Library preparation and analysis methods for preserving topological information of cell-free DNA

Abstract

Described herein are methods and systems for constructing cfDNA sequence libraries, including methods and systems for sequencing 5' and / or 3' cfDNA overhangs to identify overhang length and topographical data of the overhangs. The methods can include, for example, using cfDNA topographical data to generate cfDNA overhang sequence libraries.

Description

[0001] Cross-references to related applications

[0002] This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 433,348, filed December 16, 2022, the entire contents of which are incorporated herein by reference for all purposes. Technical Field

[0003] This document discloses methods and systems for determining and analyzing the topological information of DNA duplexes (e.g., cell-free DNA, cfDNA) and for constructing sequencing libraries to obtain such topological information. Certain aspects of this disclosure more specifically relate to methods and systems for constructing DNA libraries for determining and / or analyzing the 5' and 3' overhangs of DNA duplexes (e.g., cfDNA). Background Technology

[0004] Cell-free DNA (cfDNA) molecules are free-floating double-stranded DNA molecules (dsDNA or double-stranded DNA) present in the bloodstream, often as a result of apoptosis or necrosis, particularly in the context of disease such as cancer. These degraded linear DNA fragments are typically about 50 to 300 base pairs in length. Most commonly, cfDNA is measured for cancer screening in the early stages of disease progression by analyzing its sequence to identify cancer-related mutations.

[0005] Natural cfDNA typically has 5' or 3' ends that protrude beyond the complementary strand, forming jagged ends. Standard DNA sequencing methods can only sequence DNA with blunt ends, so traditional sequencing techniques involve removing these natural topologies during the end-repair step of conventional double-stranded library construction. Therefore, all information about the length and sequence of any jagged ends present in cfDNA, natural end sequences, gaps, and nicks, as well as the opportunity to identify any potential relevance of this information to cancer diagnosis and cancer care, is lost. Summary of the Invention

[0006] This article describes methods for determining the topological structure of cell-free DNA, and methods for preparing nucleic acid constructs for determining the topological structure of cell-free DNA. It also describes methods for detecting diseases based at least in part on the determined topological structure of cell-free DNA.

[0007] In some embodiments, methods for determining the topological structure of cell-free DNA include: extending the 3' end of the first strand of a cell-free DNA duplex with inosine bases to fill the 5' overhang of the second strand of the cell-free DNA duplex; ligating a sequencing adaptor to the cell-free DNA duplex; sequencing the first strand of the cell-free DNA duplex to produce a first sequence readout; determining one or more bases in the first sequence readout to be soft-clipped using one or more processors; soft-clipping the bases in the first sequence readout corresponding to the 3' inosine extension of the first strand of the cell-free DNA duplex using one or more processors; and determining the length or sequence of the 5' overhang of the second strand of the cell-free DNA duplex based on the soft-clipping of the 3' inosine extension of the first strand of the cell-free DNA duplex using one or more processors. The method may also include detecting the presence or absence of a disease (e.g., cancer) based on the length or sequence of the 5' overhang of the second strand of the cell-free DNA duplex using one or more processors.

[0008] A method for determining the topological structure of cell-free DNA may further include: linking a second sequencing adaptor to a 3' overhang of the second strand of a cell-free DNA duplex; extending the 3' end of the second sequencing adaptor with inosine bases to fill the 3' overhang of the second strand of the cell-free DNA duplex; linking the 3' inosine-extended end of the second sequencing adaptor to a 5' end of the first strand of the cell-free DNA duplex, wherein the linked 3' inosine-extended end provides a 5' inosine extension of the first strand of the cell-free DNA duplex; determining, by one or more processors, one or more bases to be soft-cut and linked to the 5' end read from the first sequence; soft-cutting, by one or more processors, bases corresponding to the 5' inosine extension of the first strand of the cell-free DNA duplex from the first sequence; and determining, by one or more processors, the length or sequence of the 3' overhang of the second strand of the cell-free DNA duplex based on the soft-cutting of the 5' inosine extension of the first strand of the cell-free DNA duplex. In some embodiments, the method may further include detecting the presence or absence of a disease based on the length or sequence of the 3' overhang of the second strand of the cell-free DNA duplex using one or more processors. In some embodiments, linking a second sequencing adaptor to the 3' overhang of the second strand of the cell-free DNA duplex includes: extending the 3' overhang of the second strand of the cell-free DNA duplex to provide a 3' extension, wherein the second sequencing adaptor contains a 3' overhang complementary to the 3' extension of the second strand of the cell-free DNA duplex; and linking the second sequencing adaptor to the 3' extension of the second strand of the cell-free DNA duplex. In some embodiments, the 3' overhang of the second strand of the cell-free DNA duplex is extended using nucleotide bases of the same base type.

[0009] In some embodiments of the above method, determining one or more bases to be soft-cleaved in the first sequence readout includes aligning the first sequence readout with a reference sequence and identifying unaligned portions of the first sequence readout.

[0010] In some embodiments, the method for determining the topological structure of cell-free DNA further includes sequencing the second strand of the cell-free DNA duplex to generate a second sequence readout; and using one or more processors to determine one or more bases in the second sequence readout to be soft-cut. In some embodiments, determining one or more bases in the second sequence readout to be soft-cut includes aligning the second sequence with a reference sequence and identifying unaligned portions of the second sequence readout. In some embodiments, the first and second sequence readouts are associated with a unique molecular identifier (UMI). In some embodiments, determining one or more bases in the first sequence readout to be soft-cut includes aligning the first sequence with the second sequence readout and identifying unaligned portions of the first sequence readout. In some embodiments, determining one or more bases in the second sequence readout to be soft-cut includes aligning the second sequence with the first sequence readout and identifying unaligned portions of the second sequence readout.

[0011] In some embodiments of the above method, the method further includes extending the 3' end of the second strand of the cell-free DNA duplex with inosine bases to fill the 5' overhang of the first strand of the cell-free DNA duplex; ligating a second sequencing adaptor to the cell-free DNA duplex; soft-splitting the bases corresponding to the 3' inosine extension of the second strand of the cell-free DNA duplex from the second sequence readout by one or more processors; and determining the length or sequence of the 5' overhang of the first strand of the cell-free DNA duplex based on the soft-splitting of the 3' inosine extension of the second strand of the cell-free DNA duplex by one or more processors. In some embodiments, the method further includes detecting the presence or absence of a disease based on the length or sequence of the 5' overhang of the first strand of the cell-free DNA duplex by one or more processors.

[0012] In some embodiments of the above method, the 3' end of the first strand of the extended cell-free DNA double helix includes a 3' single inosine overhang. In some embodiments, the sequencing adaptor includes a 3' cytosine overhang complementary to the 3' inosine overhang.

[0013] In some implementations, the method for determining the topology of cell-free DNA further includes amplifying the cell-free DNA duplex to link the sample index to the cell-free DNA duplex.

[0014] In some implementations of the above methods, the sequencing adaptor or second sequencing adaptor contains a sample index.

[0015] In some implementations of the above methods, the sequencing adaptor or second sequencing adaptor contains a UMI.

[0016] In some implementations of the above methods, the sequencing adaptor or second sequencing adaptor is a Y-shaped sequencing adaptor. In some implementations, the adaptor may be a full-length Y-adaptor (e.g., a Y-shaped adaptor containing an index sequence), a stubby adaptor, or a hairpin adaptor.

[0017] This document also provides a method for preparing a sequencing construct, comprising: ligating a sequencing adaptor to the 3' end of the first strand of a cell-free DNA duplex; extending the 3' end of the sequencing adaptor with inosine bases to fill the 3' end of the first strand of the cell-free DNA duplex; and ligating the 3' inosine-extended end of the sequencing adaptor to the 5' end of the second strand of the cell-free DNA duplex, wherein the ligated 3' inosine-extended end provides a 5' inosine extension of the second strand of the cell-free DNA duplex. In some embodiments, ligating the sequencing adaptor to the 3' end of the first strand of the cell-free DNA duplex comprises: extending the 3' end of the first strand of the cell-free DNA duplex to provide a 3' extension, wherein the sequencing adaptor includes a 3' end complementary to the 3' extension of the first strand of the cell-free DNA duplex; and ligating the sequencing adaptor to the 3' extension of the first strand of the cell-free DNA duplex. In some embodiments, the 3' end of the first strand of the cell-free DNA duplex is extended using nucleotide bases of the same base type.

[0018] This document also describes a method for determining the topological structure of cell-free DNA, comprising: preparing a sequencing construct according to the above method; sequencing the second strand of the cell-free DNA duplex to generate a first sequence readout; determining one or more bases in the first sequence readout to be soft-cut using one or more processors; soft-cutting bases in the first sequence readout corresponding to the 5' inosine extension of the second strand of the cell-free DNA duplex from the first sequence readout using one or more processors; and determining the length or sequence of the 3' overhang of the first strand of the cell-free DNA duplex based on the soft cutting of the 5' inosine extension of the second strand of the cell-free DNA duplex using one or more processors. In some embodiments, the method further includes detecting the presence or absence of a disease (e.g., cancer) based on the length or sequence of the 3' overhang of the first strand of the cell-free DNA duplex using one or more processors. In some embodiments, determining one or more bases in the first sequence readout to be soft-cut includes aligning the first sequence readout with a reference sequence and identifying unaligned portions of the first sequence readout.

[0019] In some embodiments of a method for determining the topological structure of cell-free DNA, the method further includes sequencing the first strand of the cell-free DNA duplex to produce a second sequence readout; and determining one or more bases in the second sequence readout to be soft-cut using one or more processors. In some embodiments, determining one or more bases in the second sequence readout to be soft-cut includes aligning the second sequence with a reference sequence and identifying unaligned portions of the second sequence readout. In some embodiments, the first and second sequence readouts are associated with a unique molecular identifier (UMI). In some embodiments, determining one or more bases in the first sequence readout to be soft-cut includes aligning the first sequence with the second sequence readout and identifying unaligned portions of the first sequence readout. In some embodiments, determining one or more bases in the second sequence readout to be soft-cut includes aligning the second sequence with the first sequence readout and identifying unaligned portions of the second sequence readout.

[0020] In some embodiments of the method for determining the topological structure of cell-free DNA, the method further includes linking a second sequencing adaptor to a 3' overhang of the second strand of the cell-free DNA duplex; extending the 3' end of the second sequencing adaptor with inosine bases to fill the 3' overhang of the second strand of the cell-free DNA duplex; and linking the 3' inosine-extended end of the second sequencing adaptor to the 5' end of the first strand of the cell-free DNA duplex, wherein the 3' inosine-extended end provides a 5' inosine extension of the first strand of the cell-free DNA duplex. In some embodiments, the method further includes sequencing the first strand of a cell-free DNA duplex to generate a second sequence readout; determining, using one or more processors, one or more bases to be soft-spliced ​​and linked to the 5' end of the second sequence readout; soft-splicing, using one or more processors, the bases corresponding to the 5' inosine extension of the first strand of the cell-free DNA duplex from the second sequence readout; and determining, using one or more processors, the length or sequence of the 3' overhang of the second strand of the cell-free DNA duplex based on the soft-splicing of the 5' inosine extension of the first strand of the cell-free DNA duplex. In some embodiments, the method further includes detecting the presence or absence of a disease using one or more processors based on the length or sequence of the 3' overhang of the second strand of the cell-free DNA duplex. In some embodiments, linking the second sequencing adaptor to the 3' overhang of the second strand of the cell-free DNA duplex includes linking a single inosine to the 3' overhang of the second strand of the cell-free DNA duplex and linking the second sequencing adaptor to the single inosine. In some embodiments, the sequencing adaptor includes a 3' cytosine overhang complementary to inosine, attached to the 3' overhang of the second strand of the cell-free DNA duplex. In some embodiments, identifying one or more bases to be soft-split and attached to the 5' end of the second sequence readout includes aligning the second sequence readout with a reference sequence and identifying any unaligned portions of the second sequence readout. In some embodiments, the first and second sequence readouts are associated with a unique molecular identifier (UMI). In some embodiments, identifying one or more bases to be soft-split and attached to the 5' end of the second sequence readout includes aligning the second sequence readout with the first sequence readout and identifying any unaligned portions of the second sequence readout. In some embodiments, identifying one or more bases to be soft-split and attached to the 5' end of the first sequence readout includes aligning the first sequence readout with the second sequence readout and identifying any unaligned portions of the first sequence readout.

