Methods for asymmetric DNA library generation and optionally integrated duplex sequencing
Inventors
ALEKSEENKO, Alisa • PELECHANO GARCIA, Vicente Jose
Assignees
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Publication Number
US-12297490-B2
Publication Date
2025-05-13
Expiration Date
Abstract
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.
Core Innovation
The invention relates to a method for generating one or more DNA products comprising a first and second index sequence (IS), where the first and second ISs have different nucleotide sequences to allow identification of the source of a PCR sample. The method uses adaptors that include a first and a second strand identifier sequence (SIS), each having a primering sequence-binding region, ligated to opposite ends of a double-stranded DNA having two ends to obtain an adapted double-stranded DNA with single-stranded 5′ overhangs at each end.
After adaptor ligation, the adapted double-stranded DNA is denatured to obtain at least one single-stranded DNA template in the presence of a first primering sequence (PS) and a blocking oligonucleotide. The temperature is decreased under conditions that promote annealing of the blocking oligonucleotide and the first PS to a first primering sequence-binding region of the single-stranded template, where the blocking oligonucleotide has an annealing temperature to the single-stranded template equal to or higher than the annealing temperature of the first PS.
Without changing the temperature, a second PS comprising a second IS is added, and then the method incubates at a temperature that allows the blocking oligonucleotide to detach from the single-stranded template and elongation from the first PS to proceed. The method then denatures to obtain at least one single-stranded DNA comprising the first IS, followed by incubation under conditions that promote annealing of the second PS to a second primering sequence-binding region and subsequent DNA polymerization to obtain one or more DNA products comprising the first and second ISs with different nucleotide sequences.
Claims Coverage
The independent claim is clm-00001. It provides a complete workflow for generating DNA products with first and second ISs that have different nucleotide sequences for identifying the source of a PCR sample, including adaptor ligation using SIS and a blocking oligonucleotide-controlled primering sequence annealing and extension sequence.
Generating indexed DNA products using distinct ISs for source identification
A method for generating one or more DNA products comprising a first and second index sequence (IS), where the first and second ISs have different nucleotide sequences and allow identification of the source of a PCR sample.
Adaptor ligation with SIS at both ends producing 5′ overhangs
Ligation of a first adaptor comprising a first strand identifier sequence (SIS) and a primering sequence-binding region to one end of a double-stranded DNA and a second adaptor comprising a second SIS and a primering sequence-binding region to the other end, where the first and second SISs have different nucleotide sequences, to obtain an adapted double-stranded DNA having a single-stranded 5′ overhang at each end.
Blocking oligonucleotide and first PS annealing during template preparation
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, and decreasing the temperature to promote annealing of the blocking oligonucleotide and the first PS to a first primering sequence-binding region of the single-stranded template with the blocking oligonucleotide having an annealing temperature equal to or higher than the annealing temperature of the first PS.
Add second PS without changing temperature then elongate after blocking detachment
Without changing the temperature, adding a second PS comprising a second IS; incubating at a temperature that allows the blocking oligonucleotide to detach from the single-stranded template and elongation from the first PS to proceed; then denaturing to obtain at least one single-stranded DNA comprising the first IS.
Second PS annealing and polymerization to obtain products carrying first and second ISs
Incubating under conditions that promote annealing of the second PS to the at least one single-stranded DNA comprising the first IS, where the second PS anneals to a second primering sequence-binding region of the single-stranded DNA and subsequent DNA polymerization obtains one or more DNA products comprising a first and second IS, the first and second ISs having different nucleotide sequences.
The claim set centers on a method that ligates SIS-containing adaptors to obtain single-stranded 5′ overhang templates, uses a blocking oligonucleotide together with a first primering sequence during controlled annealing, adds a second PS without changing temperature, allows elongation after blocking detachment, and then anneals and polymerizes with the second PS so the resulting DNA products carry both a first and a second IS with different nucleotide sequences.
Stated Advantages
Allows identification of the source of a PCR sample using the first and second IS having different nucleotide sequences.
Documented Applications
No documented applications found
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