title: 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 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.
authors.affiliation: BGI-Shenzhen, Shenzhen, Guangdong, China.
authors.lastname: Asan
authors.forename: Chunyu
authors.initials: C
authors.lastname: Geng
authors.forename: Yan
authors.initials: Y
authors.lastname: Chen
authors.forename: Kui
authors.initials: K
authors.lastname: Wu
authors.forename: Qingle
authors.initials: Q
authors.lastname: Cai
authors.forename: Yu
authors.initials: Y
authors.lastname: Wang
authors.forename: Yongshan
authors.initials: Y
authors.lastname: Lang
authors.forename: Hongzhi
authors.initials: H
authors.lastname: Cao
authors.forename: Huangming
authors.initials: H
authors.lastname: Yang
authors.forename: Jian
authors.initials: J
authors.lastname: Wang
authors.forename: Xiuqing
authors.initials: X
authors.lastname: Zhang
mesh_terms: D000465:Algorithms; D000818:Animals; D002874:Chromosome Mapping; D015894:Genome, Human; D015698:Genomic Library; D006801:Humans; D008322:Mammals / Q000235:genetics*; D016254:Mutagenesis, Insertional; D016366:Open Reading Frames; D017422:Sequence Analysis, DNA / Q000379:methods*