[0021] In some implementations of the above method, cell-free DNA duplexes are amplified to link the sample index to the cell-free DNA duplexes.

[0022] In some implementations of the above methods, the sequencing adaptor or second sequencing adaptor contains a sample index.

[0023] In some implementations of the above methods, the sequencing adaptor or second sequencing adaptor contains a UMI.

[0024] In some embodiments of the above method, the sequencing adaptor or second sequencing adaptor is a Y-shaped sequencing adaptor. In some embodiments, the adaptor may be a full-length Y adaptor (e.g., a Y-shaped adaptor containing an index sequence), a short-stalk adaptor, or a hairpin adaptor.

[0025] In some implementations of the above method, cell-free DNA duplexes are obtained from subjects suspected of having cancer or identified as having cancer.

[0026] In some implementations of any of the above methods, the method also includes treating the subject with anticancer therapy.

[0027] In some implementations of any of the above methods, the method further includes obtaining cell-free DNA duplexes from the object.

[0028] In some embodiments of any of the above methods, the method further includes obtaining cell-free DNA duplexes from a liquid biopsy sample. In some embodiments, the sample is a liquid biopsy sample and includes blood, plasma, cerebrospinal fluid, sputum, feces, urine, or saliva. In some embodiments, the cell-free DNA duplexes are circulating tumor DNA (ctDNA) duplexes.

[0029] In some implementations of any of the above methods, the sequencing adapter or second sequencing adapter includes an amplification primer binding site, a flow cell adapter sequence, or a substrate adapter sequence.

[0030] In some embodiments of any of the above methods, the method further includes amplifying the first and second strands of the cell-free DNA double helix. In some embodiments, the amplification includes polymerase chain reaction (PCR) amplification, non-PCR amplification, or isothermal amplification.

[0031] In some embodiments of any of the above methods, sequencing includes the use of massively parallel sequencing (MPS), whole genome sequencing (WGS), whole exome sequencing, targeted sequencing, direct sequencing, or Sanger sequencing. In some embodiments, sequencing includes massively parallel sequencing, and the massively parallel sequencing technology includes next-generation sequencing (NGS). In some embodiments, sequencing is performed using a next-generation sequencer.

[0032] In some embodiments of any of the above methods, the method further includes generating a report via one or more processors, the report displaying the following lengths: the 3' overhang of the first strand of the cell-free DNA duplex, the 3' overhang of the second strand of the cell-free DNA duplex, the 5' overhang of the first strand of the cell-free DNA duplex, and / or the 5' overhang of the second strand of the cell-free DNA duplex. In some embodiments, the method further includes transmitting the report to a healthcare provider. In some embodiments, the report is transmitted via a computer network or a peer-to-peer connection.

[0033] In some embodiments of any of the above methods, the method further includes generating a genomic profile of the object, the genomic profile comprising the following lengths: a 3' overhang of the first strand of the cell-free DNA duplex, a 3' overhang of the second strand of the cell-free DNA duplex, a 5' overhang of the first strand of the cell-free DNA duplex, and / or a 5' overhang of the second strand of the cell-free DNA duplex. In some embodiments, the genomic profile of the object also includes results from: comprehensive genomic profiling (CGP) tests, gene expression profiling tests, cancer hotspot group tests, DNA methylation tests, DNA fragmentation tests, RNA fragmentation tests, or any combination thereof. In some embodiments, the genomic profile of the object also includes results from nucleic acid sequencing-based tests.

[0034] This document also describes a system comprising: one or more processors; and a memory communicatively coupled to the one or more processors and configured to store instructions, which, when executed by the one or more processors, cause the system to: receive a first sequence readout obtained by: extending the 3' end of the first strand of a cell-free DNA duplex with inosine bases to fill the 5' overhang of the second strand of the cell-free DNA duplex; ligating a sequencing adaptor to the cell-free DNA duplex; and sequencing the first strand of the cell-free DNA duplex to produce the first sequence readout; determining one or more bases in the first sequence readout to be soft-cut; soft-cutting the bases in the first sequence readout corresponding to the 3' inosine extension of the first strand of the cell-free DNA duplex; and determining the length or sequence of the 5' overhang of the second strand of the cell-free DNA duplex based on the soft-cutting of the 3' inosine extension of the first strand of the cell-free DNA duplex. In some embodiments, the instructions also cause the system to detect the presence or absence of a disease (e.g., cancer) based on the length or sequence of the 5' overhang of the second strand of the cell-free DNA duplex. In some embodiments, the first sequence readout is also obtained by: linking a second sequencing adaptor to the 3' overhang of the second strand of the cell-free DNA duplex; extending the 3' end of the second sequencing adaptor with inosine bases to fill the 3' overhang of the second strand of the cell-free DNA duplex; and linking the 3' inosine-extended end of the second sequencing adaptor to the 5' end of the first strand of the cell-free DNA duplex, wherein the linked 3' inosine-extended end provides a 5' inosine extension of the first strand of the cell-free DNA duplex.

[0035] In some embodiments of the above system, when executed by one or more processors, the instructions further cause the system to: soft-shear bases corresponding to the 5' inosine extension of the first strand of the cell-free DNA duplex from a first sequence readout; and determine the length or sequence of the 3' overhang of the second strand of the cell-free DNA duplex based on the soft-shearing of the 5' inosine extension of the first strand of the cell-free DNA duplex. In some embodiments, the instructions further cause the system to detect the presence or absence of a disease based on the length or sequence of the 3' overhang of the second strand of the cell-free DNA duplex. In some embodiments, linking the second sequencing adaptor to the 3' overhang of the second strand of the cell-free DNA duplex includes: extending the 3' overhang of the second strand of the cell-free DNA duplex to provide a 3' extension, wherein the second sequencing adaptor includes a 3' overhang complementary to the 3' extension of the second strand of the cell-free DNA duplex; and linking the second sequencing adaptor to the 3' extension of the second strand of the cell-free DNA duplex. In some implementations, the 3' overhang of the second strand of the cell-free DNA duplex is extended using nucleotide bases of the same base type.

[0036] In some implementations of the above system, one or more bases in the first sequence readout are identified as to be soft-cut by a method that includes aligning the first sequence readout with a reference sequence and identifying unaligned portions of the first sequence readout.

[0037] In some embodiments of the above system, when executed by one or more processors, the instructions further cause the system to: receive a second sequence readout obtained by sequencing the second strand of a cell-free DNA duplex; and identify one or more bases in the second sequence readout to be soft-cut. In some embodiments, one or more bases in the second sequence readout are identified as to be soft-cut by a method comprising aligning the second sequence with a reference sequence and identifying unaligned portions of the second sequence readout. In some embodiments, the first and second sequence readouts are associated by a unique molecular identifier (UMI). In some embodiments, one or more bases in the first sequence readout are identified as to be soft-cut by a method comprising aligning the first sequence with the second sequence readout and identifying unaligned portions of the first sequence readout. In some embodiments, one or more bases in the second sequence readout are identified as to be soft-cut by a method comprising aligning the second sequence with the first sequence readout and identifying unaligned portions of the second sequence readout. In some embodiments, the second sequence readout is further obtained by extending the 3' end of the second strand of the cell-free DNA duplex with inosine bases to fill the 5' overhang of the first strand of the cell-free DNA duplex, and by linking the second sequencing adaptor to the cell-free DNA duplex. In some embodiments, when the instructions are executed by one or more processors, the system further causes to: soft-cleave the bases corresponding to the 3' inosine extension of the second strand of the cell-free DNA duplex from the second sequence readout; and determine the length or sequence of the 5' overhang of the first strand of the cell-free DNA duplex based on the soft-cleavage of the 3' inosine extension of the second strand of the cell-free DNA duplex.

[0038] In some embodiments of the above system, the 3' end of the first strand of the extended cell-free DNA duplex includes a single 3' inosine overhang. In some embodiments, the sequencing adaptor includes a 3' cytosine overhang complementary to the single 3' inosine overhang.

[0039] This document also describes a system comprising: one or more processors; and a memory communicatively coupled to the one or more processors and configured to store instructions, which, when executed by the one or more processors, cause the system to: receive a first sequence readout obtained by: attaching a sequencing adaptor to the 3' overhang of the first strand of a cell-free DNA duplex; extending the 3' end of the sequencing adaptor with inosine bases to fill the 3' overhang of the first strand of the cell-free DNA duplex; and extending the 3' end of the sequencing adaptor with inosine bases. The extended end is attached to the 5' end of the second strand of the cell-free DNA duplex, wherein the attached 3' inosine-extended end provides the 5' inosine extension of the second strand of the cell-free DNA duplex; one or more bases to be soft-cut in the first sequence readout are identified; the bases corresponding to the 5' inosine extension of the second strand of the cell-free DNA duplex are soft-cut from the first sequence readout; and the length or sequence of the 3' overhang of the first strand of the cell-free DNA duplex is determined based on the soft cutting of the 5' inosine extension of the second strand of the cell-free DNA duplex. In some embodiments, the instructions also cause the system to detect the presence or absence of a disease (e.g., cancer) based on the length or sequence of the 3' overhang of the first strand of the cell-free DNA duplex.

[0040] In some embodiments of the above system, connecting the second sequencing adaptor to the 3' overhang of the second strand of the cell-free DNA duplex includes: extending the 3' overhang of the second strand of the cell-free DNA duplex to provide 3' extension, wherein the second sequencing adaptor includes a 3' overhang complementary to the 3' extension of the second strand of the cell-free DNA duplex; and connecting the second sequencing adaptor to the 3' extension of the second strand of the cell-free DNA duplex. In some embodiments, the 3' overhang of the second strand of the cell-free DNA duplex is extended using nucleotide bases of the same base type.

[0041] In some implementations of the above system, one or more bases in the first sequence readout are identified as to be soft-cut by a method that includes aligning the first sequence readout with a reference sequence and identifying unaligned portions of the first sequence readout.

[0042] In some embodiments of the above system, when executed by one or more processors, the instructions further cause the system to: receive a second sequence readout obtained by sequencing the second strand of a cell-free DNA duplex, and identify one or more bases in the second sequence readout to be soft-cut. In some embodiments, one or more bases in the second sequence readout are identified as to be soft-cut by a method comprising aligning the second sequence with a reference sequence and identifying unaligned portions of the second sequence readout. In some embodiments, the first and second sequence readouts are associated by a unique molecular identifier (UMI). In some embodiments, one or more bases in the first sequence readout are identified as to be soft-cut by a method comprising aligning the first sequence with the second sequence readout and identifying unaligned portions of the first sequence readout. In some embodiments, one or more bases in the second sequence readout are identified as to be soft-cut by a method comprising aligning the second sequence with the first sequence readout and identifying unaligned portions of the second sequence readout. In some embodiments, the second sequence readout is further obtained by: linking a second sequencing adaptor to the 3' overhang of the second strand of the cell-free DNA duplex; extending the 3' end of the second sequencing adaptor with inosine bases to fill the 3' overhang of the second strand of the cell-free DNA duplex; and linking the 3' inosine-extended end of the second sequencing adaptor to the 5' end of the first strand of the cell-free DNA duplex, wherein the 3' inosine-extended end provides the 5' inosine extension of the first strand of the cell-free DNA duplex. In some embodiments, when the instructions are executed by one or more processors, the system further causes to: soft-cleave bases corresponding to the 3' inosine extension of the first strand of the cell-free DNA duplex from the second sequence readout; and determine the length or sequence of the 5' overhang of the second strand of the cell-free DNA duplex based on the soft-cleavage of the 3' inosine extension of the first strand of the cell-free DNA duplex. In some embodiments, linking the second sequencing adaptor to the 3' overhang of the second strand of the cell-free DNA duplex includes linking a monosine to the 3' overhang of the second strand of the cell-free DNA duplex and linking the second sequencing adaptor to the monosine. In some embodiments, the sequencing adaptor includes a 3' cytosine overhang complementary to the monosine linked to the 3' overhang of the second strand of the cell-free DNA duplex.

