Methods for asymmetric DNA library generation and optionally integrated duplex sequencing
Methods and products are disclosed for asymmetrically adapting fragmented nucleic acids for next generation sequencing, including providing strand identifier sequences and index sequences to identify the source strand and sample, respectively, of the fragmented nucleic acids. The methods and products allow for efficient and reliable detection of low-frequency mutations including in subpopulations of cells within a subject and also for the amplification of the fragmented nucleic acids when there is a low yield of isolated fragmented nucleic acids.
1. A method for generating one or more DNA products comprising a first and second index sequence (IS), the first and second ISs having different nucleotide sequences and allowing for identification of the source of a PCR sample, said method comprising:
(I) ligating, in a reaction mixture, a first adaptor comprising a first strand identifier sequence (SIS) and a primering sequence-binding region to one end of a double-stranded DNA having two ends, and a second adaptor comprising a second SIS and a primering sequence-binding region to the other end of the double-stranded DNA, the first and second SISs having different nucleotide sequences, to obtain an adapted double-stranded DNA having a single-stranded 5′ overhang at each end;
(II) denaturing the adapted double-stranded DNA to obtain at least one single-stranded DNA template in the presence of a first primering sequence (PS) and a blocking oligonucleotide;
(III) decreasing the temperature under conditions that promote: (A) annealing of (i) the blocking oligonucleotide and (ii) the first PS comprising a first index sequence (IS) to the at least one single-stranded template, wherein (i) the blocking oligonucleotide and (ii) the first PS anneal to a first primering sequence-binding region of the at least one single-stranded template, the blocking oligonucleotide having an annealing temperature to the single-stranded template equal to or higher than the annealing temperature of the first PS;
(IV) without changing the temperature, adding to the reaction mixture a second PS comprising a second IS;
(V) incubating at a temperature that allows the blocking oligonucleotide to detach from the at least one single-stranded template and elongation from the first PS to proceed;
(VI) denaturing to obtain at least one single-stranded DNA comprising the first IS; and
(VII) incubating under conditions that promote annealing of the second PS to the at least one single-stranded DNA comprising the first IS, wherein the second PS anneal to a second primering sequence-binding region of the at least one single-stranded DNA and subsequent DNA polymerization to obtain one or more DNA products comprising a first and second IS, the first and second ISs having different nucleotide sequences.
2. The method according to claim 1 , wherein the ligating in (I) proceeds at a temperature of 12-25° C.
3. The method according to claim 1 , wherein the denaturing in (II) proceeds at a temperature of 85-105° C.
4. The method according to claim 1 , wherein temperature at which the blocking oligonucleotide anneals to the single-stranded template in (III) is in the range of 44-52° C.
5. The method according to claim 1 , wherein the temperature that allows the blocking oligonucleotide to detach from the single-stranded template and elongation from the second PS to proceed in (V) is in the range of 60-75° C.
6. The method according to claim 1 , wherein the denaturing in (VI) proceeds at a temperature of 85-105° C.
7. The method according to claim 1 , wherein the conditions that promote annealing of the second PS to the at least one single-stranded DNA comprising the first IS and subsequent DNA polymerization in (VII) comprise incubating at a temperature higher than the annealing temperature at which the blocking oligonucleotide anneals to the at least one single-stranded template.
8. The method according to claim 7 , wherein the temperature higher than the annealing temperature at which the blocking oligonucleotide anneals to the at least one single-stranded template is in the range of 57-65° C.
9. The method according to claim 1 , which comprises performing (I)-(III) in a reaction mixture lacking at least one of dATP, dTTP, dCTG, or dGTP, with the proviso that when dTTP is lacking dUTP is also lacking.
10. The method according to claim 9 , which further comprises adding to the reaction mixture in (IV) the dNTP or dNTPs lacking in (I)-(III).
11. The method according to claim 1 , which comprises partially elongating the adapted double-stranded DNA in the presence of a dNTP mixture lacking at least one of dATP, dTTP, dCTG, or dGTP, with the proviso that when dTTP is lacking dUTP is also lacking, wherein the partial elongation occurs after the ligating in (I).