Abstract
Second generation sequencing has been widely used to sequence whole genomes. Though various paired-end sequencing methods have been developed to construct the long scaffold from contigs derived from shotgun sequencing, the classical paired-end sequencing of the Bacteria Artificial Chromosome (BAC) or fosmid libraries by the Sanger method still plays an important role in genome assembly. However, sequencing libraries with the Sanger method is expensive and time-consuming. Here we report a new strategy to sequence the paired-ends of genomic libraries with parallel pyrosequencing, using a Chinese amphioxus (Branchiostoma belcheri) BAC library as an example. In total, approximately 12,670 non-redundant paired-end sequences were generated. Mapping them to the primary scaffolds of Chinese amphioxus, we obtained 413 ultra-scaffolds from 1,182 primary scaffolds, and the N50 scaffold length was increased approximately 55 kb, which is about a 10% improvement. We provide a universal and cost-effective method for sequencing the ultra-long paired-ends of genomic libraries. This method can be very easily implemented in other second generation sequencing platforms.
Highlights
Generation, massively parallel sequencing, notable for its high throughput and cost-efficiency, has accelerated the pace of genome sequencing of new species
We developed a novel and universal high throughput method to sequence the paired-ends of a genomic library as an ultra-long span paired-end library preparation method (Figure 1)
The plasmids are fragmented by hydrodynamic shearing, and the size distribution of the fragments are kept within 61 kb of the vector backbone of the genomic DNA library
Summary
Massively parallel sequencing, notable for its high throughput and cost-efficiency, has accelerated the pace of genome sequencing of new species. Even billions, of reads are generated in a single instrument run, but the sequence read length it obtained is shorter than that of the traditional Sanger method [1,2]. Various spans of paired end reads are used to resolve assembly of the shotgun reads into a fine map of the genome [4,5,6,7]. The larger the span of a paired-end library, the lower the level of uniformity of the fragments and the lower the efficiency of circularization. The spans of these libraries are limited and usually range from 800 bp to 20 kb, sometimes up to 40 kb, which depend on the size of the first fragmentation
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