[0043] In some implementations of any of the above systems, the sequencing adaptor or second sequencing adaptor contains a UMI.

[0044] In some implementations of any of the above systems, the sequencing adaptor or second sequencing adaptor is a Y-shaped sequencing adaptor. In some implementations, the adaptor may be a full-length Y-adaptor (e.g., a Y-shaped adaptor containing an index sequence), a short-stalk adaptor, or a hairpin adaptor.

[0045] In some embodiments of any of the above systems, the system also includes a nucleic acid amplifier configured to amplify cell-free DNA duplexes to link sample indexes with cell-free DNA duplexes. In some embodiments, the nucleic acid amplifier is a thermal cycler.

[0046] In some implementations of any of the above systems, cell-free DNA duplexes are obtained from subjects suspected of having cancer or identified as having cancer.

[0047] In some implementations of any of the above systems, cell-free DNA duplexes are obtained from the object.

[0048] In some implementations of any of the above systems, cell-free DNA duplexes are obtained from liquid biopsy samples.

[0049] In some implementations of any of the above systems, the sample is a liquid biopsy sample and includes blood, plasma, cerebrospinal fluid, sputum, feces, urine, or saliva.

[0050] In some implementations of any of the above systems, the cell-free DNA double strand is a circulating tumor DNA (ctDNA) double strand.

[0051] In some implementations of any of the above systems, the sequencing adaptor or second sequencing adaptor includes an amplification primer binding site, a flow cell adaptor sequence, or a base adaptor sequence.

[0052] In some embodiments of any of the above systems, the system further includes a nucleic acid amplifier configured to amplify the first and second strands of cell-free DNA duplexes. In some embodiments, the amplification includes polymerase chain reaction (PCR) amplification, non-PCR amplification, or isothermal amplification.

[0053] In some embodiments of any of the above systems, the system further includes a sequencer configured to sequence the first strand and / or the second strand of a cell-free DNA duplex. In some embodiments, the sequencer is configured for massively parallel sequencing (MPS), whole-genome sequencing (WGS), whole-exome sequencing, targeted sequencing, direct sequencing, or Sanger sequencing. In some embodiments, the sequencer is configured for massively parallel sequencing, and the massively parallel sequencing technology includes next-generation sequencing (NGS). In some embodiments, the sequencer is a next-generation sequencer.

[0054] In some embodiments of any of the above systems, the instructions, when executed by one or more processors, also cause the system to generate a report displaying the following lengths: the 3' overhang of the first strand of the cell-free DNA duplex, the 3' overhang of the second strand of the cell-free DNA duplex, the 5' overhang of the first strand of the cell-free DNA duplex, and / or the 5' overhang of the second strand of the cell-free DNA duplex. In some embodiments, the instructions, when executed by one or more processors, also cause the system to transmit the report to a healthcare provider. In some embodiments, the report is transmitted via a computer network or peer-to-peer connection.

[0055] In some embodiments of any of the above systems, the instructions, when executed by one or more processors, further cause the system to generate a genomic profile of the object by one or more processors, the genomic profile comprising the following lengths: a 3' overhang of the first strand of the cell-free DNA duplex, a 3' overhang of the second strand of the cell-free DNA duplex, a 5' overhang of the first strand of the cell-free DNA duplex, and / or a 5' overhang of the second strand of the cell-free DNA duplex. In some embodiments, the genomic profile of the object also includes results from: a comprehensive genome profiling (CGP) test, a gene expression profiling test, a cancer hotspot group test, a DNA methylation test, a DNA fragmentation test, an RNA fragmentation test, or any combination thereof. In some embodiments, the genomic profile of the object also includes results from nucleic acid sequencing-based tests.

[0056] This document also describes a non-transitory computer-readable storage medium storing one or more programs comprising instructions that, when executed by one or more processors of a system, cause the system to: receive a first sequence readout obtained by: extending the 3' end of the first strand of a cell-free DNA duplex with inosine bases to fill the 5' overhang of the second strand of the cell-free DNA duplex; ligating a sequencing adaptor to the cell-free DNA duplex; and sequencing the first strand of the cell-free DNA duplex to produce the first sequence readout; determining one or more bases in the first sequence readout to be soft-cut; soft-cutting from the first sequence readout the bases corresponding to the 3' inosine extension of the first strand of the cell-free DNA duplex; and determining the length or sequence of the 5' overhang of the second strand of the cell-free DNA duplex based on the soft-cutting of the 3' inosine extension of the first strand of the cell-free DNA duplex. In some embodiments, the instructions also cause the system to detect the presence or absence of a disease (e.g., cancer) based on the length or sequence of the 5' overhang of the second strand of the cell-free DNA duplex.

[0057] In some embodiments of the aforementioned storage medium, the first sequence readout is further obtained by: linking a second sequencing adaptor to the 3' overhang of the second strand of the cell-free DNA duplex; extending the 3' end of the second sequencing adaptor with inosine bases to fill the 3' overhang of the second strand of the cell-free DNA duplex; and linking the 3' inosine-extended end of the second sequencing adaptor to the 5' end of the first strand of the cell-free DNA duplex, wherein the linked 3' inosine-extended end provides the 5' inosine extension of the first strand of the cell-free DNA duplex. In some embodiments, when the instructions are executed by one or more processors, the system further causes to: soft-splice the bases corresponding to the 5' inosine extension of the first strand of the cell-free DNA duplex during the first sequence readout; and determine the length or sequence of the 3' overhang of the second strand of the cell-free DNA duplex based on the soft-splicing of the 5' inosine extension of the first strand of the cell-free DNA duplex. In some implementations, the instructions also enable the system to detect the presence or absence of a disease based on the length or sequence of the 3' overhang of the second strand of the cell-free DNA duplex.

[0058] In some embodiments of the aforementioned storage medium, linking the second sequencing adaptor to the 3' overhang of the second strand of the cell-free DNA duplex includes: extending the 3' overhang of the second strand of the cell-free DNA duplex to provide 3' extension, wherein the second sequencing adaptor includes a 3' overhang complementary to the 3' extension of the second strand of the cell-free DNA duplex; and linking the second sequencing adaptor to the 3' extension of the second strand of the cell-free DNA duplex. In some embodiments, the 3' overhang of the second strand of the cell-free DNA duplex is extended using nucleotide bases of the same base type.

[0059] In some embodiments of the aforementioned storage medium, one or more bases in the first sequence readout are identified as to be soft-cut by a method comprising aligning the first sequence readout with a reference sequence and identifying any unaligned portions of the first sequence readout.

[0060] In some embodiments of the aforementioned storage medium, when executed by one or more processors, the instructions further cause the system to: receive a second sequence readout obtained by sequencing the second strand of a cell-free DNA duplex, and identify one or more bases in the second sequence readout to be soft-cut. In some embodiments, one or more bases in the second sequence readout are identified as to be soft-cut by a method comprising aligning the second sequence with a reference sequence and identifying unaligned portions of the second sequence readout. In some embodiments, the first and second sequence readouts are associated by a unique molecular identifier (UMI). In some embodiments, one or more bases in the first sequence readout are identified as to be soft-cut by a method comprising aligning the first sequence with the second sequence readout and identifying unaligned portions of the first sequence readout. In some embodiments, one or more bases in the second sequence readout are identified as to be soft-cut by a method comprising aligning the second sequence with the first sequence readout and identifying unaligned portions of the second sequence readout. In some embodiments, the second sequence readout is further obtained by extending the 3' end of the second strand of the cell-free DNA duplex with inosine bases to fill the 5' overhang of the first strand of the cell-free DNA duplex, and by linking the second sequencing adaptor to the cell-free DNA duplex. In some embodiments, when the instructions are executed by one or more processors, the system further causes to: soft-cleave the bases corresponding to the 3' inosine extension of the second strand of the cell-free DNA duplex from the second sequence readout; and determine the length or sequence of the 5' overhang of the first strand of the cell-free DNA duplex based on the soft-cleavage of the 3' inosine extension of the second strand of the cell-free DNA duplex.

[0061] In some embodiments of the aforementioned storage medium, the 3' end of the first strand of the extended cell-free DNA double helix includes a single 3' inosine overhang. In some embodiments, the sequencing adaptor includes a 3' cytosine overhang complementary to the single 3' inosine overhang.

[0062] This document also describes a non-transitory computer-readable storage medium storing one or more programs comprising instructions that, when executed by one or more processors of a system, cause the system to: receive a first sequence readout obtained by: attaching a sequencing adaptor to a 3' overhang of a first strand of a cell-free DNA duplex; extending the 3' end of the sequencing adaptor with inosine bases to fill the 3' overhang of the first strand of the cell-free DNA duplex; and attaching the 3' inosine-extended end of the sequencing adaptor to a 5' end of a second strand of the cell-free DNA duplex, wherein the attached 3' inosine-extended end provides a 5' inosine extension of the second strand of the cell-free DNA duplex; determining one or more bases to be soft-cut in the first sequence readout; soft-cutting bases corresponding to the 5' inosine extension of the second strand of the cell-free DNA duplex from the first sequence readout; and determining the length or sequence of the 3' overhang of the first strand of the cell-free DNA duplex based on the soft-cutting of the 5' inosine extension of the second strand of the cell-free DNA duplex. In some implementations, the instructions also enable the system to detect the presence or absence of a disease (e.g., cancer) based on the length or sequence of the 3' overhang of the first strand of the cell-free DNA duplex.

[0063] In some embodiments of the aforementioned storage medium, linking the second sequencing adaptor to the 3' overhang of the second strand of the cell-free DNA duplex includes: extending the 3' overhang of the second strand of the cell-free DNA duplex to provide 3' extension, wherein the second sequencing adaptor includes a 3' overhang complementary to the 3' extension of the second strand of the cell-free DNA duplex; and linking the second sequencing adaptor to the 3' extension of the second strand of the cell-free DNA duplex. In some embodiments, the 3' overhang of the second strand of the cell-free DNA duplex is extended using nucleotide bases of the same base type.

[0064] In some embodiments of the aforementioned storage medium, one or more bases in the first sequence readout are identified as to be soft-cut by a method comprising aligning the first sequence readout with a reference sequence and identifying any unaligned portions of the first sequence readout.

[0065] In some embodiments of the aforementioned storage medium, when executed by one or more processors, the instructions further cause the system to: receive a second sequence readout obtained by sequencing the second strand of a cell-free DNA duplex, and identify one or more bases in the second sequence readout to be soft-cut. In some embodiments, one or more bases in the second sequence readout are identified as to be soft-cut by a method comprising aligning the second sequence with a reference sequence and identifying unaligned portions of the second sequence readout. In some embodiments, the first and second sequence readouts are associated by a unique molecular identifier (UMI). In some embodiments, one or more bases in the first sequence readout are identified as to be soft-cut by a method comprising aligning the first sequence with the second sequence readout and identifying unaligned portions of the first sequence readout. In some embodiments, one or more bases in the second sequence readout are identified as to be soft-cut by a method comprising aligning the second sequence with the first sequence readout and identifying unaligned portions of the second sequence readout. In some embodiments, the second sequence readout is further obtained by: linking a second sequencing adaptor to the 3' overhang of the second strand of the cell-free DNA duplex; extending the 3' end of the second sequencing adaptor with inosine bases to fill the 3' overhang of the second strand of the cell-free DNA duplex; and linking the 3' inosine-extended end of the second sequencing adaptor to the 5' end of the first strand of the cell-free DNA duplex, wherein the 3' inosine-extended end provides the 5' inosine extension of the first strand of the cell-free DNA duplex. In some embodiments, when the instructions are executed by one or more processors, the system further causes to: soft-cleave bases corresponding to the 3' inosine extension of the first strand of the cell-free DNA duplex from the second sequence readout; and determine the length or sequence of the 5' overhang of the second strand of the cell-free DNA duplex based on the soft-cleavage of the 3' inosine extension of the first strand of the cell-free DNA duplex. In some embodiments, linking the second sequencing adaptor to the 3' overhang of the second strand of the cell-free DNA duplex includes linking a single inosine to the 3' overhang of the second strand of the cell-free DNA duplex, and linking the second sequencing adaptor to inosine. In some embodiments, the sequencing adaptor includes a 3' cytosine overhang complementary to the single inosine linked to the 3' overhang of the second strand of the cell-free DNA duplex.

