High throughput genome sequencing on dna arrays

Abstract

The present invention is directed to methods and compositions for acquiring nucleotide sequence information of target sequences using adaptors interspersed in target polynucleotides. The sequence information can be new, e.g. sequencing unknown nucleic acids, re-sequencing, or genotyping. The invention preferably includes methods for inserting a plurality of adaptors at spaced locations within a target polynucleotide or a fragment of a polynucleotide. Such adaptors may serve as platforms for interrogating adjacent sequences using various sequencing chemistries, such as those that identify nucleotides by primer extension, probe ligation, and the like. Encompassed in the invention are methods and compositions for the insertion of known adaptor sequences into target sequences, such that there is an interruption of contiguous target sequence with the adaptors. By sequencing both “upstream” and “downstream” of the adaptors, identification of entire target sequences may be accomplished.

1 . (canceled) 
     
     
         2 . A method of preparing an array of fragments of a target nucleic acid to be sequenced, the method comprising:
 (a) providing a plurality of double-stranded DNA molecules each containing a target sequence from the target nucleic acid;   (b) introducing a nick into one of the strands in each of the double-stranded DNA molecules at an initial site, exposing a free 5′ strand and a free 3′ strand at the site;   (c) treating the nicked DNA molecules with a DNA polymerase so as to extend the free 3′ strand and displace or degrade the free 5′ strand, thereby moving the nick along the nicked strand to a new position downstream from the initial site;   (d) disabling the DNA polymerase;   (e) cleaving the DNA molecules at the new position, thereby forming a gap in both strands of the DNA molecules;   (f) inserting a downstream oligonucleotide adaptor into the gap of each DNA molecule to form a nucleic acid construct; and   (g) arraying the constructs on a solid surface for nucleic acid sequencing.   
     
     
         3 . The method of  claim 2 , wherein step (b) comprises inserting an initial oligonucleotide adaptor into the DNA molecules at or near the initial site. 
     
     
         4 . The method of  claim 3 , wherein the initial adaptor comprises a recognition site for a nicking enzyme, and step (b) further comprises contacting the DNA molecules with the nicking enzyme so as to introduce the nick in or near the initial adaptor. 
     
     
         5 . The method of  claim 3 , wherein the initial adaptor is inserted into the DNA molecules such that at one end of the adaptor, only one of the two strands is ligated to the rest of the DNA molecule, thereby introducing the nick into the opposite strand. 
     
     
         6 . The method of  claim 3 , wherein the initial adaptor is not an interspersed adaptor. 
     
     
         7 . The method of  claim 2 , wherein the new position of the nick following step (c) is a few hundred bases downstream from the initial site. 
     
     
         8 . The method of  claim 2 , wherein the DNA molecules are circularized after step (f) such that the downstream adaptor connects to a site upstream from the initial site of the nick. 
     
     
         9 . The method of  claim 2 , wherein the DNA molecules are amplified before or after step (g). 
     
     
         10 . The method of  claim 2 , wherein the amplified constructs are arrayed on the surface in step (g) in a regular pattern such that at least 70 percent of the constructs are optically resolvable 
     
     
         11 . The method of  claim 2 , further comprising determining at least part of the sequence of the target nucleic acid from the DNA molecules arrayed on the surface. 
     
     
         12 . An iterative process for introducing multiple oligonucleotide adaptors into double-stranded DNA fragments in a predetermined order, but at spacings that are not precisely known in advance,
 wherein each of the fragments contains both a target sequence from a target nucleic acid to be sequenced and an initial oligonucleotide adaptor that is upstream from at least part of the target sequence,   wherein the process comprises:   (a) introducing a nick into one of the strands in each of the double-stranded fragments at an initial site that is within or near the initial adaptor, exposing a free 5′ strand and a free 3′ strand at the site;   (b) treating the nicked fragments with a DNA polymerase so as to extend the free 3′ strand and displace or degrade the free 5′ strand, thereby moving the nick along the nicked strand to a new position downstream from the initial site;   (c) inserting another oligonucleotide adaptor into the fragments at the new position;   (d) repeating steps (a), (b), and (c) using the most recently inserted adaptor to introduce the next nick until all of the multiple adaptors are inserted into the DNA fragments.   
     
     
         13 . A method for preparing an array of fragments of a target nucleic acid to be sequenced, the method comprising:
 (a) providing a plurality of double-stranded circular DNA molecules, each containing:
 (i) a target sequence from the target nucleic acid, 
 (ii) a first adaptor sequence, and 
 (iii) a nick in one strand of the circular DNA within or nearby the first adaptor sequence, thereby forming an exposed 5′ end and an exposed 3′ end in the strand; 
   (b) treating the nicked fragments with a DNA polymerase so as to extend the strand at the 3′ end and displace or degrade the strand at the 5′ end, thereby moving the nick along the strand to a new position;   (c) disabling the DNA polymerase;   (d) cleaving the circular DNA molecules at the new position, thereby forming linear DNA molecules that each comprise:
 (i) the first adaptor sequence, 
 (ii) a first mate-pair arm, and 
 (iii) a second mate-pair arm; and subsequently 
   (e) arraying the DNA molecules on a solid surface for nucleic acid sequencing.   
     
     
         14 . The method of  claim 13 , wherein reaction conditions of the nick translation in step (b) is controlled so that the nick is moved by an average distance of 100 to 200 bases. 
     
     
         15 . The method of  claim 13 , wherein the linear DNA comprises 10 to 50 bases of adaptor sequence and 30 to 300 bases of sequence from the target nucleic acid. 
     
     
         16 . The method of  claim 13 , wherein the nick was introduced into the circular DNA by contacting the circles with a nicking enzyme that binds to a recognition sequence contained within the first adaptor sequence. 
     
     
         17 . The method of  claim 13 , wherein the nick was introduced into the circular DNA by inserting the first adaptor into the circular DNA such that at one end of the adaptor, only one of the two strands is ligated to the rest of the circular DNA, thereby introducing the nick into the opposite strand. 
     
     
         18 . The method of  claim 13 , wherein a second oligonucleotide adaptor is inserted into the linear DNA at the new position. 
     
     
         19 . The method of  claim 13 , wherein the second adaptor is a single-stranded oligonucleotide that is ligated to the strand of the circular DNA in which the nick resides, and a cut is made in the opposite strand of the circular DNA, thereby cleaving the circular DNA. 
     
     
         20 . The method of  claim 13 , further comprising determining at least part of the sequence of the target nucleic acid by sequencing the DNA molecules arrayed on the surface. 
     
     
         21 . The method of  claim 20 , wherein the part of the sequence is determined by a process that includes sequencing by synthesis.