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.
Claims20
A method of fragmenting genomic DNA in a sample, the method comprising:
- (a) dividing the sample into a plurality of separate aliquots;
- (b) amplifying genomic DNA in the separate aliquots to produce a plurality of amplicons, wherein the amplifying is conducted with a population of dNTPs containing dNTP analogs, whereby a number of nucleotides in the amplicons are dNTP analogs;
- (c) removing the dNTP analogs from the amplicons to form gapped DNA;
- and
- (d) treating the gapped DNA such that gaps on opposite strands converge, thereby creating blunt-ended DNA fragments.
A method of fragmenting a sample of DNA, comprising:
- (a) dividing the sample into a plurality of separate aliquots;
- (b) amplifying DNA in the separate aliquots to form a plurality of amplicons, wherein the amplifying is conducted with a population of dNTPs, wherein the population of dNTPs comprises:
- (i) a predetermined ratio of dUTP to dTTP, such that a number of thymines in the DNA are replaced by uracils, and
- (ii) a predetermined ratio of 5-methyl dCTP to dCTP, such that a number of cytosines are replaced by 5-methyl cytosines,
- (c) removing the uracils and the 5-methyl cytosines from the amplicons to form gapped DNA,
- (d) treating the gapped DNA such that gaps on opposite strands converge, thereby creating blunt-ended DNA fragments.
A method of fragmenting a sample of double-stranded target DNA, the method comprising:
- (a) dividing the sample into separate aliquots,
- (b) amplifying DNA in the separate aliquots to form a plurality of amplicons, wherein
- (i) the amplifying is conducted with a population of dNTPs that comprises dNTP analogs, such that a number of nucleotides in the DNA are dNTP analogs, and
- (ii) the amplifying is conducted in the presence of an additive selected from glycogen, dimethyl sulfoxide (DMSO), Extreme Thermostable Single-Stranded DNA Binding Protein (ET SSB), trimethylglycine (TMG) (betaine), and any combination thereof,
- (c) removing the dNTP analogs from the amplicons to form gapped DNA,
- (d) treating the gapped DNA acid to translate the gaps until gaps on opposite strands converge, thereby creating blunt-ended DNA fragments.
A method for reducing GC bias when sequencing a sample of genomic DNA, wherein the sequencing comprises:
- (i) dividing the sample into a plurality of separate aliquots;
- (ii) amplifying genomic DNA in the separate aliquots to produce a plurality of amplicons,
- (iii) combining the amplicons from a plurality of the aliquots into a mixture;
- (iv) obtaining sequence reads from amplicons in the mixture;
- and
- (v) assembling the sequence reads into assembled sequence information by a process that comprises determining the mixtures of origin from which the sequence reads were obtained;
- wherein the method of reducing GC bias comprises:
- (a) conducting the amplifying in step (ii) in the presence of a combination of nucleotides containing dNTP analogs, such that some of the nucleotides incorporated into the amplicons are dNTP analogs;
- and then
- (b) removing the dNTP analogs from the amplicons to form gapped DNA.
Dependencies are inferred from the claim text.