[0066] In some implementations of the aforementioned storage medium, the sequencing adaptor or second sequencing adaptor contains a UMI.

[0067] In some embodiments of the aforementioned storage medium, the sequencing adaptor or second sequencing adaptor is a Y-shaped sequencing adaptor. In some embodiments, the adaptor may be a full-length Y-adaptor (e.g., a Y-shaped adaptor containing an index sequence), a short-stalk adaptor, or a hairpin adaptor.

[0068] In some embodiments of the aforementioned storage medium, cell-free DNA duplexes are obtained from individuals suspected of having cancer or identified as having cancer.

[0069] In some embodiments of the aforementioned storage medium, cell-free DNA duplexes are obtained from the object.

[0070] In some embodiments of the storage medium described above, cell-free DNA double strands are obtained from liquid biopsy samples. For example, in some embodiments, the sample is a liquid biopsy sample and includes blood, plasma, cerebrospinal fluid, sputum, feces, urine, or saliva.

[0071] In some embodiments of the aforementioned storage medium, the cell-free DNA double strand is a circulating tumor DNA (ctDNA) double strand.

[0072] In some implementations of the aforementioned storage medium, the sequencing adaptor or second sequencing adaptor includes an amplification primer binding site, a flow cell adaptor sequence, or a substrate adaptor sequence.

[0073] In some embodiments of the aforementioned storage medium, sequencing includes the use of massively parallel sequencing (MPS), whole-genome sequencing (WGS), whole-exome sequencing, targeted sequencing, direct sequencing, or Sanger sequencing. In some embodiments, sequencing includes massively parallel sequencing, and the massively parallel sequencing technology includes next-generation sequencing (NGS). In some embodiments, sequencing is performed using a next-generation sequencer.

[0074] In some embodiments of the aforementioned storage medium, the instructions, when executed by one or more processors, further cause the system to generate a report displaying the following lengths: the 3' overhang of the first strand of the cell-free DNA duplex, the 3' overhang of the second strand of the cell-free DNA duplex, the 5' overhang of the first strand of the cell-free DNA duplex, and / or the 5' overhang of the second strand of the cell-free DNA duplex. In some embodiments, the instructions, when executed by one or more processors, further cause the system to transmit the report to a healthcare provider. In some embodiments, the report is transmitted via a computer network or peer-to-peer connection.

[0075] In some embodiments of the aforementioned storage medium, when executed by one or more processors, the instructions further cause the system to generate a genomic profile of the object via one or more processors, the genomic profile comprising the following lengths: a 3' overhang of the first strand of the cell-free DNA duplex, a 3' overhang of the second strand of the cell-free DNA duplex, a 5' overhang of the first strand of the cell-free DNA duplex, and / or a 5' overhang of the second strand of the cell-free DNA duplex. In some embodiments, the genomic profile of the object further comprises results from: comprehensive genome profiling (CGP) tests, gene expression profiling tests, cancer hotspot group tests, DNA methylation tests, DNA fragmentation tests, RNA fragmentation tests, or any combination thereof. In some embodiments, the genomic profile of the object further comprises results from nucleic acid sequencing-based tests. Attached Figure Description

[0076] Various aspects of the disclosed methods, apparatus, and systems are specifically set forth in the appended claims. A better understanding of the features and advantages of the disclosed methods, apparatus, and systems will be obtained by referring to the following detailed description and accompanying drawings of exemplary embodiments, wherein:

[0077] Figure 1A An exemplary process for preparing sequencing constructs and determining the 5' overhang length of a DNA duplex molecule (e.g., cfDNA molecule) according to some embodiments is shown.

[0078] Figure 1B It shows Figure 1A An exemplary embodiment of the process for preparing sequencing constructs and determining the 5' overhang length of a DNA double-stranded molecule (e.g., cfDNA molecule) is shown. In this exemplary method, the 5' overhang in the cfDNA is filled with deoxyinosine, and then an adaptor is ligated to the end of the cfDNA. Optionally, the cfDNA is PCR amplified prior to sequencing. The sequences are then aligned, and their overhang length and sequence are analyzed.

[0079] Figure 2A An exemplary method is shown, according to some embodiments, for preparing a sequencing construct that can be used to determine the length of the 3' overhang of a DNA duplex (e.g., a cfDNA molecule).

[0080] Figure 2B An exemplary method for determining the length of the 3' overhang of a DNA double helix (e.g., a cfDNA molecule) according to some embodiments is shown.

[0081] Figure 3AExemplary methods for preparing sequencing constructs that can be used to determine the length of 5' and / or 3' protrusions of DNA duplex molecules (e.g., cfDNA molecules) according to some embodiments are shown, as well as procedures for determining the length of 5' and / or 3' protrusions of DNA duplex molecules.

[0082] Figure 3B Examples of implementation schemes are shown. Figure 3A An exemplary embodiment of a method for preparing sequencing constructs and determining the length of the 5' and / or 3' overhangs of a DNA double-stranded molecule (e.g., a cfDNA molecule) is shown. In this exemplary method, the 5' overhangs in the cfDNA are filled with deoxyinosine. One adaptor is attached to the 3' overhang of the bottom strand, and another adaptor is attached to the opposite end of the molecule. The 3' overhangs of the cfDNA are then filled with deoxyinosine. Optionally, the cfDNA is PCR amplified prior to sequencing. The sequences are then aligned, and their overhang lengths and sequences are analyzed.

[0083] Figure 3C Examples of implementation schemes are shown. Figure 3A Another exemplary embodiment of the method shown for preparing sequencing constructs and determining the length of the 5' and / or 3' overhangs of DNA duplex molecules (e.g., cfDNA molecules). Figure 3C The aspects of the method illustrated herein can be readily applied to other embodiments of the method described herein, including... Figure 1A , 1B The process is illustrated in 2A and 2B. In this exemplary method, the 5' overhang in the cfDNA is filled with deoxyinosine, and then a first adaptor is ligated to the cfDNA at the deoxyinosine-filled end. The 3' overhang in the cfDNA is extended with a single type of nucleotide (denoted by "Y"), a second adaptor is ligated to the cfDNA, and the 3' overhang is filled with deoxyinosine. Optionally, the cfDNA is PCR amplified prior to sequencing. The sequences are then aligned, and their overhang lengths and sequences are analyzed.

[0084] Figure 4 The process for determining the topological structure of cfDNA according to some implementation schemes is shown.

[0085] Figure 5 An exemplary readout processing matrix for extracting data from paired-end sequencing is shown according to some implementation schemes.

[0086] Figure 6 An exemplary computing device or system according to one embodiment of the present disclosure is shown.

[0087] Figure 7Exemplary computer systems or computer networks are shown as examples of some of the systems described herein. Detailed Implementation

[0088] This article describes methods and systems for determining the topological structure (particularly the length and sequence of the 5' and / or 3' overhangs) of DNA duplex (e.g., cell-free DNA) molecules. Standard methods for evaluating cfDNA fail to adequately capture the 5' and 3' overhang information to provide a complete description of the cfDNA molecule's topological information or characteristics, which can provide information related to the onset, progression, diagnosis, and treatment of various diseases, particularly cancer. This lack of information not only hinders our understanding of disease progression but also impedes our ability to utilize this information to improve human health (e.g., early detection of diseases such as cancer, any association with treatment efficacy, etc.).

[0089] The methods and systems described herein provide for the generation and analysis of sequencing libraries that capture 5' and / or 3' overhangs, including the length and / or sequence of the natural ends of DNA double-stranded molecules. In particular, the methods and systems described herein can also generate and analyze sequence libraries of cfDNA collected from patients with early-stage cancer or suspected cancer. Therefore, the methods and systems provide solutions to the limitations of other methods for analyzing cfDNA, enabling better capture and utilization of the information present in cfDNA molecules.

[0090] Therefore, in one aspect, a method for determining the topological structure of cell-free DNA includes: extending the 3' end of the first strand of a cell-free DNA duplex with inosine bases to fill the 5' overhang of the second strand of the cell-free DNA duplex; ligating a sequencing adaptor to the cell-free DNA duplex; sequencing the first strand of the cell-free DNA duplex to produce a first sequence readout; determining one or more bases to be soft-cut in the first sequence readout using one or more processors; soft-cutting the bases corresponding to the 3' inosine extension of the first strand of the cell-free DNA duplex from the first sequence readout using one or more processors; and determining the length or sequence of the 5' overhang of the second strand of the cell-free DNA duplex based on the soft-cutting of the 3' inosine extension of the first strand of the cell-free DNA duplex using one or more processors. The method may further include: linking a second sequencing adaptor to the 3' overhang of the second strand of a cell-free DNA duplex; extending the 3' end of the second sequencing adaptor with inosine bases to fill the 3' overhang of the second strand of the cell-free DNA duplex; linking the 3' inosine-extended end of the second sequencing adaptor to the 5' end of the first strand of the cell-free DNA duplex, wherein the linked 3' inosine-extended end provides the 5' inosine extension of the first strand of the cell-free DNA duplex; determining, by one or more processors, one or more bases to be soft-cut and linked to the 5' end of the first sequence readout; soft-cutting, by one or more processors, the bases corresponding to the 5' inosine extension of the first strand of the cell-free DNA duplex from the first sequence readout; and determining, by one or more processors, the length or sequence of the 3' overhang of the second strand of the cell-free DNA duplex based on the soft-cutting of the 5' inosine extension of the first strand of the cell-free DNA duplex. In some embodiments, the method further includes detecting the presence or absence of a disease (e.g., cancer) based on the length or sequence of the 5' overhangs of the first and / or second strands of the cell-free DNA duplex using one or more processors. In some embodiments, the method further includes detecting the presence or absence of a disease (e.g., cancer) based on the length or sequence of the 3' overhangs of the first and / or second strands of the cell-free DNA duplex using one or more processors.

[0091] In another aspect, there is a method for preparing a sequencing construct, which includes linking a sequencing adaptor to the 3' overhang of the first strand of a cell-free DNA duplex; extending the 3' end of the sequencing adaptor with inosine bases to fill the 3' overhang of the first strand of the cell-free DNA duplex; and linking the 3' inosine-extended end of the sequencing adaptor to the 5' end of the second strand of the cell-free DNA duplex, wherein the linked 3' inosine-extended end provides a 5' inosine extension of the second strand of the cell-free DNA duplex. The method may further include analyzing the sequencing construct to determine the topology of the cell-free DNA duplex, for example, by sequencing the second strand of the cell-free DNA duplex to generate a first sequence readout; determining one or more bases in the first sequence readout to be soft-cut by one or more processors; soft-cutting bases in the first sequence readout corresponding to the 5' inosine extension of the second strand of the cell-free DNA duplex by one or more processors; and determining the length or sequence of the 3' overhang of the first strand of the cell-free DNA duplex based on the soft cutting of the 5' inosine extension of the second strand of the cell-free DNA duplex by one or more processors. In some embodiments, the method further includes detecting the presence or absence of a disease (e.g., cancer) by one or more processors based on the length or sequence of the 5' overhang of the first and / or second strands of the cell-free DNA duplex. In some embodiments, the method further includes detecting the presence or absence of a disease (e.g., cancer) by one or more processors based on the length or sequence of the 3' overhang of the first and / or second strands of the cell-free DNA duplex.

[0092] Systems and computer-readable storage media for carrying out the methods described herein are also described.

[0093] definition

[0094] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0095] Unless the context clearly indicates otherwise, nouns without quantifiers as used in this specification and the appended claims mean one / more / a. Unless otherwise stated, any reference to "or" herein is intended to cover "and / or / or".

[0096] "About" and "approximately" should generally be interpreted as an acceptable degree of error for the quantity being measured, taking into account the nature or precision of the measurement. An exemplary degree of error is within 20 percent (%) of a given value or range of values, typically within 10 percent, and more usually within 5 percent.

