High throughput genome sequencing on DNA arrays
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 - 82 . (canceled)
83 . A method for making a random array comprising a solid substrate comprising a surface, said method comprising:
(a) providing a library of circular templates, where said circular templates comprise:
(i) a first target sequence of a target polynucleotide;
(ii) a first adaptor;
(iii) a second target sequence of said target polynucleotide; an
(iv) a second interspersed adaptor, wherein said second interspersed adaptor is located between said first target sequence and said second target sequence, and wherein said first target sequence and said second target sequence are contiguous segments in said target polynucleotide;
(b) generating concatemers from said library of circular templates such that said concatemers comprise multiple copies of one of said library of circular templates;
(c) randomly disposing said concatemers on said surface, wherein said surface comprises discrete regions, and wherein said concatemers are randomly disposed on said discrete regions such that at least 70 percent of said concatemers are individually resolvable and such that at least 70 percent of said concatemers have a nearest neighbor distance of 200 nm or greater, thereby making said random array.
84 . The method of claim 83 , wherein said surface comprises silica.
85 . The method of claim 84 , wherein said silica comprises functional moieties.
86 . The method of claim 85 , wherein said functional moieties are a member selected from: amines, silanes and hydroxyl.
87 . The method of claim 86 , wherein said functional moieties are amines.
88 . The method of claim 83 , wherein said concatemers are immobilized on said discrete regions through a noncovalent interaction.
89 . (canceled)
90 . The method of claim 83 , wherein said concatemers are disposed on said surface at a density of at least 1000 molecules per μm 2 .
91 . The method of claim 83 , wherein said circular templates further comprise a third target sequence and a third interspersed adaptor, wherein said third interspersed adaptor is located between said second target sequence and said third target sequence.
92 . The method of claim 91 , wherein said circular templates further comprise a fourth target sequence and a fourth interspersed adaptor, wherein said fourth interspersed adaptor is located between said third target sequence and said fourth target sequence.
93 . The method of claim 83 , wherein said library of circular templates is formed by a method comprising:
(a) providing first circular polynucleotides comprising said first adaptor and a fragment of said target polynucleotide, wherein said first adaptor comprises a recognition site for a restriction enzyme;
(b) cleaving said first circular polynucleotides with said restriction enzyme to form first linear constructs, wherein said restriction enzyme binds to said recognition site within said first adaptor and cleaves said fragment at a distance from said recognition site, wherein said first linear construct comprises said first adaptor interposed between said first and second target sequences;
(c) ligating said second interspersed adaptor to said first linear constructs to form second linear constructs;
(d) circularizing said second linear constructs to create second circular polynucleotides, wherein said second circular polynucleotides comprise said first adaptor, said first target sequence, said second interspersed adaptor, said second target sequence in that order, such that nucleotides immediately adjacent to each terminus of said second interspersed adaptor represent a contiguous segment of said fragment.
94 . The method of claim 93 , wherein said restriction enzyme cleaves said fragment at a distance of 2 to 20 nucleotides from said recognition site.
95 . The method of claim 93 , wherein said restriction enzyme cleaves said fragment at a distance of 18 to 25 nucleotides from said recognition site.
96 . The method of claim 83 , wherein said at least 70 percent of said concatemers randomly disposed on said discrete regions have a nearest neighbor distance of 500 nm or greater.
97 . The method of claim 83 , wherein said at least 70 percent of said concatemers randomly disposed on said discrete regions have a nearest neighbor distance of 700 nm or greater.
98 . The method of claim 83 , wherein said surface further comprises inert inter-regional areas between said discrete regions and wherein said concatemers do not bind to said inert inter-regional areas.
99 . The method of claim 83 , wherein said first adaptor and said second interspersed adaptor have known sequences and said first and second target sequences are unknown genomic sequences.
100 . The method of claim 83 , wherein said discrete regions have a size from about 125 nm to about 250 nm.
101 . The method of claim 83 , wherein said discrete regions have a size from about 200 nm to about 500 nm.
102 . The method of claim 83 , wherein substantially all of said discrete regions have a single concatemer attached thereto.