Polony sequencing methods
We describe ultra-high throughput polony genome sequencing that can permit, for example, generating raw data to re-sequencing the human genome in about one week (including library prep and sequencing) at a reasonable cost. The methods described herein include one or more of the following: (1) increasing polony sequencing read length, (2) improving library construction and emulsions protocols, (3) increasing bead density and/or moving to alternative clonal amplication strategies (other than emulsion PCR or ePCR), (4) extending software capabilities to allow SNP calls from our new sequencing raw data, (5) Dual Primer Emulsion PCR, and (6) diagnostic method exploiting one or more of the foregoing.
1. A method for obtaining the sequence of a polynucleotide, the method comprising:
providing a polynucleotide library comprising a plurality of polynucleotide fragments immobilized to a substrate, wherein providing the polynucleotide library comprises:
fragmenting a nucleic acid molecule, thereby producing a plurality of nucleic acid fragments;
ligating a first end PCR primer to a first end of at least one fragment, and a second end PCR primer to a second end of the at least one fragment; and
sequencing the ends of the at least one fragment, thereby identifying each of the at least one fragment as part of a known sequence;
hybridizing a first probe having known nucleic acid sequences to at least a portion of the immobilized polynucleotide library fragments;
identifying at least a portion of the first probe hybridized to the immobilized polynucleotide library fragments;
assigning to each hybridized fragment a nucleic acid sequence that is the complement of a plurality of nucleotides of the nucleic acid sequence of the first probe hybridized to the immobilized polynucleotide library fragment, wherein the assigning does not require a ligation step;
hybridizing a second probe having known nucleic acid sequences to at least a portion of the immobilized polynucleotide library fragments;
identifying at least a portion of the second probe hybridized to the immobilized polynucleotide library fragments; and
assigning to each hybridized fragment a nucleic acid sequence that is the complement of a plurality of nucleotides of the nucleic acid sequence of the second probe hybridized to the immobilized polynucleotide library fragment, wherein the assigning does not require a ligation step.
2. The method of claim 1 further comprising amplifying the plurality of library polynucleotide fragments.
3. The method of claim 2 wherein amplifying the plurality of library polynucleotide fragments comprises:
providing a plurality of substrates, at least one of the substrates comprising a first end PCR primer and a second end PCR primer affixed to the substrate,
wherein providing a polynucleotide library comprising a plurality of polynucleotide fragments comprises:
fragmenting a nucleic acid molecule, thereby producing a plurality of nucleic acid fragments;
ligating a first end PCR primer to a first end of at least one fragment, and a second end PCR primer to a second end of the at least one fragment, wherein the PCR primers comprise a recognition site for a restriction enzyme that cuts remotely from its recognitions site; and
digesting the at least one fragment; and
amplifying the digested fragment; and
wherein ligating the first end PCR primer to the polynucleotide fragment of the library, or ligating the second end PCR primer to the polynucleotide fragment of the library, comprises affixing the library fragment to the substrate via an affixed end PCR primer;
suspending the plurality of substrates in an emulsion wherein at least one emulsion droplet comprises:
one substrate to which one library fragment is affixed; and
free PCR components; and
amplifying the library fragment.