Entangled mate sequencing
First Claim
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1. A method of determining nucleotide sequence identities of heterogeneous, tethered nucleic acid template sequences comprising the steps of:
- providing a mosaic target nucleic acid sequence including a first template nucleic acid sequence and a second template nucleic acid sequence, wherein the first and second template nucleic acid sequences are tethered and heterogeneous and of relatively homogeneous length;
simultaneously reading the first template nucleic acid sequence and the second template nucleic acid sequence to obtain a mixed entangled sequencing signal representative of each of the first template nucleic acid sequence and the second template nucleic acid sequence; and
disentangling the mixed sequencing signal into its constituent individual template sequences by matching the mixed entangled sequencing signal to its closest match in a reference collection of known sequence signatures wherein the mixed entangled sequencing signal is obtained by annealing a first primer sequence to a portion of the first template nucleic acid sequence and a second primer sequence to a portion of the second template nucleic acid sequence;
extending the annealed primers simultaneously; and
sequentially determining the identity of each set of two bases extended from the 3′
ends of the first and second primers to obtain mixed base signatures and using said mixed base signatures to obtain a mixed nucleic acid sequence signature of the tethered first and second template nucleic acid sequences, wherein the identity of each set of two bases extended from the 3′
ends of the first and second primers is sequentially determined by nucleotide addition sequencing.
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Abstract
Methods and compositions are provided for performing a set of N DNA sequencing reaction cycles whereby sequence information is obtained for approximately 2*N nucleotide bases.
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Citations
21 Claims
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1. A method of determining nucleotide sequence identities of heterogeneous, tethered nucleic acid template sequences comprising the steps of:
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providing a mosaic target nucleic acid sequence including a first template nucleic acid sequence and a second template nucleic acid sequence, wherein the first and second template nucleic acid sequences are tethered and heterogeneous and of relatively homogeneous length; simultaneously reading the first template nucleic acid sequence and the second template nucleic acid sequence to obtain a mixed entangled sequencing signal representative of each of the first template nucleic acid sequence and the second template nucleic acid sequence; and disentangling the mixed sequencing signal into its constituent individual template sequences by matching the mixed entangled sequencing signal to its closest match in a reference collection of known sequence signatures wherein the mixed entangled sequencing signal is obtained by annealing a first primer sequence to a portion of the first template nucleic acid sequence and a second primer sequence to a portion of the second template nucleic acid sequence; extending the annealed primers simultaneously; and sequentially determining the identity of each set of two bases extended from the 3′
ends of the first and second primers to obtain mixed base signatures and using said mixed base signatures to obtain a mixed nucleic acid sequence signature of the tethered first and second template nucleic acid sequences, wherein the identity of each set of two bases extended from the 3′
ends of the first and second primers is sequentially determined by nucleotide addition sequencing. - View Dependent Claims (2, 21)
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3. A method of determining nucleotide sequence identities of tethered, heterogeneous nucleic acid template sequences comprising the steps of:
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providing a heterogeneous library of mosaic target nucleic acid sequences, in which a plurality include a first template nucleic acid sequence and a second template nucleic acid sequence, wherein the first and second template nucleic acid sequences are tethered and heterogeneous and of relatively homogeneous length; simultaneously reading the first template nucleic acid sequence and the second template nucleic acid sequence to obtain a mixed entangled sequencing signal representative of each of the first template nucleic acid sequence and the second template nucleic acid sequence; and disentangling the mixed sequencing signal into its constituent individual template sequences by matching the mixed entangled sequencing signal to its closest match in a reference collection of known sequence signatures wherein the mixed entangled sequencing signal is obtained by annealing a first primer sequence to a portion of the first template nucleic acid sequence and a second primer sequence to a portion of the second template nucleic acid sequence; extending the annealed primers simultaneously; and sequentially determining the identity of each set of two bases extended from the 3′
ends of the first and second primers to obtain mixed base signatures and using said mixed base signatures to obtain a mixed nucleic acid sequence signature of the tethered first and second template nucleic acid sequences, wherein the identity of each set of two bases extended from the 3′
ends of the first and second primers is sequentially determined by nucleotide addition sequencing. - View Dependent Claims (4, 5, 6, 7, 8)
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9. A method of determining nucleotide sequence identities of two tethered nucleic acid sequences comprising the steps of:
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providing a heterogeneous library of mosaic target nucleic acid sequences, in which a plurality include a first template nucleic acid sequence and a second template nucleic acid sequence, wherein the first and second template nucleic acid sequences are tethered and heterogeneous and of relatively homogeneous length; simultaneously reading the first template nucleic acid sequence, the second template nucleic acid sequence and the third template nucleic acid sequence to obtain a mixed entangled sequencing signal representative of each of the first template nucleic acid sequence the second template nucleic acid sequence and the third template nucleic acid sequence; and disentangling the mixed sequencing signal into its constituent individual template sequences by matching the mixed entangled sequencing signal to its closest match in a reference collection of known sequence signatures wherein the mixed entangled sequencing signal is obtained by annealing a first primer sequence to a portion of the first template nucleic acid sequence and a second primer sequence to a portion of the second template nucleic acid sequence; extending the annealed primers simultaneously; and sequentially determining the identity of each set of two bases extended from the 3′
ends of the first and second primers to obtain mixed base signatures and using said mixed base signatures to obtain a mixed nucleic acid sequence signature of the tethered first and second template nucleic acid sequences, wherein the identity of each set of two bases extended from the 3′
ends of the first and second primers is sequentially determined by nucleotide addition sequencing. - View Dependent Claims (10, 11, 12, 13, 14, 15)
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16. A method of identifying a cell having heterogeneous nucleic acid sequences comprising the steps of:
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providing a cell having a mosaic target nucleic acid sequence including a first barcode sequence and a second barcode sequence, wherein the first and second barcode sequences are tethered and heterogeneous and of relatively homogeneous length; simultaneously reading the first barcode sequence and the second barcode sequence to obtain a mixed entangled sequencing signal representative of each of the first barcode sequence and second barcode sequence; disentangling the mixed sequencing signal into its constituent individual barcode sequences by matching the mixed entangled sequencing signal to its closest match in a reference collection of known sequence signatures; wherein the mixed entangled sequencing signal is obtained by annealing a first primer sequence to a portion of the first template nucleic acid sequence and a second primer sequence to a portion of the second template nucleic acid sequence; extending the annealed primers simultaneously; sequentially determining the identity of each set of two bases extended from the 3′
ends of the first and second primers to obtain mixed base signatures and using said mixed base signatures to obtain a mixed nucleic acid sequence signature of the tethered first and second template nucleic acid sequences, wherein the identity of each set of two bases extended from the 3′
ends of the first and second primers is sequentially determined by nucleotide addition sequencing; anddetermining the nucleotide sequence identity of one or both of the tethered first and second barcode sequences to identify the cell from the mixed nucleic acid barcode sequence signature. - View Dependent Claims (17, 18, 19, 20)
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Specification