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US 2011/0033854 A1Published application
Methods and compositions for long fragment read sequencing
AssigneeComplete Genomics, Inc.
InventorsRadoje Drmanac, Brock A. Peters, Andrei Alexeev, Peter Hong
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Abstract
The present invention is directed to methods and compositions for long fragment read sequencing. The present invention encompasses methods and compositions for preparing long fragments of genomic DNA, for processing genomic DNA for long fragment read sequencing methods, as well as software and algorithms for processing and analyzing sequence data.
Claims27
A method of fragmenting a double-stranded target nucleic acid, said method comprising:
- (a) providing genomic DNA;
- (b) dividing said DNA into a plurality of separate aliquots;
- (c) amplifying said DNA in said separate aliquots to produce a plurality of amplicons, wherein said amplifying is conducted with a population of dNTPs, wherein said population of dNTPs comprises dNTP analogs, such that a number of nucleotides in said amplicons are replaced by said dNTP analogs;
- (d) removing said dNTP analogs from said amplicons to form gapped DNA;
- (e) treating said gapped DNA to translate said gaps until gaps on opposite strands converge, thereby creating blunt-ended DNA fragments;
- wherein substantially every fragment in a separate aliquot is non-overlapping with every other fragment of the same aliquot.
3 more elements
A method of fragmenting a double-stranded target nucleic acid, said method comprising:
- (a) providing genomic DNA;
- (b) dividing said DNA into a first tier of separate aliquots;
- (c) amplifying said DNA in said separate aliquots to form a plurality of amplicons, wherein said amplifying is conducted with a population of dNTPs, wherein said population of dNTPs comprises:
- (i) a predetermined ratio of dUTP to dTTP, such that a number of thymines in said DNA are replaced by uracils;
- (ii) a predetermined ratio of 5-methyl dCTP to dCTP, such that a number of cytosines are replaced by 5-methyl cytosines;
- (d) removing said uracils and said 5-methyl cytosines from said amplicons to form gapped DNA;
- (e) treating said gapped DNA to translate said gaps until gaps on opposite strands converge, thereby creating blunt-ended DNA fragments,
- wherein said blunt-ended fragments have less GC bias and less coverage bias as compared to fragments generated in the absence of 5-methyl cytosine.
5 more elements
A method of fragmenting a double-stranded target nucleic acid, said method comprising:
- (a) providing genomic DNA;
- (b) dividing said DNA into separate aliquots;
- (c) amplifying said DNA in said separate aliquots to form a plurality of amplicons, wherein:
- (i) said amplifying is conducted with a population of dNTPs that comprises dNTP analogs, such that a number of nucleotides in said DNA are replaced by said dNTP analogs;
- and
- (ii) said amplifying is conducted in the presence of an additive selected from glycogen, DMSO, ET SSB, betaine, and any combination thereof;
- (c) removing said dNTP analogs from said amplicons to form gapped DNA;
- (d) treating said gapped DNA acid to translate said gaps until gaps on opposite strands converge, thereby creating blunt-ended DNA fragments,
- wherein said blunt-ended fragments have less GC bias as compared to fragments generated in the absence of said additive.
6 more elements
A method of obtaining sequence information from a genome, said method comprising:
- (a) providing a population of first fragments of said genome;
- (b) preparing emulsion droplets of said first fragments, such that each emulsion droplet comprises a subset of said population of first fragments;
- (c) obtaining a population of second fragments within each emulsion droplet, such that said second fragments are shorter than said first fragments;
- (d) combining said emulsion droplets of said second fragments with emulsion droplets of adaptor tags;
- (e) ligating said second fragments with said adaptor tags to form tagged fragments;
- (f) combining said tagged fragments into a single mixture;
- (g) obtaining sequence reads from said tagged fragments, wherein said sequence reads include sequence information from said adaptor tags and said fragments to identify fragments from the same emulsion droplet, thereby providing sequence information for said genome.
4 more elements
Dependencies are inferred from the claim text.