Method and kit for identifying molecular markers of disease

A method for identifying at least one RNA fragment comprising a 3′ phosphate or 2′/3′ cyclic phosphate (3′P RNA) as a molecular marker of a disease from a biological sample of a subject suffering from the disease, wherein the method comprises the following steps:

• (a) phosphorylating the at least one 3′P RNA contained in the biological sample at the 5′ end obtaining at least one phosphorylated RNA fragment;
• (b) ligating the 3′ end of the at least one phosphorylated RNA fragment to the 5′ end of an RNA-based adapter obtaining at least one first ligation product, wherein the RNA-based adapter has formula (I):
• 5′ OH—Nx-C1-L1-Az-PR1-Ny-C2-B—OH 3′ (I)
• wherein
• N is a ribonucleotide,
• x and y are integer numbers independently selected from 1 to 20,
• L1 is an oligoribonucleotide sequence having a length comprised between 15 and 30,
• A is an abasic site or a spacer allowing the arrest of a retrotranscriptase enzyme activity,
• z is an integer number from 1 to 5,
• PR1 is a first portion of a first nucleic acid domain of a sequencing platform adapter construct having a length comprised between 10 and 80,
• B is none, or a ribonucleotide having a 2′-fluoro-base, or a ribonucleotide having a modified nucleobase conferring nuclease resistance, and
• C1 and C2 are none or a barcode sequence comprising up to 20 nucleotides, provided that at least one between C1 and C2 is none;
• (c) self-ligating the at least one first ligation product to form at least one circular RNA molecule;
• (d) performing a reverse transcription of the at least one circular RNA molecule obtaining at least one single strand cDNA molecule comprising the sequence of the at least one 3′P RNA, wherein the reverse transcription is carried out using a reverse transcription primer having formula (II):
• 5′ OH-R2-Nz-D1-OH 3′ (II)
• wherein
• D1 is the reverse complement deoxyoligoribonucleotide of L1, wherein complementarity of D1 to L1 is comprised between 60% and 100%,
• N is a ribonucleotide,
• z is an integer number from 1 to 20, and
• R2 is a second nucleic acid domain of the sequencing platform adapter construct having a length comprised between 10 and 50;
• (e) performing a PCR amplification of the at least one single strand cDNA molecule obtaining at least one amplification product, wherein the PCR amplification is carried out alternatively:
• (i) in two sequential steps, wherein:
• the first PCR amplification is carried out using a first pair of primers, the first forward primer and the first reverse primer having formula (III) and (IV), respectively:
• 5′ OH-T1-T2-OH 3′ (III)
• 5′ OH-T3-OH 3′ (IV)
• wherein
• T1 is a second portion of the first nucleic acid domain of the sequencing platform adapter construct having a length comprised between 10 and 50,
• T2 is a first DNA oligonucleotide sequence having a length comprised between 10 and 30 annealing on at least one part of the first portion of the first nucleic acid domain of the sequencing platform adapter construct, and
• T3 is a second DNA oligonucleotide sequence having a length comprised between 10 and 50 annealing on at least one part of the second nucleic acid domain of the sequencing platform adapter construct;
• the second PCR amplification is carried out using a second pair of primers, the second forward primer and the second reverse primer having formula (V) and (VI), respectively:
• 5′ OH-Q1-Q2-Q3-OH 3′ (V)
• 5′ OH-Q4-Q5-Q6-OH 3′ (VI)
• wherein
• Q1 is a third nucleic acid domain of the sequencing platform adapter construct having a length comprised between 10 and 50,
• Q2 is a fourth nucleic acid domain of the sequencing platform adapter construct having a length comprised between 6 and 20,
• Q3 is a third DNA oligonucleotide sequence having a length comprised between 10 and 50 annealing on at least one part of the first nucleic acid domain of the sequencing platform adapter construct,
• Q4 is a fifth nucleic acid domain of the sequencing platform adapter construct having a length comprised between 10 and 50,
• Q5 is a sixth nucleic acid domain of the sequencing platform adapter construct having a length comprised between 6 and 20,
• Q6 a fourth DNA oligonucleotide sequence having a length comprised between 10 and 50 annealing on at least one part of the second nucleic acid domain of the sequencing platform adapter construct;
• or
• (ii) in one single step using a third pair of primers, the third forward primer and the third reverse primer having formula (VII) and (VIII), respectively:
• 5′ OH-Q1-Q2-Q7-OH 3′ (VII)
• 5′ OH-Q4-Q5-Q6-OH 3′ (VIII)
• wherein
• Q1, Q2, Q4, Q5 and Q6 have the meaning set forth above, and
• Q7 is a DNA oligonucleotide sequence having a length comprised between 10 and 50, comprising at the 5′ end a second portion of the first nucleic acid domain of the sequencing platform adapter construct and annealing at the 3′ end on at least one part of the first portion of the first nucleic acid domain of the sequencing platform adapter construct (PR1 of formula (I));
• (f) sequencing the at least one amplification product obtaining the sequence of the at least one 3′P RNA comprised in the at least one single strand cDNA molecule;
• (g) repeating steps (a) to (f) on at least one control sample, wherein the control sample is a biological sample of a healthy, treated or non-treated subject;
• (h) calculating for the at least one 3′P RNA contained in the amplification products obtained from the biological sample and the control sample:
• (i) a number of counts, (ii) a Dart-RNAseq p-value, (iii) a cleavage pattern and (iv) a Dart-RNAseq fold change of a normalized parameter based on the number of counts in the biological sample versus the control sample according to the following equation:
• Dart
• ⁢
• ‐
• ⁢
• RNAseq
• ⁢
• fold
• ⁢
• change
• =
• (
• nCounts
• Treat
• nCounts
• CTRL
• )
• wherein
• nCountsTreat is the normalized parameter based on the number of counts for the 3′P RNA determined in the biological sample,
• nCountsCTRL is the normalized parameter based on the number of counts for the 3′P RNA determined in the control sample;
• wherein the at least one 3′P RNA is the at least one molecular marker of the disease if:
• the number of counts is ≥200,
• the Dart RNAseq p-value is ≤0.05,
• the cleavage pattern is ≤40% per-base cleavage frequencies along the 3′P RNA length and ≥60% per-base cleavage frequencies on the 5′ and 3′ ends of the 3′P RNA, and
• the Dart RNAseq fold change is ≥2 or ≤0.5.