Paired-End Sequencing of Long-Range DNA Fragments for De Novo Assembly of Large, Complex Mammalian Genomes by Direct Intra-Molecule Ligation

Abstract

Background: The relatively short read lengths from next generation sequencing (NGS) technologies still pose a challenge for de novo assembly of complex mammal genomes. One important solution is to use paired-end (PE) sequence information experimentally obtained from long-range DNA fragments (>1 kb). Here, we characterize and extend a long-range PE library construction method based on direct intra-molecule ligation (or molecular linker-free circularization) for NGS. Results: We found that the method performs stably for PE sequencing of 2- to 5- kb DNA fragments, and can be extended to 10-20 kb (and even in extremes, up to ~35 kb). We also characterized the impact of low quality input DNA on the method, and develop a whole-genome amplification (WGA) based protocol using limited input DNA (<1 μg). Using this PE dataset, we accurately assembled the YanHuang (YH) genome, the first sequenced Asian genome, into a scaffold N50 size of >2 Mb, which is over100-times greater than the initial size produced with only small insert PE reads(17 kb). In addition, we mapped two 7- to 8- kb insertions in the YH genome using the larger insert sizes of the long-range PE data. Conclusions: In conclusion, we demonstrate here the effectiveness of this long-range PE sequencing method and its use for the de novo assembly of a large, complex genome using NGS short reads. © 2012 Asan et al.