JOURNAL ARTICLE

Paired-end sequencing of long-range DNA fragments for de novo assembly of large, complex Mammalian genomes by direct intra-molecule ligation

Asan, Chunyu Geng, Yan Chen, Kui Wu, Qingle Cai, Yu Wang, Yongshan Lang, Hongzhi Cao, Huangming Yang, Jian Wang, Xiuqing Zhang
PloS One 2012, 7 (9): e46211
23029438

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 over 100-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.

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