[0097] As used herein, the terms “comprising” (and any form or variation thereof), “having” (and any form or variation thereof), “including” (and any form or variation thereof), or “containing” (and any form or variation thereof) are inclusive or open-ended and do not exclude additional, undocumented additives, components, integers, elements, or method steps.

[0098] The terms “individual,” “patient,” or “object” as used herein are used interchangeably and refer to any single animal for which treatment is desired, such as a mammal (including, for example, non-human animals such as dogs, cats, horses, rabbits, zoo animals, cattle, pigs, sheep, and non-human primates). In some specific embodiments, the individual, patient, or object referred to herein is a human being.

[0099] The terms “cancer” and “tumor” are used interchangeably in this article. These terms refer to cells that possess the hallmarks of cancerous cells, such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, and the presence of certain characteristic morphological features. Cancer cells typically take the form of tumors, but such cells can exist alone in animals or can be non-tumorigenic cancer cells, such as leukemia cells. These terms include solid tumors, soft tissue tumors, or metastatic lesions. The term “cancer” as used in this article includes both premalignant and malignant cancers.

[0100] As used in this article, “treatment” (and its grammatical variations) refers to a clinical intervention (e.g., administration of anticancer agents or anticancer therapy) that attempts to alter the natural course of the individual being treated. The desired effects of treatment include, but are not limited to, preventing disease recurrence, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, improving or mitigating the disease state, and alleviating or improving prognosis.

[0101] Where a range of values ​​is provided, it should be understood that every intermediate value between the upper and lower limits of the range, as well as any other specified value or intermediate value within the specified range, is included within the scope of this disclosure. Where the specified range includes an upper or lower limit, the range excluding any of the included limits is also included in this disclosure.

[0102] The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter. This description is presented to enable those skilled in the art to make and use the invention, and is provided in the context of the patent application and its claims. Various modifications to the described embodiments will be apparent to those skilled in the art, and the general principles herein can be applied to other embodiments. Therefore, the invention is not intended to be limited to the illustrated embodiments, but rather to be consistent with the widest scope consistent with the principles and features set forth herein.

[0103] Figure 1 to Figure 7 Processes and systems with various implementations are illustrated. In some exemplary processes, modules may optionally be combined, the order of some modules may optionally be changed, and some modules may optionally be omitted. In some instances, additional steps may be performed in combination with the exemplary processes. Therefore, the operations shown (and described in more detail below) are exemplary in nature and should not be considered limiting.

[0104] All publications, patents, and patent applications mentioned herein are incorporated herein by reference in their entirety. In the event of any conflict between any incorporated references and this disclosure, this disclosure shall prevail.

[0105] Methods for determining the topology of protruding ends

[0106] This document provides methods for generating sequencing constructs for DNA duplexes (e.g., cfDNA molecules) and analyzing said sequencing constructs to determine the topological structure of the DNA duplex molecule. For example, the ends of the cfDNA may include 5' and / or 3' overhangs, and the length and / or sequence of such overhangs can be determined according to the methods described herein. For example, the method may include filling overhang vacancies with nucleotides or analogues thereof (e.g., by using inosine nucleotides (e.g., deoxyinosine)). An adaptor can then be attached to the ends of the cfDNA duplex molecule, and the first and / or second strands can be sequenced, processed (e.g., soft-cleaving inosine bases), and the cfDNA topology, including the length and sequence of the 5' and / or 3' overhangs, can be analyzed.

[0107] As further described herein, the method may include generating sequencing constructs for DNA duplex molecules (e.g., cfDNA). These sequencing constructs may be prepared to analyze 5' overhangs (e.g., to determine length and / or sequence), 3' overhangs (e.g., to determine length and / or sequence), or both 5' and 3' overhangs. Some embodiments are described with reference to single DNA duplex molecules (e.g., single cfDNA duplexes), but it should be understood that the method can be applied to constructing sequencing libraries in which multiple DNA duplex molecules, which may have different topologies, can be prepared and / or analyzed in parallel.

[0108] In some cases, one or both strands of the cfDNA duplex may contain 5' and / or 3' overhangs. Sequencing construct generation and analysis methods utilize nucleic acid analogs (e.g., deoxyinosine (dI)) to fill 5' and / or 3' overhang vacancies, thereby preserving overhang sequence and length data for further downstream applications. Deoxyinosine may be incorporated or added during elongation by a polymerase, including but not limited to any standard or high-fidelity formulation of: Taq polymerase and its variants (e.g., LongAmp Taq, etc.). Various DNA polymerases can be used, including Taq, Hemo Klen Taq, OneTaq, Bst DNA polymerase, Bsu DNA polymerase, Phi29 DNA polymerase, and T7 DNA polymerase. Other polymerases, such as Pfu, KOD, or Tth DNA polymerase, can also be used.

[0109] Additional enzymes can be used to incorporate the sequencing adaptor to each end of the cfDNA duplex. For example, in some embodiments, a ligase can be used to ligate the sequencing adaptor to the cfDNA end via enzymatic ligation. In some embodiments, a terminal transferase (e.g., terminal deoxynucleotidyl transferase) can be used to extend the 3' overhang of the cfDNA molecule to produce a nucleotide tail with a known sequence that pairs with the adaptor. In some embodiments, the adaptor contains a sequence complementary to the extended sequence produced by the terminal transferase at the 3' overhang of the cfDNA molecule. The terminal transferase can ligate one or more (e.g., multiple nucleotide bases) to the 3' overhang of the cfDNA duplex molecule. The ligated bases can be, for example, typical bases (e.g., A, C, T, or G). In some embodiments, the ligated bases belong to the same nucleotide base type. The adaptor may contain a sequencing primer binding site, and in some embodiments may also contain a sample index sequence and / or a UMI sequence. Once the adaptor is attached to the 5' and 3' ends of the cfDNA duplex, the first and / or second strands of the cfDNA duplex can be sequenced to produce a first readout and / or a second readout.

[0110] Once a sequencing construct or sequencing library has been prepared, one or both strands of a DNA duplex molecule can be sequenced to produce one or more sequence reads. Sequencing can be performed by any suitable method. In some embodiments of this method, sequencing is performed by next-generation sequencing. As used herein, “next-generation sequencing” (or “NGS”) may also be referred to as “massively parallel sequencing” (or “MPS”) and refers to any sequencing method that determines the nucleotide sequence of an individual nucleic acid molecule (e.g., as in single-molecule sequencing) or a clonal amplification proxy for an individual nucleic acid molecule in a high-throughput manner. Next-generation sequencing methods are known in the art and are described, for example, in Metzker, M. (2010) Nature Biotechnology Reviews 11:31-46, which is incorporated herein by reference. Other examples of sequencing methods suitable for use in implementing the methods and systems disclosed herein are described, for example, in International Patent Application Publication No. WO 2012 / 092426. In some embodiments, sequencing may include, for example, whole genome sequencing (WGS), whole exome sequencing, targeted sequencing, or direct sequencing. In some implementations, sequencing may be performed using, for example, Sanger sequencing. In some cases, sequencing may include paired-end sequencing technology, which allows sequencing of both ends of a fragment and produces high-quality, mappable sequence data for use in detecting, for example, cfDNA 5' and / or 3' overhang sequences.

[0111] The disclosed methods and systems can be implemented using sequencing platforms such as Roche 454, Illumina Solexa, ABI-SOLiD, ION Torrent, Complete Genomics, Pacific Bioscience, Helicos, and / or Polonator. In some embodiments, sequencing may include Illumina MiSeq sequencing. In some embodiments, sequencing may include Illumina HiSeq sequencing. In some embodiments, sequencing may include Illumina NovaSeq sequencing. Optimized methods for sequencing nucleic acids extracted from samples are described in more detail in, for example, International Patent Application Publication No. WO 2020 / 236941, the entire contents of which are incorporated herein by reference.

[0112] Incorporating dI to fill overhang vacancies provides a clear boundary in each sequence readout—where the cfDNA sequence ends and the dI-filled sequence begins at the overhang vacancy. During amplification (e.g., PCR amplification), dI is converted into one or more nucleotides (e.g., A, T, G, C). During alignment, the nucleotides representing the serrated ends (dI-filled) will not be aligned (mapped) to the reference genomic sequence, and thus will identify where the original cfDNA sequence (e.g., fragment) ends and where the dI-filled sequence begins. dI incorporation also results in erroneous nucleotide pairings, which can be identified via sequence alignment. Calculating the removed dI sequence via soft shearing provides information about the length of the 5' and / or 3' overhang sequences for each cfDNA molecule. When combined with sequence alignment, the sequences of the 5' and / or 3' overhangs can be determined, as described further in detail below.

[0113] Sequencing readouts can be analyzed to identify one or more bases in the readout to be soft-spliced. As further described herein, the method may include incorporation of inosine bases during the preparation of the sequencing construct. Soft shearing can be based on the presence of inosine extension, which indicates a protrusion on the complementary strand. Therefore, the length of a protrusion in a strand can be determined based on the soft shearing of one or more inosine extensions in the complementary strand. Given the length of the protrusion and the sequence adjacent to the protrusion, the sequence of the protrusion itself can be determined.

[0114] Identifying one or more bases to be soft-sliced ​​in a sequence readout may involve aligning the readout with a reference sequence or a complementary strand sequence. The complementary strand can be identified using standard molecular barcode (i.e., unique molecular identifier (UMI)) techniques. These UMI techniques can be exogenous unique identifiers or non-unique identifiers; for example, the exogenous barcode added to the molecule is not unique, but after alignment, other molecular characteristics identified from the readout (e.g., coordinates, a portion of the endogenous sequence, etc.) are combined with the non-unique barcode to form the UMI.

[0115] Alignment is the process of matching a query sequence readout with one or more other sequence readouts or a reference sequence. Alignment may also include mapping the sequence readout, such as mapping it to a location within the reference sequence, such as a genomic location or locus. In some embodiments, the sequence readout may be aligned with a known reference sequence (e.g., a wild-type sequence). In some embodiments, the reference sequence may be obtained from a database of human genomes (e.g., the HG19 human reference genome) or cancer mutations (e.g., COSMIC). Sequence alignment methods for sequence readouts are described, for example, in Trapnell, C. and Salzberg, SL Nature Biotech., 2009, 27:455-457. Optimizations of sequence alignment are described in the art, for example, as described in International Patent Application Publication No. WO 2012 / 092426. Further descriptions of sequence alignment methods are provided, for example, in International Patent Application Publication No. WO 2020 / 236941, the entire contents of which are incorporated herein by reference.

[0116] In some embodiments, the methods and systems disclosed herein may integrate multiple individually tuned alignment methods or algorithms to optimize base calling performance in sequencing methods, particularly those relying on massively parallel sequencing (MPS). In some embodiments, the disclosed methods and systems may include the use of one or more global alignment algorithms. In some embodiments, the disclosed methods and systems may include the use of one or more local alignment algorithms. Some examples of alignment algorithms that can be used include, but are not limited to, the Burrows-Wheeler Alignment (BWA) software package (see, for example, Li, et al. (2009), “Fast and Accurate Short Read Alignment with Burrows-Wheeler Transform”, Bioinformatics 25:1754-60; Li, et al. (2010), “Fast and Accurate Long-Read Alignment with Burrows-Wheeler Transform”, Bioinformatics sepub.PMID:20080505), the Smith-Waterman algorithm (see, for example, Smith, et al. (1981), “Identification of Common Molecular Subsequences”, J. Molecular Biology 147(1):195–197), and the Striped Smith-Waterman algorithm (see, for example, Farrar (2007), “Striped Smith–Waterman Speeds Database Searches Six Times Over Other SIMD Implementations, Bioinformatics 23(2):156-161), Needleman-Wunsch algorithm (Needleman, et al. (1970) "A General Method Applicable to the Search for Similarities in the Amino Acid Sequence of Two Proteins", J. Molecular Biology 48(3):443–53), or any combination thereof.

