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Systems for and methods of hybrid pyrosequencing

  • US 8,901,043 B2
  • Filed: 07/06/2012
  • Issued: 12/02/2014
  • Est. Priority Date: 07/06/2011
  • Status: Expired due to Fees
First Claim
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1. A method for identifying a nucleic acid sequence in a sample by hybrid pyrosequencing, the method comprising:

  • (a) selecting a nucleotide to be dispensed to a sample comprising a primed DNA template and reagents sufficient for sequencing-by-synthesis of a de novo nucleic acid sequence to be identified, wherein selecting comprises querying a database of non-sequence parameters and/or comparing the de novo sequence to a library of candidate sequences, further wherein the query of the database of non-sequence parameters comprises using an algorithm to determine whether the non-sequence parameters comprising incidence of infection and organism type or diagnostic symptoms and organism type, or frequency of the nucleotide in the library of candidate sequences has the strongest predictive value, and selecting the nucleotide based on the strongest predictive value;

    (b) controlling a dispenser to dispense the selected nucleotide to the sample;

    (c) detecting a response signal to the dispensation;

    (d) determining whether the dispensed nucleotide was incorporated or not incorporated;

    (e) repeating steps (a)-(d) in response to determining that the dispensed nucleotide was not incorporated;

    (f) in response to determining that the dispensed nucleotide was incorporated;

    (i) comparing the de novo sequence to the library of candidate sequences; and

    (ii) excluding candidate sequences that do not match the de novo sequence; and

    (g) repeating steps (a)-(f) until the de novo sequence is uniquely identified, wherein a sequence and/or nonsequence parameter database is queried during each reaction cycle.

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