Differential tagging of rna for preparation of a cell-free dna/rna sequencing library

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.

1 .- 39 . (canceled) 
     
     
         40 . 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.   
     
     
         41 . The method of  claim 40 , wherein the reverse transcribing comprises amplifying the cDNA to produce double-stranded tagged cDNA. 
     
     
         42 . The method of  claim 41 , wherein the amplifying comprises degrading the RNA of the hybrid molecules to produce single-stranded cDNA. 
     
     
         41 . The method of  claim 40 , wherein the sequencing comprises amplifying the cDNA to produce double-stranded tagged cDNA. 
     
     
         44 . The method of  claim 40 , wherein the primer comprises a random sequence. 
     
     
         45 . The method of  claim 40 , wherein the binding pair comprises biotin and streptavidin. 
     
     
         46 . The method of  claim 45 , wherein the first member of the binding pair is biotin, and the second member of the binding pair is streptavidin. 
     
     
         47 . The method of  claim 40 , wherein the cleavage site comprises uracil, and cleaving the cleavage site comprises exposing the cleavage site to a uracil DNA glycosylase and a DNA glycosylase-lyase endonuclease. 
     
     
         48 . The method of  claim 40 , wherein the reverse transcribing comprises extension of cDNA along a template-switch oligonucleotide (TSO). 
     
     
         49 . The method of  claim 48 , wherein the TSO comprises a universal switch primer sequence. 
     
     
         50 . The method of  claim 40 , wherein the sequencing comprises joining sequencing adapters to the tagged cDNA and the DNA. 
     
     
         51 . The method of  claim 40 , wherein the sample is blood, a blood fraction, plasma, serum, saliva, sputum, urine, semen, transvaginal fluid, cerebrospinal fluid, or stool. 
     
     
         52 . The method of  claim 51 , wherein the sample is blood or a blood fraction. 
     
     
         53 . The method of  claim 40 , further comprising identifying presence or absence of a condition of a subject based on the cDNA-derived sequences and the DNA-derived sequences. 
     
     
         54 . The method of  claim 53 , wherein the condition is cancer. 
     
     
         55 . The method of  claim 53 , further comprising treating the subject based on the cDNA-derived sequences and the DNA-derived sequences. 
     
     
         56 .- 57 . (canceled)