[0117] The disclosed methods and systems can be implemented using soft-splitting analysis. The first and / or second sequences can be aligned with a reference sequence, or the first sequence can be aligned with the second readout, and vice versa. 5' and / or 3' overhang sequences with inosine-filled gaps will not align with the reference sequence, and therefore these inosine-filled gaps are computationally pruned from the first and / or second readouts of the first and / or second sequences of the cfDNA molecule. The lengths of the 5' and / or 3' overhangs can be calculated based on the number of misaligned inosine bases that have been soft-splitted. This information can then be further processed to identify additional DNA topological features (including 5' and / or 3' overhang sequences) and to analyze their association with diseases (e.g., cancer).

[0118] Figure 1A Exemplary methods for determining the topology of cell-free DNA (e.g., for determining the length of the 5' overhang of a cell-free DNA duplex) according to some embodiments are shown. Figure 1A As shown at position 102, the method includes extending the 3' end of the first strand of the cell-free DNA duplex with inosine bases to fill the 5' overhang of the second strand of the cell-free DNA duplex. In some embodiments, inosine is deoxyinosine (dI). At position 104, the method includes ligating a sequencing adaptor to the cell-free DNA duplex. In some embodiments, ligating the sequencing adaptor includes linking the sequencing adaptor to the cell-free DNA duplex. The sequencing adaptor may be, for example, a Y-shaped sequencing adaptor. In some embodiments, the adaptor may be a full-length Y-adaptor (e.g., a Y-shaped adaptor containing an index sequence), a short-stalk adaptor, or a hairpin adaptor. In some embodiments, preparing the sequencing construct may include amplifying the first and second strands of the cell-free DNA duplex, for example, prior to sequencing. The amplification process may allow the sample index to be ligated to the cell-free DNA duplex. However, in some embodiments, the sample index is included in the sequencing adaptor, so that the sample index does not need to be incorporated into the downstream amplification process. In some implementations, amplification includes polymerase chain reaction (PCR) amplification, non-PCR amplification, or isothermal amplification.

[0119] At position 106, the method includes sequencing the first strand of a cell-free DNA double-strand to produce a first sequence readout. In some embodiments, sequencing includes next-generation sequencing (“NGS”). In some embodiments, sequencing includes paired-end sequencing. In some embodiments, the sequencing adaptor or second sequencing adaptor contains an amplification primer binding site, a flow cell adaptor sequence, or a basement adaptor sequence. In some embodiments, sequencing includes using massively parallel sequencing (MPS), whole-genome sequencing (WGS), whole-exome sequencing, targeted sequencing, direct sequencing, or Sanger sequencing. In some embodiments, sequencing includes massively parallel sequencing, and the massively parallel sequencing technology includes next-generation sequencing (NGS). In some embodiments, sequencing is performed using a next-generation sequencer.

[0120] exist Figure 1A At position 108, the method further includes determining one or more bases in the first sequence readout to be soft-cleaved by one or more processors. Determining one or more bases in the first sequence readout to be soft-cleaved may include, for example, comparing the first sequence readout with a reference sequence and identifying unaligned portions of the first sequence readout. Unaligned portions of the first sequence readout may be associated with inosine bases in the extended first strand. Because these inosine bases are artificially generated, they cannot be aligned with a reference sequence or complementary strand, and therefore indicate protrusions in the complementary strand. Therefore, these unaligned bases in the 3' portion of the sequence readout can be calculably identified for soft cleaving.

[0121] In some embodiments, the method includes sequencing the second strand of a cell-free DNA duplex to produce a second sequence readout. One or more bases in the first sequence readout to be soft-cut can be determined by aligning the first sequence (i.e., for the first strand) with the second sequence readout (i.e., for the second strand) and identifying unaligned portions of the first sequence readout. Matching the first and second sequence readouts may include using a common UMI between the first and second sequence readouts. Unaligned 3' portions of the sequence readout can be identified for soft cutting.

[0122] exist Figure 1A At position 110, the method includes reading from the first sequence by one or more processors soft-splitting bases corresponding to the 3' inosine extension of the first strand of the cell-free DNA duplex. At position 112, the method includes determining the length or sequence of the 5' overhang of the second strand of the cell-free DNA duplex based on soft-splitting of the 3' inosine extension of the first strand of the cell-free DNA duplex by one or more processors.

[0123] Figure 1B It shows Figure 1AAn exemplary implementation of the process described herein. Figure 1B In the example shown, the double-stranded DNA duplex includes 5' overhangs on both the top and bottom strands, although the method can be performed with 5' overhangs on only one strand or both strands. In the example shown at 114, the 3' ends of the top and bottom strands are extended using an inosine base (e.g., deoxyinosine) to fill the 5' overhangs. Optionally, a polymerase capable of linking a single nucleotide (e.g., an inosine base) to the 3' end (e.g., a blunt end) of the DNA duplex molecule can be used to produce a single 3' nucleotide overhang (e.g., a single 3' inosine overhang), such as... Figure 1B As shown at 116. The polymerase can be, for example, Taq polymerase. In another embodiment, a terminal transferase can be used to ligate one or more (e.g., multiple) nucleotide bases to the 3' overhang of the cfDNA duplex molecule. The ligated bases can be, for example, typical bases (e.g., A, C, T, or G). The 3' overhang can improve the ligation efficiency of the sequencing adaptor, which may contain a 3' overhang complementary to the 3' overhang of the cfDNA molecule. In some embodiments, the ligated bases belong to the same nucleotide base type. The sequencing adaptor can then be ligated to the DNA duplex molecule at 118. Optionally, if a tail is included at the 3' end, the adaptor may contain a 3' overhang complementary to the tail. For example, if a single inosine tail is included at the 3' end, the adaptor may contain a 3' overhang containing a cytosine base, such as... Figure 1B As shown in the illustration. This exemplary embodiment further illustrates an optional step involving the amplification of the first strand of a cell-free DNA duplex, such as by polymerase chain reaction (PCR). In this example, the amplification process adds a sample index to both the first and second strands of the cell-free DNA duplex. In some embodiments, the sequencing adaptor contains a unique molecular identifier (UMI) that allows pairing of the first strand (top) and the second strand (bottom).

[0124] Once the sequencing construct is prepared, such as Figure 1BAs shown in the top section, nucleic acid molecules can be sequenced at position 122 to provide sequencing readouts. Optionally, the sequencing construct can be amplified prior to sequencing, as shown at position 120, for example by PCR, which allows for the incorporation of index sequences, such as sample indexes. In the exemplary embodiment shown, the sequencing readout is aligned at position 124, for example, with a reference sequence. In another example, the top and bottom strands of the same DNA duplex molecule are aligned with each other. Alignment may include the use of a unique molecular identifier (which may be included, for example, in a sequencing adaptor) that allows the top and bottom strands to match each other. Portions in the sequence readout that cannot be aligned with the reference sequence or complementary strand can be identified for soft splicing, as shown at position 126. That is, bases that cannot be aligned with the reference sequence or complementary strand can be identified as inosine bases used for artificially extending the 3' end. Soft-spliced ​​bases can be used to determine the length of the 5' overhang in the complementary strand, since inosine bases are used to fill the 5' overhang.

[0125] In some embodiments, a method for determining the topology of cell-free DNA is provided, comprising: extending the 3' end of a first strand of a cell-free DNA duplex with inosine bases to fill the 5' overhang of a second strand of the cell-free DNA duplex; ligating a sequencing adaptor to the cell-free DNA duplex; sequencing the first strand of the cell-free DNA duplex to produce a first sequence readout; determining, by one or more processors, one or more bases in the first sequence readout to be soft-spliced; soft-splicing, by one or more processors, the bases in the first sequence readout corresponding to the 3' inosine extension of the first strand of the cell-free DNA duplex; and determining, by one or more processors, the length or sequence of the 5' overhang of the second strand of the cell-free DNA duplex based on the soft-splicing of the 3' inosine extension of the first strand of the cell-free DNA duplex. In some embodiments, extending the 3' end of the first strand of the cell-free DNA duplex includes forming a single 3' inosine overhang. In some implementations, the sequencing adaptor includes a 3' nucleotide overhang (e.g., a cytosine overhang) that is complementary to a single 3' inosine overhang.

[0126] In some embodiments, the method further includes detecting the presence or absence of a disease based on the length or sequence of the 5' overhang of the second strand of the cell-free DNA duplex using one or more processors. In some embodiments, the disease is cancer.

[0127] Alternatively or as an alternative, the methods described herein may include preparing sequencing constructs for analyzing the 3' overhangs of DNA molecules, such as... Figure 2AAs shown at 202, the method includes attaching a sequencing adaptor (e.g., by ligating) to the 3' overhang of the first strand of a cell-free DNA duplex. In some embodiments, the sequencing adaptor is directly attached to the 3' overhang of the first strand. In other embodiments, the 3' overhang of the first strand may be extended by one or more bases (i.e., providing a 3' extension or tail). The sequencing adaptor may contain a 3' overhang complementary to the 3' extension of the first strand of the cell-free DNA duplex. Including an overhang complementary to the extension or tail attached to the 3' overhang in the adaptor improves the efficiency of the ligation reaction. The 3' extension or tail may be, for example, a single inosine. Inosine is an analog of guanine and therefore preferentially pairs with cytosine bases (although it may also pair with other typical nucleotide bases (i.e., adenine, thymine, or guanine)). If a single inosine tail is included at the 3' end, the adaptor may contain, for example, a 3' overhang containing a cytosine base complementary to the inosine base.

[0128] In some implementations, one or more enzymes with terminal transferase and ligase activities are used to ligate the sequencing adaptor to the 3' overhang of the first strand of the cell-free DNA duplex, wherein the enzyme extends the 3' overhang and ligates the sequencing adaptor to the extended 3' overhang. For example, xGen can be used. TM Adaptase TM The process is performed using a module containing enzymes with terminal transferase and ligase activities. The terminal transferase produces a 3' tail, which may be referred to as an "adaptase tail" or "AdT". In some embodiments, the 3' tail provided by the terminal transferase activity contains multiple bases of the same base type. The sequencing adaptor contains a 3' overhang complementary to the 3' overhang of the DNA duplex molecule. This process is carried out in... Figure 3C This is further illustrated in the text.

[0129] exist Figure 2A At position 204, the method further includes extending the 3' end of the sequencing adaptor with inosine bases to fill the 3' overhang of the first strand of the cell-free DNA duplex. In some embodiments, inosine is deoxyinosine (dI). At position 206, the 3' inosine-extended end of the sequencing adaptor is attached (e.g., via ligation) to the 5' end of the second strand of the cell-free DNA duplex. This results in the attached 3' inosine-extended end of the sequencing adaptor providing a 5' inosine extension of the second strand of the cell-free DNA duplex. That is, the inosine extension is located between the sequencing adaptor and the original second strand of the DNA duplex molecule.

[0130] Figure 2BAn exemplary method for determining the topological structure of cell-free DNA (e.g., for determining the length of the 3' overhang of a cell-free DNA duplex) is shown, the method being based on Figure 2A The method for preparing the sequencing construct is shown above. At 208, the second strand of the cell-free DNA double helix is ​​sequenced to produce a first sequence readout. In some embodiments, sequencing includes next-generation sequencing (“NGS”). In some embodiments, sequencing includes paired-end sequencing. In some embodiments, the sequencing adaptor or second sequencing adaptor contains an amplification primer binding site, a flow cell adaptor sequence, or a base adaptor sequence. In some embodiments, sequencing includes using massively parallel sequencing (MPS), whole-genome sequencing (WGS), whole-exome sequencing, targeted sequencing, direct sequencing, or Sanger sequencing. In some embodiments, sequencing includes massively parallel sequencing, and the massively parallel sequencing technology includes next-generation sequencing (NGS). In some embodiments, sequencing is performed using a next-generation sequencer.

[0131] exist Figure 2B At position 210, the method further includes determining one or more bases in the first sequence readout to be soft-cleaved by one or more processors. Determining one or more bases in the first sequence readout to be soft-cleaved may include, for example, comparing the first sequence readout with a reference sequence and identifying unaligned portions of the first sequence readout. Unaligned portions of the first sequence readout may be associated with inosine bases in the extended first strand. Because these inosine bases are artificially generated, they cannot be aligned with a reference sequence or the complementary strand, and therefore indicate protrusions in the complementary strand. Thus, these unaligned bases in the 5' portion of the sequence readout can be identified for soft cleaving.

