Systems for and Methods of Hybrid Pyrosequencing

Abstract

The systems and methods of the invention provide a guided approach to pyrosequencing (i.e., hybrid pyrosequencing). A de novo nucleic acid sequence may compared to a library of possible results and the next nucleotide to be dispensed is selected based on the comparison of the de novo sequence and the library of possible results. In another example, at least the first nucleotide to be dispensed is selected based on a query of a database(s) of non-sequence parameters (e.g., incidence of infection, diagnostic symptoms, sample source) and subsequent dispensations determined based on a comparison of the de novo sequence and the library of possible results (e.g., candidate sequences). The systems and methods of the invention may be performed using a droplet actuator.

1 - 20 . (canceled) 
     
     
         21 . A system for identifying a nucleic acid sequence in a sample by hybrid pyrosequencing, the system comprising at least one controller configured to:
 (a) select 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 the selection is based on a query of a database of non-sequence parameters and/or a comparison of 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) control a dispenser to dispense the selected nucleotide to the sample;   (c) control a detector to detect a response signal to the dispensation;   (d) determine whether the dispensed nucleotide was incorporated or not incorporated;   (e) repeat the select, dispense, detect, and determine functions in response to determining that the dispensed nucleotide was not incorporated;   (f) compare the de novo sequence to the library of candidate sequences and   (g) exclude candidate sequences that do not match the de novo sequence in response to determining that the dispensed nucleotide was incorporated; and   (h) repeat the select, dispense, detect, determine, compare, and exclude functions until the de novo sequence is uniquely identified, wherein a sequence and/or nonsequence parameter database is queried during each reaction cycle.   
     
     
         22 . The system of  claim 21 , further comprising a droplet actuator, wherein the at least one controller is configured to:
 (a) control the dispenser to dispense:
 (i) a first droplet comprising the primed DNA template immobilized on a bead; 
 (ii) a second droplet comprising the selected nucleotide and detection reagents; and 
 (iii) a third droplet comprising enzyme reagents; and 
   (b) cause the droplet actuator to:
 (i) combine the first, second, and third droplets; 
 (ii) transport the combined droplet to a detector location; and 
 (iii) transport the droplets to a washing location for wash of the beads prior to returning to the dispense function. 
   
     
     
         23 . The system of  claim 22 , wherein the detection reagents comprise ammonium persulfate and luciferin and the enzyme reagents comprise DNA polymerase, adenosine 5′ phosphosulfate, and luciferase. 
     
     
         24 . The system of  claim 22 , wherein the bead is a magnetically responsive bead. 
     
     
         25 . The system of  claim 21 , wherein identification of the nucleic acid sequence in the sample identifies one or more organisms in the sample. 
     
     
         26 . The system of  claim 25 , wherein the organism is a bacteria, virus, or fungi. 
     
     
         27 . The system of  claim 21 , wherein identification of the nucleic acid sequence in the sample rules out one or more organisms in the sample. 
     
     
         28 . The system of  claim 21 , wherein identification of the nucleic acid sequence in the sample determines the nucleotide sequence of an unknown organism in the sample. 
     
     
         29 . The system of  claim 21 , wherein the sample is a biological sample or an environmental sample. 
     
     
         30 . The system of  claim 21 , wherein the nucleic acid sequence is a human immunodeficiency virus (HIV) sequence, a methicillin-resistant  Staphylococcus aureus  (MRSA) sequence, an influenza A sequence, or a 16S or 28S ribosomal DNA sequence of a known or an unknown organism. 
     
     
         31 . The system of  claim 21 , wherein the database of non-sequence parameters comprises one or more of source of the sample, incidence of infection and organism type, or diagnostic symptoms and organism type. 
     
     
         32 . (canceled) 
     
     
         33 . The system of  claim 21 , wherein the at least one controller is configured to select a first nucleotide to be dispensed based in part on the query of a database of non-sequence parameters and in part on frequency of the nucleotide in the library of candidate sequences. 
     
     
         34 . The system of  claim 21 , wherein the at least one controller is configured to select the nucleotide to be dispensed, subsequent to a first nucleotide to be dispensed, based on the comparison of the de novo sequence to the library of candidate sequences to select a most probable nucleotide for that position and to decrease the library of candidate sequences. 
     
     
         35 . The system of  claim 21 , wherein the at least one controller is configured to transition to a directed sequencing approach wherein the de novo sequence is uniquely identified to near 100% certainty, and wherein the at least one controller is configured to dispense the nucleotide to the sample in an order dictated by the de novo sequence uniquely identified to near 100% certainty. 
     
     
         36 . The system of  claim 21 , wherein the at least one controller is configured to transition to a de novo sequencing approach in response to all the candidate sequences in the library of candidate sequences being excluded, and wherein the at least one controller is configured to dispense the nucleotides A, G, C, and T to the sample in a predetermined order to identify the nucleotide sequence. 
     
     
         37 . The system of  claim 21 , wherein the at least one controller is configured to query an additional database of candidate sequences in response to all the candidate sequences in the library of candidate sequences being excluded. 
     
     
         38 . The system of  claim 21 , wherein the at least one controller is configured to control the dispenser to dispense multiple nucleotides simultaneously to reduce the library of candidate sequences. 
     
     
         39 . The system of  claim 38 , wherein multiple nucleotides are 2 or 3 nucleotides. 
     
     
         40 . The system of  claim 21 , and wherein the at least one controller is configured to distinguish between a first similar sequence and a second similar sequence in the library of candidate sequences based on a determination of the most probable nucleotide to follow in each of the first similar sequence and the second similar sequence. 
     
     
         41 . A computer program product for identifying a nucleic acid sequence in a sample by hybrid pyrosequencing, the computer program comprising a computer readable storage medium having computer readable program code embodied therewith, the computer readable program code comprising:
 (a) computer readable program code configured to select 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 the selection is based on a query of a database of non-sequence parameters and/or a comparison of 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) computer readable program code configured to control a dispenser to dispense the selected nucleotide to the sample;   (c) computer readable program code configured to receive signal data that is responsive to the dispensation;   (d) computer readable program code configured to determine whether the dispensed nucleotide was incorporated or not incorporated;   (e) computer readable program code configured to repeat the select, control, receive, and determine functions in response to determining that the dispensed nucleotide was not incorporated;   (f) computer readable program code configured to compare the de novo sequence to the library of candidate sequences, and to exclude candidate sequences that do not match the de novo sequence in response to determining that the dispensed nucleotide was incorporated; and   (g) computer readable program code configured to repeat the select, control, receive, determine, compare, and exclude functions until the de novo sequence is uniquely identified, wherein a sequence and/or nonsequence parameter database is queried during each reaction cycle.