (>100,000 bases) to accurately assemble complex repeat rich genomes are needed. We have successfully developed 40 kb mate-pair NGS libraries by designing and constructing a novel pNGS fosmid system. Our results show that ~70% pNGS fosmid paired-end sequences can be obtained by either Illumina or 454 sequencing, which is significantly better than existing long-span mate-pair systems. Directly pooled pNGS fosmids sequencing of daunting genomes adds additional application. We have also developed a clone free long-span mate-pair NGS library construction technology for 10-300kb inserts.
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