[0132] In some embodiments, the method includes sequencing the second strand of a cell-free DNA duplex to produce a second sequence readout. One or more bases in the first sequence readout to be soft-cut can be determined by aligning the first sequence (i.e., for the first strand) with the second sequence readout (i.e., for the second strand) and identifying unaligned portions of the first sequence readout. Matching the first and second sequence readouts may include using a common UMI between the first and second sequence readouts. Unaligned 5' portions of the sequence readout can be identified for soft cutting.

[0133] exist Figure 2BAt position 212, the method includes soft-cleaving bases from sequence readout using one or more processors, specifically soft-cleaving bases from the first sequence readout corresponding to the 5' inosine extension of the second strand of the cell-free DNA duplex. At position 214, the method includes determining the length of the 3' overhang of the first strand of the cell-free DNA duplex based on soft-cleaving of the 5' inosine extension of the second strand of the cell-free DNA duplex using one or more processors. The sequence of the 3' overhang can be determined based on the length of the 3' overhang of the first strand of the cell-free DNA duplex and the sequence of the first strand of the cell-free DNA.

[0134] In some embodiments, a method for preparing a sequencing construct is provided, comprising: ligating a sequencing adaptor to a 3' overhang of a first strand of a cell-free DNA duplex; extending the 3' end of the sequencing adaptor with inosine bases to fill the 3' overhang of the first strand of the cell-free DNA duplex; and ligating the 3' inosine-extended end of the sequencing adaptor to a 5' end of a second strand of the cell-free DNA duplex, wherein the ligated 3' inosine-extended end provides a 5' inosine extension of the second strand of the cell-free DNA duplex. In some embodiments, ligating the sequencing adaptor to the 3' overhang of the first strand of the cell-free DNA duplex comprises: extending the 3' overhang of the first strand of the cell-free DNA duplex to provide a 3' extension, wherein the sequencing adaptor includes a 3' overhang complementary to the 3' extension of the first strand of the cell-free DNA duplex; and ligating the sequencing adaptor to the 3' extension of the first strand of the cell-free DNA duplex. In some embodiments, the 3' overhang of the first strand of the cell-free DNA duplex is extended using nucleotide bases of the same base type.

[0135] In some embodiments, a method for determining the topological structure of cell-free DNA is provided, comprising: preparing a sequencing construct as described herein; sequencing the second strand of a cell-free DNA duplex to produce a first sequence readout; determining, by one or more processors, one or more bases in the first sequence readout to be soft-cut; soft-cutting, by one or more processors, bases in the first sequence readout corresponding to the 5' inosine extension of the second strand of the cell-free DNA duplex ...

[0136] In some embodiments, the method includes: ligating a second sequencing adaptor to a 3' overhang of a second strand of a cell-free DNA duplex; extending the 3' end of the second sequencing adaptor with inosine bases to fill the 3' overhang of the second strand of the cell-free DNA duplex; and ligating the 3' inosine-extended end of the second sequencing adaptor to a 5' end of a first strand of the cell-free DNA duplex, wherein the 3' inosine-extended end provides a 5' inosine extension of the first strand of the cell-free DNA duplex. In some embodiments, the method further includes sequencing the first strand of the cell-free DNA duplex to produce a second sequence readout; determining, by one or more processors, one or more bases to be soft-spliced ​​and ligated to the 5' end of the second sequence readout; soft-splicing, by one or more processors, the bases corresponding to the 5' inosine extension of the first strand of the cell-free DNA duplex from the second sequence readout; and determining, by one or more processors, the length or sequence of the 3' overhang of the second strand of the cell-free DNA duplex based on the soft-splicing of the 5' inosine extension of the first strand of the cell-free DNA duplex. In some embodiments, linking the second sequencing adaptor to the 3' overhang of the second strand of the cell-free DNA duplex includes linking a single inosine to the 3' overhang of the second strand of the cell-free DNA duplex, and linking the second sequencing adaptor to inosine. In some embodiments, the sequencing adaptor includes a 3' cytosine overhang complementary to the single inosine linked to the 3' overhang of the second strand of the cell-free DNA duplex.

[0137] In some embodiments, the method includes amplifying the first and second strands of the cell-free DNA duplex as described above. In some embodiments, the method includes sequencing as described above. In some embodiments, determining one or more bases to be soft-cleaved in the first sequence readout includes aligning the first and / or sequence readouts as described above.

[0138] In some embodiments, the method further includes detecting the presence or absence of a disease based on the length or sequence of the 3' overhang of the first strand of the cell-free DNA duplex using one or more processors. In some embodiments, the method further includes detecting the presence or absence of a disease based on the length or sequence of the 3' overhang of the second strand of the cell-free DNA duplex using one or more processors. In some embodiments, the disease is cancer.

[0139] In some cases, DNA double-stranded molecules contain both 5' and 3' overhangs. The methods presented in this paper can be used to generate sequencing constructs and analyze such constructs to determine the topological structure of both the 5' and 3' overhangs of the DNA double-stranded molecules. Figure 3AA method is illustrated for determining the topology of a DNA duplex (e.g., cfDNA), such as for determining the 3' and 5' overhang lengths of a DNA duplex molecule (e.g., a cell-free DNA duplex molecule). As shown at 302, the method includes extending the 3' end of the first strand of the DNA duplex with an inosine base to fill the 5' overhang of the second strand of the cell-free DNA duplex. In some embodiments, inosine is deoxyinosine (dI). The 3' end of the first strand of the DNA duplex molecule can be extended using a polymerase. Optionally, a polymerase capable of linking a single nucleotide (e.g., an inosine base) to the 3' end (e.g., a blunt end) of the DNA duplex molecule can be used to generate a single 3' nucleotide overhang (e.g., a single 3' inosine overhang). The polymerase can be, for example, Taq polymerase. In another embodiment, a terminal transferase can be used to link one or more (e.g., multiple) nucleotide bases to the 3' overhang end of the cfDNA duplex molecule. The linked bases can be, for example, typical bases (e.g., A, C, T, or G). A 3' overhang can improve the ligation efficiency of the sequencing adaptor, which may contain a 3' overhang complementary to the 3' overhang of the cfDNA molecule. In some embodiments, the linked bases belong to the same nucleotide base type.

[0140] At 304, the method includes ligating a first sequencing adaptor and a second sequencing adaptor to a DNA duplex molecule. In some embodiments, ligating the sequencing adaptor includes ligating the sequencing adaptor to a cell-free DNA duplex. The sequencing adaptor may be, for example, a Y-shaped sequencing adaptor. In some embodiments, the adaptor may be a full-length Y-adaptor (e.g., a Y-shaped adaptor containing an index sequence), a short-stalked adaptor, or a hairpin adaptor. Figure 3B and Figure 3C As shown, the first sequencing adaptor can be ligated to the end of a DNA double-stranded molecule having a 5' overhang. The first and sequence sequencing adaptors can be used in the same reaction (e.g., as shown in...). Figure 3B (as shown) or in a sequential reaction (e.g., as shown) Figure 3B (As shown in the image) is linked to the DNA double-stranded molecule. Figure 3BIn the exemplary process shown, the first sequencing adaptor contains a 3' overhang complementary to the 3' tail on the first strand of the DNA duplex molecule. Similarly, the second sequencing adaptor contains a 3' overhang complementary to the 3' tail on the second strand of the DNA duplex molecule. As described above, a single 3' nucleotide overhang (e.g., a single 3' inosine overhang) can be generated using a polymerase capable of linking a single nucleotide (e.g., an inosine base) to the 3' end (e.g., a blunt end) of the DNA duplex molecule. Inosine is an analogue of guanine and therefore preferentially pairs with cytosine bases (although it can pair with other typical nucleotide bases (i.e., adenine, thymine, or guanine)). For example, if the inosine tail is contained at the 3' end, then the adaptor may contain a 3' overhang containing a single nucleotide (e.g., a cytosine base) to preferentially complement the inosine base. The polymerase may be, for example, Taq polymerase. In another embodiment, a terminal transferase may be used to ligate one or more (e.g., multiple) nucleotide bases to the 3' overhang of the cfDNA duplex molecule. The ligated base may be, for example, a typical base (e.g., A, C, T, or G). The 3' overhang can improve the ligation efficiency of the sequencing adaptor, which may contain a 3' overhang complementary to the 3' overhang of the cfDNA molecule. In some embodiments, the ligated bases belong to the same nucleotide base type.

[0141] In some embodiments, the first and second sequencing adaptors are sequentially ligated to the DNA duplex molecule, for example, the second sequencing adaptor is ligated to the DNA duplex molecule after the first sequencing adaptor is ligated to the DNA duplex molecule. The first sequencing adaptor can be ligated as described above. To ligate the second sequencing adaptor, an enzyme with a terminal transferase can be used to extend the 3' overhang of the second strand of the DNA molecule, such as... Figure 3C As shown in the image. For example, this can be achieved using xGen. TM Adaptase TM The sequencing adaptor is performed using a module containing enzymes with terminal transferase and ligase activities. The terminal transferase produces a 3' tail, which may be referred to as a "ligase tail" or "AdT". In some embodiments, the 3' tail provided by the terminal transferase activity contains multiple bases of the same base type. The sequencing adaptor contains a 3' overhang that is complementary to the 3' bases of the 3' overhangs of the DNA duplex molecule.

[0142] exist Figure 3AAt position 306, the method further includes extending the 3' end of the sequencing adaptor with inosine bases to fill the 3' overhang of the first strand of the cell-free DNA duplex. In some embodiments, inosine is deoxyinosine (dI). At position 308, the 3' inosine-extended end of the sequencing adaptor is attached to the 5' end of the second strand of the cell-free DNA duplex (e.g., by ligation). This results in the attached 3' inosine-extended end of the sequencing adaptor providing a 5' inosine extension of the second strand of the cell-free DNA duplex. That is, the inosine extension is located between the sequencing adaptor and the original second strand of the DNA duplex molecule.

[0143] In some implementations, the sequencing construct is amplified, for example, prior to sequencing. The amplification process allows the sample index to be ligated to the cell-free DNA duplex. However, in some implementations, the sample index is included in the sequencing adaptor, eliminating the need to incorporate the sample index during downstream amplification. In some implementations, amplification includes polymerase chain reaction (PCR) amplification, non-PCR amplification, or isothermal amplification.

[0144] The first strand of the DNA duplex molecule is sequenced at position 310 to produce a first sequence readout. In some embodiments, both the first and second strands of the DNA duplex molecule are sequenced to produce a first and a second sequence readout, respectively. In some embodiments, sequencing includes next-generation sequencing (“NGS”). In some embodiments, sequencing includes paired-end sequencing. In some embodiments, the sequencing adaptor or second sequencing adaptor contains an amplification primer binding site, a flow cell adaptor sequence, or a basement adaptor sequence. In some embodiments, sequencing includes using massively parallel sequencing (MPS), whole-genome sequencing (WGS), whole-exome sequencing, targeted sequencing, direct sequencing, or Sanger sequencing. In some embodiments, sequencing includes massively parallel sequencing, and the massively parallel sequencing technology includes next-generation sequencing (NGS). In some embodiments, sequencing is performed using a next-generation sequencer.

[0145] At 312, the method includes using one or more processors to determine one or more bases linked to the 5' and / or 3' ends of the first sequence readout to be soft-split. As described above, during amplification (e.g., PCR amplification), inosine bases are converted into one or more nucleotides (e.g., A, T, G, C). During alignment, the nucleotides representing inosine bases will not be aligned (mapped) to a reference genomic sequence, and thus will identify where the original cfDNA sequence (e.g., fragment) ends and where the inosine base sequence of the gap begins. Therefore, determining one or more bases in the first sequence readout to be soft-split may include, for example, aligning the first sequence readout to a reference sequence and identifying unaligned portions of the first sequence readout. Unaligned portions of the first sequence readout (i.e., unaligned nucleotides) may be associated with inosine bases in the extended first strand. Because these nucleotides representing inosine bases are artificially generated, they are not aligned to a reference sequence or complementary strand, and thus indicate gaps in the complementary strand. Therefore, these unaligned nucleotide bases in the 5' and / or 3' portions of the sequence readout can be identified for soft cleavage.

