Differential tagging of rna for preparation of a cell-free dna/rna sequencing library
AssigneeGrail, LLC
InventorsMatthew Larson, H. John Kim, Nick Eattock, Arash Jamshidi
Life of this patent
Estimating the term…
Abstract
In various aspects, the present disclosure provides methods, compositions, reactions mixtures, kits, and systems for sequencing both RNA and DNA from a single source sample. In some embodiments, RNA is treated so as to differentiate RNA sequences from DNA sequences derived from the same sample. In some embodiments, the RNA and DNA are cell-free polynucleotides.
Claims26
A method of distinguishing sequences of RNA and DNA in a sample, the method comprising:
- a. obtaining a sample comprising both RNA and DNA;
- b. reverse transcribing the RNA to produce cDNA/RNA hybrid molecules;
- c. degrading the RNA of the hybrid molecules to produce single-stranded cDNA;
- d. preferentially joining a tag oligonucleotide comprising a tag sequence to the single-stranded cDNA in a reaction comprising a single-stranded DNA ligase to produce tagged cDNA;
- and
- e. sequencing the DNA and the tagged cDNA;
- wherein the reverse transcribing, preferentially joining, and sequencing are performed in the presence of the DNA.
-57. (canceled)
A method of distinguishing sequences of RNA and DNA in a sample, the method comprising:
- a. obtaining a sample comprising both RNA and DNA;
- b. joining a tag oligonucleotide comprising a tag sequence to the RNA in a reaction comprising an RNA ligase to produce tagged RNA;
- c. reverse transcribing the tagged RNA to produce tagged cDNA;
- and
- d. sequencing the DNA and the tagged cDNA;
- wherein the joining, reverse transcribing, and sequencing are performed in the presence of the DNA.
A method of sequencing cell-free nucleic acids comprising DNA and RNA from a single biological sample, the method comprising:
- a. obtaining a sample comprising the cell-free nucleic acids;
- b. reverse transcribing the RNA to produce cDNA/RNA hybrid molecules by extending a primer, wherein the primer is covalently joined to a first member of a binding pair via a cleavage site;
- c. separating the cDNA from the DNA by binding the first member of the binding pair to a substrate comprising a second member of the binding pair;
- d. cleaving the cleavage site;
- and
- e. sequencing the cDNA and the DNA after the separating.
Dependencies are inferred from the claim text.