Methods for accurate parallel quantification of nucleic acids in dilute or non-purified samples

A method for the high-throughput detection of one or more target nucleotide sequence in a plurality of samples, the method comprising the steps of:

• (i) providing for each target nucleotide sequence in each of the samples:
• a first probe, a second probe and a bridge oligo or a plurality of oligonucleotides capable of annealing to each other to form a bridge oligo complex,
• wherein the first probe comprises, starting from the 5′ end of the molecule, a first bridge oligo-specific sequence, optionally a first sequence barcode, and a first target specific portion at the 3′ end of first probe;
• and wherein the second probe comprises, starting from the 5′ end of the molecule, a second target specific portion, optionally a second sequence barcode, and a second bridge oligo-specific sequence at the 3′ end of second probe;
• and wherein the bridge oligo or bridge oligo complex contains sequences complementary to the first bridge oligo-specific sequence and the second bridge oligo-specific sequence in the first probe and the second probe, respectively, and optionally a third barcode;
• and wherein at least one of the first sequence barcode or the second sequence barcode or the third barcode is present in the first probe or the second probe or the bridge oligo or bridge oligo complex, respectively;
• and wherein at least one of the first probe or the second probe or the bridge oligo or bridge oligo complex comprises a first capture moiety,
• (ii) contacting, for each of the one or more target nucleotide sequence, the first probe and the second probe with, each of the samples in a separate tube, the bridge oligo or plurality of oligonucleotides capable of forming a bridge oligo complex and allow self-annealing into a plurality of ligation complexes;
• (iii) contacting nucleic acids present in each of the samples to be tested for the target nucleotide sequences with the ligation complexes;
• (iv) allowing the first target specific portion and the second target specific portion of the respective first probe and the second probe to hybridize to essentially adjacent sections on the target sequence, thereby forming a hybridization complex;
• (v) bringing the hybridization complex in contact with a solid support comprising a second capture moiety and allowing the first capture moiety and the second capture moiety to interact such that the hybridization complexes become linked to the solid support;
• (vi) separating the solid-support-linked hybridization complexes from components of the samples that are not linked to the solid-support;
• (vii) ligating the probes in the hybridization complexes to provide ligated ligation complexes;
• (viii) prior to step (vii) optionally pooling the ligated ligation complexes from the plurality of samples;
• (ix) amplifying nucleic acids from the one or more ligated ligation complexes;
• (x) subjecting the nucleic acids obtained in step (ix) to high-throughput sequencing technology to determine the barcode sequence(s);
• (xi) identifying the presence and/or number of the target nucleotide sequence in the plurality of samples by determination of at least part of the first target specific portion and/or the second target specific portion, and/or at least part of the first barcode and/or the second barcode, and/or at least part of the third barcode;
• and
• (xii) optionally pooling the hybridization complexes from the plurality of samples or optionally pooling the ligated ligation complexes from the plurality of samples.