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EP-4248447-B1 - ENCODED ASSAYS

EP4248447B1EP 4248447 B1EP4248447 B1EP 4248447B1EP-4248447-B1

Inventors

  • VAN ROOYEN, PIETER
  • BERTI, LORENZO
  • BRODIN, Jeffrey
  • EIDSON, DONALD BRIAN
  • STONE, Gavin
  • POLLACK, MICHAEL
  • ECKHARDT, ALLEN

Dates

Publication Date
20260506
Application Date
20211123

Claims (15)

  1. A method of conducting a methylation specific assay for a set of targets, the method comprising: (a) providing a sample comprising a set of targets; (b) subjecting the sample to a recognition event, in which one or more targets in the set of targets in the sample is hybridized to a recognition element comprising a code from a set of codes, thereby yielding a set of coded targets comprising the one or more targets and the recognition element; (c) subjecting each recognition element of the set of coded targets to a methylation specific transformation event, in which a molecular transformation of each recognition element of the set of coded targets produces a circular recognition element, thereby yielding a set of circular recognition elements comprising the code; (d) subjecting each circular recognition element to rolling circle amplification to produce a set of amplified codes; (e) subjecting each amplified code of the set of amplified codes to a detection event, wherein the detection event determines the presence of the coded targets.
  2. The method of claim 1, further comprising: (f) estimating a quantity of the target of the set of coded targets, wherein the estimating comprises counting the codes of the amplified codes of the set of amplified codes comprising the coded target.
  3. The method of claim 1 or 2, wherein each code from the set of codes has a length of 3 to 75 nucleotides.
  4. The method of any one of claims 1-3, wherein each code from the set of codes is generated from a 4-ary nucleotide alphabet of adenosine (A), cytosine (C), guanine (G) and thymine (T).
  5. The method of any one of claims 1-3, wherein each code from the set of codes is generated from a 3-ary nucleotide alphabet of a set of three selected from the group consisting of A, C, G and T.
  6. The method of any one of claims 1-5, wherein the recognition element comprises one or more universal primers.
  7. The method of any one of claims 1-6, wherein the transformation event comprises ligating termini of the recognition element in the presence of the target but not in the absence of the target.
  8. The method of any one of claims 1-7, further comprising subjecting the set of amplified codes to an exonuclease cleanup step.
  9. The method of any one of claims 1-8, wherein the target of the set of targets comprises one or more mutated nucleic acid sequences.
  10. The method of claim 9, wherein the one or more mutated nucleic acid sequences comprises: (a) one or more point mutations; (b) one or more substitutions; (c) one or more insertions; (d) one or more deletions; (e) one or more copy number variations; or (f) any combination of (a) to (e).
  11. The method of any one of claims 1-8, wherein the target of the set of targets is a methylated nucleic acid sequence.
  12. The method of any one of claims 1-11, wherein the detection event comprises obtaining a fluorescence measurement.
  13. The method of any one of claims 1-12, wherein the set of coded targets further comprises one or more sequencing adapters for next generation sequencing, and wherein the method is a multiplexed method further comprising performing next generation sequencing.
  14. The method of any one of claims 1-13, wherein the detection event comprises imaging with a fluorescence optical measurement system.
  15. The method of any one of claims 1-14, wherein the method is performed on an assay substrate comprising wells.

Description

1. FIELD OF THE INVENTION The invention relates to methods of conducting a methylation specific encoded assay, in which a target analyte is detected based on association of the target with a code, and detection of the code as a surrogate for detection of the target analyte. Methods, systems, cartridges, reagents, and kits for carrying out the encoded assays are also disclosed. 2. BACKGROUND OF THE INVENTION Many assays require high-level of sensitivity and specificity, and are associated with low signal level. Low signal requires amplification (e.g., PCR, immunostaining cascades, and the like) resulting in complex and lengthy protocols, high-level of background and other biases limiting the performance of the assay. There is a need in the art for assays that are easier to read and detect at higher sensitivity than the analyte itself. Krzywkowski, et al, "Simultaneous Single-Cell In Situ Analysis of Human Adenovirus Type 5 DNA and mRNA Expression Patterns in Lytic and Persistent Infection", J. Virology (2017), vol. 91, no. 11, describes a padlock probe-based rolling-circle amplification technique that enables simultaneous detection and analysis of HAdV-5 genomic DNA and virus-encoded mRNAs in individual infected cells. WO 2017/177017 A1 provides compositions, methods and systems for quantifying target sequences and identifying target sequence variants. WO 2018/160397 A1 provides a circular proximity ligation assay in which proximity-probes are employed as bridges to connect two free oligonucleotides via a dual ligation event, resulting in the formation of a circle. The circles can then be quantified by e.g. qPCR. Diep, et al, "Library-free methylation sequencing with bisulfite padlock probes", Nature Methods (2012), vol. 9, no. 3 describes bisulfite padlock probes with a design algorithm to generate efficient padlock probes, a library-free protocol that reduces sample-preparation cost and time and is compatible with automation, and a bioinformatics pipeline to obtain both methylation levels and genotypes from sequencing of bisulfite-converted DNA. 3. SUMMARY OF THE INVENTION The invention provides a method of conducting a methylation specific assay for a set of targets as defined in the appended claims. The method may also include subjecting the amplified codes to a cleanup step. The method may also include counting detected codes and estimating target quantity-based counts of detected codes, e.g., estimating target quantity based on a correlation of counts of detected codes and target quantity in the sample analyzed. In some cases, each code from the set of codes has a length ranging from 3 to 100 nucleotides. In some cases, each code from the set of codes has a length ranging from 3 to 75 nucleotides. In some cases, each code from the set of codes is a predetermined code. In some cases, each code from the set of codes is selected to avoid interaction with other assay components. In some cases, each code from the set of codes is selected to ensure that it differs from each other code from the set of codes. In some cases, each code from the set of codes is homopolymer free. In some cases, each code from the set of codes is generated from a 4-ary nucleotide alphabet of A, C, G and T. In some cases, the code is generated using a 4-state encoding trellis with 3 transitions per state. In some cases, each code from the set of codes is generated from a 3-ary nucleotide alphabet of a set of three of A, C, G and T. In some cases, the code is generated using a 4-state encoding trellis with 3 transitions per state. In certain configurations, the recognition elements includes one or more universal primers linked to the code. In certain configurations, the recognition elements includes a TaqMan sequence linked to the code. In certain configurations, each of the recognition elements include one or more unique molecular identifier sequences linked to the code. In certain configurations, the recognition event includes binding a padlock probe of a set of padlock probes to each target of the set of targets. In some cases, each of the coded targets of the set of coded targets includes a target bound to one of the padlock probes of the set of padlock probes. In certain configurations, the set of targets include nucleic acid targets. In some cases, the recognition element includes a padlock probe including a code. In certain configurations, the padlock probe includes terminal primer sequences complimentary to a segment of one or more targets of the set of targets. In certain configurations, a first one of the terminal primer sequences includes a terminal one or more nucleotides complementary to one or more target nucleotides; a second one of the terminal primer sequences includes one or more nucleotides complementary to a nucleotide adjacent to the one or more target nucleotides; when the one or more target nucleotides is/are present, the first one of the terminal primer sequences and second one of the terminal primer sequences