Whole Genome Mapping by DNA Sequencing With Linked-Paired-End Library

Abstract

The present invention relates to innovative means of generating sequence-linked DNA fragments and subsequent uses of such linked DNA fragments for de novo haplotype-resolved whole genome mapping and massively parallel sequencing. In various embodiments described herein, the methods of the invention relate to methods of generating paired-end nucleic acid fragment sharing common linker nucleic acid sequences using a nicking endonuclease, a T7 endonuclease, a restriction enzyme, or a transposase, methods of analyzing the nucleotides sequences from the linked-paired-end sequenced fragments, and methods of de novo whole genome mapping. Thus, the methods of this invention allow establishing sequence contiguity across the whole genome, and achieving high-quality, low-cost de novo assembly of complex genomes.

1 .- 16 . (canceled) 
     
     
         17 . A method of generating a linked-paired-end nucleic acid fragment from a DNA sample, the method comprising:
 a. contacting a double-stranded DNA template having a first and a second DNA strand with a restriction endonuclease to form overhang double stranded cuts of the DNA at a sequence-specific location; and,   b. conducting a base extension reaction on the fragments of the overhang DNA thereby generating blunt end double-stranded DNA fragments that shares common linker sequences between each other.   
     
     
         18 . The method of  claim 17 , wherein the nicking endonuclease is type II endonuclease. 
     
     
         19 . The type II endonuclease of  claim 18 , wherein the endonuclease is from subtype IIb. 
     
     
         20 . The method of  claim 19 , wherein the endonuclease comprises one or more endonucleases selected from the group consisting of BcgI, Bsp24I, BaeI, CjeI, and CjePI. 
     
     
         21 . The method of  claim 20 , wherein the endonuclease enzyme is BaeI. 
     
     
         22 . The method of  claim 17 , wherein the base extension reaction comprises contacting the first DNA strand with a polymerase, one or more nucleotides, and a ligase. 
     
     
         23 . The method of  claim 17 , wherein the generated fragments are 50 kb or less and the adjacent linker fragments share at least one selected from the group consisting of 100 bp or more and 50 bp or more. 
     
     
         24 . The method of  claim 17 , wherein the linked-paired-end fragments are sequenced with at least one high throughput next generation sequencing platform selected from the group consisting of Illumina sequencing, SOLiD sequencing, 454 pyrosequencing and Ion Torrent semiconductor sequencing. 
     
     
         25 . The method of  claim 24 , wherein the library preparation for sequencing comprises:
 a. circularizing the linked-paired-end fragments;   b. fragmenting and size selecting the fragments of interest; and,   c. ligating adaptors at a location selected from the group consisting of both ends of the fragments for paired-end sequencing and one end of the fragments for singled-end sequencing.   
     
     
         26 . A method of whole genome mapping, the method comprising
 a. constructing a linked-paired-end sequencing library wherein the linked DNA fragments are used; and,   b. mapping the sequence reads of the sequenced libraries to the reference genome map.   
     
     
         27 . The method of  claim 26 , wherein the genome mapping comprises a haplotype sequence assembly. 
     
     
         28 . The method of  claim 27 , wherein the haplotype comprises the human major histocompatibility (MHC) region. 
     
     
         29 . A method of analyzing computationally the nucleotides sequences from linked-paired-end sequenced fragments, the method comprising performing a pairwise comparison of all overlap nucleotides sequence read, and correlating the nucleotides sequence read to a sequence assembly, a genetic or cytogenetic map, a structural pattern, a structural variation, physiological characteristic, a methylation pattern, an epigenomic pattern, a location of a CpG island, a single nucleotide polymorphism (SNP), a copy number variation (CNV), or a combination thereof.