Method for multiplexed detection of nucleic acids using spectrally encoded beads

A method for characterizing a population of microbial strains in a sample, wherein different microbial strains in the population comprise polynucleotides with different microbe-identifying sequences, the method comprising:

• (i) amplifying polynucleotides obtained from the sample to form a plurality of amplicons, wherein the amplicons comprise microbe-identifying sequences;
• (ii) combining the amplicons with a plurality of microbeads,
• wherein each microbead of the plurality of microbeads has a lanthanide spectral signature and a plurality of copies of a capture polynucleotide immobilized on the microbead,
• wherein each of the plurality of copies of the capture polynucleotide immobilized on any one microbead of the plurality of microbeads comprises a same predetermined sequence
• that is paired with the lanthanide spectral signature of the one microbead, such that the plurality of copies of the capture polynucleotide immobilized on the one microbead can be identified based on the lanthanide spectral signature of the one microbead, wherein the plurality of microbeads comprises one or more subpopulations, each characterized by a particular lanthanide spectral signature, such that microbeads of different subpopulations have different lanthanide spectral signatures,
• wherein, for each microbead of at least some of the plurality of microbeads, the plurality of copies of the capture polynucleotide immobilized on the microbead comprises a sequence substantially complementary to a microbe-identifying sequence of one or more of the plurality of amplicons,
• wherein the combining is conducted under conditions in which at least some of the plurality of amplicons hybridize to the plurality of copies of the capture polynucleotide immobilized on at least some of the plurality of microbeads, thereby producing captured amplicons, and
• wherein the plurality of amplicons is labeled with one or more signal-generating moieties prior to, simultaneously with, or after being captured onto at least some of the plurality of microbeads, such that microbeads comprising immobilized signal-generating moieties are produced;
• wherein the magnitude of signal detected from an individual microbead corresponds to the amount of the plurality of amplicons captured on the individual microbead,
• (iii) measuring the magnitude of the signal from the microbeads comprising immobilized signal-generating moieties;
• (iv) for each of the plurality of microbeads, determining the lanthanide spectral signature, thereby determining the microbe-identifying sequences of the captured amplicons;
• (v) generating a pattern of measured amount of hybridization of the captured amplicons to individual capture polynucleotides;
• (vi) for each microbial strain of a plurality of microbial strains, comparing the pattern of hybridization measured in (v) to a predicted pattern of hybridization of amplicons to each of the individual capture polynucleotides, the predicted pattern of hybridization corresponding to the microbial strain, wherein predicting hybridization of the amplicons to a capture polynucleotide comprises calculating the Gibbs free energy of hybridization;
• (vii) correlating the pattern of hybridization measured in (v) to the predicted pattern of hybridization for each microbial strain of the plurality of microbial strains to identify the microbial strain that has a predicted pattern of hybridization that has the strongest correlation with the pattern of hybridization measured in (v);
• and
• (viii) determining that the microbial strain identified in (vii) is present in the population of microbial strains in the sample if the strongest correlation is greater than a predetermined threshold.