MULTI-REGION NUCLEIC ACID ANALYSIS
Provided herein are methods for detecting two or more genomic regions of interest in a target in an assay. Also provided are sets of multi-region coded recognition elements and uses thereof to detect two or more genomic regions of interest in a target.
1 . A method for detecting two or more genomic regions of interest in a target of a set of targets in an assay, the method comprising:
(a) subjecting the set of targets to a hybridization event, wherein each target of the set of targets is uniquely recognized by and hybridized to at least one coded recognition element from a set of coded recognition elements, wherein each coded recognition element comprises:
(i) a first target-specific binding site that flanks a first genomic region of interest binding site to a first of the two or more genomic regions of interest in the target of the set of targets;
(ii) a second target-specific binding site that flanks a second genomic region of interest binding site to a second of the two or more genomic regions of interest in the target of the set of targets; and
(iii) a code from a set of codes, wherein each code from the set of codes comprises at least one segment encoding one or more symbols that correspond to a sequence of one or more nucleotides, and wherein each code from the set of codes is unique for each coded recognition element from the set of coded recognition elements;
(b) subjecting the coded recognition elements from the set of coded recognition elements to a ligation event to yield a set of ligated coded recognition elements; and
(c) performing an amplification reaction on the ligated coded recognition elements; and
(d) detecting the two or more genomic regions of interest associated with the amplified ligated coded recognition elements; wherein the detecting comprises
(e) decoding the codes of the amplified ligated coded recognition elements by recording a signal produced in response to interrogation of each segment of the codes of the amplified ligated recognition elements by one or more hybridization probes, and upon completion of the interrogation determining a probability of the presence of each of the codes of the amplified ligated recognition elements by applying a soft decision probabilistic decoding algorithm to the recorded signal, wherein the presence of the code is indicative of the presence of the target, thereby assaying for the two or more genomic regions of interest in the target of the set of targets.
2 . The method of claim 1 , further comprising performing (a) to (e) with at least 100 coded recognition elements comprising to predict a presence of a plurality of target nucleic acids.
3 . The method of claim 1 , further comprising performing (a) to (e) with at least 1,000 coded recognition elements to predict a presence of a plurality of target nucleic acids.
4 . The method of claim 1 , further comprising introducing a bridge element to the target nucleic acid, wherein the bridge element hybridizes to a region of the target nucleic acid between the first genomic region of interest binding site and the second genomic region of interest binding site, and wherein the ligating in (c) further comprises ligating the bridge element to the 3′ end and 5′ end of the coded recognition element.
5 . The method of claim 1 , wherein the amplification reaction comprises performing rolling circle amplification to generate concatemeric amplification products.
6 . The method of claim 5 , further comprising:
(a) cleaving the concatemeric amplification products to yield a plurality of unit length monomer fragments each comprising a copy of the code;
(b) re-circularizing the unit length monomer fragments to generate re-circularized monomers; and
(c) amplifying the re-circularized monomers in a second rolling circle amplification reaction to produce multiple rolling circle amplification products of the re-circularized monomers.
7 . The method of claim 1 , further comprising subjecting the amplified coded recognition elements to an exonuclease reaction.
8 . The method of claim 1 , wherein the presence of the target nucleic acid is informative for:
(a) pathogen detection;
(b) leveraging variable regions within pseudogenes to disambiguate a genotype;
(c) identifying methylation events from bisulfite converted DNA or from non-treated samples; or
(d) any combination of (a), (b) and (c).
9 . The method of claim 1 , wherein each code comprises four to sixteen segments.
10 . The method of claim 1 , wherein at least one of the segments is interrogated more than one time by hybridization with one or more hybridization probes, wherein the one or more hybridization probes comprises an optical label or a fluorescent label.
11 . The method of claim 10 , wherein the one or more hybridization probes comprises one optical label of at least four different optical labels or one fluorescent label of at least four different fluorescent labels.
12 . The method of claim 1 , further comprising performing (a) to (e) for hundreds of targets comprising said target.
13 . The method of claim 1 , wherein the two or more genomic regions of interest comprise two or more point mutations, two or more substitutions, two or more insertions, two or more deletions, two or more copy number variations, or any combination thereof.
14 . The method of claim 1 , wherein each code from the set of codes is a predetermined code based, at least in part, to avoid assay component interactions; and wherein the code is homopolymer free.
15 . A set of multi-region coded recognition elements, wherein a multi-region coded recognition element of the set of multi-region coded recognition elements comprises:
(a) a first target-specific binding site that flanks a first genomic region of interest binding site;
(b) a second target-specific binding site that flanks a second genomic region of interest binding site; and
(c) a code from a set of codes, wherein the code is a soft decodable code comprising at least one segment encoding one or more symbols that correspond to a sequence of one or more nucleotides.
16 . The set of multi-region coded recognition elements of claim 15 , wherein the set of multi-region coded recognition elements are padlock probes or molecular inversion probes.
17 . The set of multi-region coded recognition elements of claim 15 , further comprising a bridge element that, when bound to a target, is disposed between the first genomic region of interest binding site and the second genomic region of interest binding site of the multi-region coded recognition element.
18 . The set of multi-region coded recognition elements of claim 15 , wherein the set of multi-region coded recognition elements comprises at least 10 multi-region coded recognition elements.
19 . The set of multi-region coded recognition elements of claim 15 , wherein the set of multi-region coded recognition elements comprises at least 100 multi-region coded recognition elements.
20 . The set of multi-region coded recognition elements of claim 17 , wherein the multi-region coded recognition elements in the set of multi-region coded recognition elements are hybridized to the first target specific binding site, the first genomic region of interest, the second target specific binding site, and the second genomic region of interest of a target of interest and comprises a contiguous nucleic acid molecule as a result of a ligation or gap-filing ligation of a 5′ probe arm of the multi-region coded recognition elements, the bridge element, and a 3′ probe arm of each of the multi-region coded recognition elements.