[0146] In some embodiments, the method includes sequencing the second strand of a cell-free DNA duplex to produce a second sequence readout. One or more bases in the first sequence readout to be soft-cut can be determined by aligning the first sequence (i.e., for the first strand) with the second sequence readout (i.e., for the second strand) and identifying unaligned portions of the first sequence readout. Matching the first and second sequence readouts may include using a common UMI between the first and second sequence readouts. Unaligned 5' and / or 3' portions of the sequence readout can be identified for soft cutting.

[0147] At 314, the method includes reading from the first sequence by one or more processors soft-splitting bases corresponding to the 3' inosine extension and / or 5' inosine extension of the first strand of the cell-free DNA duplex. At 316, the method includes determining the length or sequence of the 5' overhang of the second strand of the cell-free DNA duplex based on soft-splitting of the 3' inosine extension of the first strand of the cell-free DNA duplex, and / or determining the length or sequence of the 3' overhang of the second strand of the cell-free DNA duplex based on soft-splitting of the 5' inosine extension of the first strand of the cell-free DNA duplex.

[0148] In some embodiments, a method for determining the topological structure of cell-free DNA is provided, comprising: extending the 3' end of the first strand of a cell-free DNA duplex with inosine bases to fill the 5' overhang of the second strand of the cell-free DNA duplex; ligating a sequencing adaptor to the cell-free DNA duplex; sequencing the first strand of the cell-free DNA duplex to produce a first sequence readout; determining, by one or more processors, one or more bases in the first sequence readout to be soft-cut; soft-cutting, by one or more processors, the bases in the first sequence readout corresponding to the 3' inosine extension of the first strand of the cell-free DNA duplex; and determining, by one or more processors, the length or sequence of the 5' overhang of the second strand of the cell-free DNA duplex based on the soft-cutting of the 3' inosine extension of the first strand of the cell-free DNA duplex. In some embodiments, the method further includes linking a second sequencing adaptor to a 3' overhang of the second strand of a cell-free DNA duplex; extending the 3' end of the second sequencing adaptor with inosine bases to fill the 3' overhang of the second strand of the cell-free DNA duplex; linking the 3' inosine-extended end of the second sequencing adaptor to a 5' end of the first strand of the cell-free DNA duplex, wherein the linked 3' inosine-extended end provides a 5' inosine extension of the first strand of the cell-free DNA duplex; determining, by one or more processors, one or more bases to be soft-cut and linked to the 5' end of the first sequence readout; soft-cutting, by one or more processors, the bases corresponding to the 5' inosine extension of the first strand of the cell-free DNA duplex from the first sequence readout; and determining, by one or more processors, the length or sequence of the 3' overhang of the second strand of the cell-free DNA duplex based on the soft-cutting of the 5' inosine extension of the first strand of the cell-free DNA duplex. In some embodiments, the method includes extending the 3' end of the second strand of the cell-free DNA duplex with inosine bases to fill the 5' overhang of the first strand of the cell-free DNA duplex; ligating a second sequencing adaptor to the cell-free DNA duplex; soft-splitting the bases corresponding to the 3' inosine extension of the second strand of the cell-free DNA duplex from the second sequence readout by one or more processors; and determining the length or sequence of the 5' overhang of the first strand of the cell-free DNA duplex based on the soft-splitting of the 3' inosine extension of the second strand of the cell-free DNA duplex by one or more processors.

[0149] In some embodiments, linking the second sequencing adaptor to the 3' overhang of the second strand of the cell-free DNA duplex includes: extending the 3' overhang of the second strand of the cell-free DNA duplex to provide 3' extension, wherein the second sequencing adaptor includes a 3' overhang complementary to the 3' extension of the second strand of the cell-free DNA duplex; and linking the second sequencing adaptor to the 3' extension of the second strand of the cell-free DNA duplex. In some embodiments, the 3' overhang of the second strand of the cell-free DNA duplex is extended using nucleotide bases of the same base type.

[0150] In some embodiments, the method further includes detecting the presence or absence of a disease based on the length or sequence of the 5' overhang of the second strand of the cell-free DNA duplex using one or more processors. In some embodiments, the method further includes detecting the presence or absence of a disease based on the length or sequence of the 3' overhang of the second strand of the cell-free DNA duplex using one or more processors. In some embodiments, the method further includes detecting the presence or absence of a disease based on the length or sequence of the 5' overhang of the first strand of the cell-free DNA duplex using one or more processors. In some embodiments, the method further includes detecting the presence or absence of a disease based on the length or sequence of the 3' overhang of the first strand of the cell-free DNA duplex using one or more processors. In some embodiments, the method further includes detecting the presence or absence of a disease based on the length or sequence of the 3' overhang of the second strand of the cell-free DNA duplex using one or more processors. In some embodiments, the disease is cancer.

[0151] In some embodiments, the method includes amplifying the first and second strands of the cell-free DNA duplex as described above. In some embodiments, the method includes sequencing as described above. In some embodiments, determining one or more bases to be soft-cleaved in the first sequence readout includes aligning the first and / or second sequence readouts as described above.

[0152] Figure 4 A process for determining the topology of cfDNA according to some embodiments is illustrated. Sequence readouts obtained by the methods described herein are received at one or more processors. Figure 4In the process illustrated, Readout 1 and Readout 2 represent readouts sequenced from both ends of the single strand of the duplex. Preprocessing of the sequence readouts prior to further analysis may include, for example, trimming poly-G regions (e.g., dark cycles), adaptor sequences, and / or portions of readouts considered low-quality (e.g., based on sequencing quality scores below a predetermined threshold, such as Q10 or lower, Q20 or lower, or Q30 or lower). If the sequencing readout contains an adaptase tail (i.e., a 3' tail added by a terminal transferase), this adaptase tail may be trimmed. The resulting sequencing readouts (i.e., after preprocessing) may be further analyzed to determine one or more bases in the sequencing readout to be soft-cut, for example, by aligning the sequence readout to a reference sequence. Based on the alignment, one or more bases may be identified for soft cutting, and the length of the soft cut (i.e., the "jagger" length) may be measured. This length indicates the protruding ends in the complementary strand. The genomic contents of the complementary strand may be extracted to provide the sequence of the protruding ends.

[0153] Figure 5 An exemplary readout processing matrix for extracting data from paired-end sequencing, according to some embodiments, is shown, which includes Figure 3A and Figure 3B The sequence constructs and analysis methods shown.

[0154] In some embodiments of any of the methods described herein, DNA double-stranded molecules are obtained from an individual. The method may also include generating a DNA double-stranded molecule overhang profile for the individual, which contains information about the overhangs of multiple DNA double-stranded molecules. For example, for multiple DNA double-stranded molecules, this information may include the lengths of the 3' overhangs of the first strand of the DNA double-stranded molecule, the lengths of the 5' overhangs of the first strand of the DNA double-stranded molecule, the lengths of the 3' overhangs of the second strand of the DNA double-stranded molecule, and / or the lengths of the 5' overhangs of the second strand of the DNA double-stranded molecule. In some embodiments, the information includes the sequence of the 5' or 3' overhang. In some embodiments, the information includes (1) the ratio of the length of the 5' overhang of the first strand of the DNA double-stranded molecule to the length of the 3' overhang of the first strand of the DNA double-stranded molecule, and (2) the ratio of the length of the 5' overhang of the first strand of the DNA double-stranded molecule to the length of the 3' overhang of the second strand of the DNA double-stranded molecule.

[0155] (3) The ratio of the length of the 5' overhang of the first strand of the DNA double-stranded molecule to the length of the 5' overhang of the second strand of the DNA double-stranded molecule; (4) The ratio of the length of the 3' overhang of the first strand of the DNA double-stranded molecule to the length of the 5' overhang of the second strand of the DNA double-stranded molecule; or (5) The ratio of the length of the 3' overhang of the first strand of the DNA double-stranded molecule to the length of the 3' overhang of the second strand of the DNA double-stranded molecule. In some embodiments, the information includes (1) the ratio of the length of the 5' overhang of the first strand of the DNA double-stranded molecule, the length of the 3' overhang of the first strand of the DNA double-stranded molecule, the length of the 5' overhang of the second strand of the DNA double-stranded molecule, the length of the 3' overhang of the second strand of the DNA double-stranded molecule, and (2) the length of the double strand.

[0156] The method may further include comparing the overhang spectrum of a DNA double-stranded molecule with that of a reference DNA double-stranded molecule, for example, using one or more processors. In some embodiments, the reference DNA double-stranded molecule overhang spectrum is based on DNA double-stranded molecules from a normal sample, multiple normal samples, or a synthetically produced normal sample. In some embodiments, the reference DNA double-stranded molecule overhang spectrum is based on DNA double-stranded molecules from samples obtained from an individual with cancer or multiple individuals with cancer. In some embodiments, the reference DNA double-stranded molecule overhang spectrum is based on DNA double-stranded molecules from samples obtained from an individual with an abnormal fetus or multiple individuals with an abnormal fetus. In some embodiments, the reference DNA double-stranded molecule overhang spectrum is based on DNA double-stranded molecules from samples obtained from an individual who has received a stable transplant or multiple individuals who have received a stable transplant. In some embodiments, the reference DNA double-stranded molecule overhang spectrum is based on DNA double-stranded molecules from a matched normal sample obtained from the individual. In some embodiments, the reference DNA double-stranded molecule overhang spectrum is based on DNA double-stranded molecules from a previous sample obtained from the individual. The previous sample can be a normal baseline or a disease state baseline (i.e., a DNA duplex molecule overhang spectrum representing a previous state of the disease state).

[0157] In some embodiments, the method described herein further includes generating a report via one or more processors, the report indicating the lengths of: the 3' overhang of the first strand of the cell-free DNA duplex, the 3' overhang of the second strand of the cell-free DNA duplex, the 5' overhang of the first strand of the cell-free DNA duplex, and / or the 5' overhang of the second strand of the cell-free DNA duplex, or other DNA duplex molecule overhang profile information. In some embodiments, the method further includes transmitting the report to a healthcare provider. In some embodiments, the report is transmitted via a computer network, a peer-to-peer connection, or to an application programming interface (API).

[0158] In some embodiments, the method further includes generating a genomic profile of the object, the genomic profile comprising the following lengths: a 3' overhang of the first strand of the cell-free DNA duplex, a 3' overhang of the second strand of the cell-free DNA duplex, a 5' overhang of the first strand of the cell-free DNA duplex, and / or a 5' overhang of the second strand of the cell-free DNA duplex. In some embodiments, the genomic profile of the object also includes results from: comprehensive genome profiling (CGP) tests, gene expression profiling tests, cancer hotspot group tests, DNA methylation tests, DNA fragmentation tests, RNA fragmentation tests, or any combination thereof. In some embodiments, the genomic profile of the object also includes results from nucleic acid sequencing-based tests.

[0159] DNA topological information, such as the length and / or sequence information of 3' and / or 5' overhangs, determined according to the methods described herein, can be used to detect the presence, potential presence, or absence of a disease (e.g., cancer). For example, this is particularly useful for determining the presence or absence of disease in patients with early-stage cancer or those with low cancer levels, or for detecting disease recurrence. For example, in some embodiments, the individual's circulating tumor DNA (ctDNA) content is less than 1%, less than 0.8%, less than 0.5%, less than 0.3%, less than 0.1%, or less than 0.05%.

[0160] sample

[0161] The disclosed methods and systems can be used with any of a variety of samples (also referred to herein as specimens) containing nucleic acids (e.g., DNA) collected from a subject (e.g., a patient). Some examples of specimens include, but are not limited to, liquid biopsy specimens, blood specimens (e.g., peripheral whole blood specimens), plasma specimens, serum specimens, lymph specimens, saliva specimens, sputum specimens, urine specimens, gynecological fluid specimens, circulating tumor cell (CTC) specimens, cerebrospinal fluid (CSF) specimens, pericardial fluid specimens, pleural fluid specimens, ascites (peritoneal fluid) specimens, stool (or feces) specimens, or other body fluid, secretion, and / or excretion specimens (or specimens derived therefrom).

[0162] In some implementations, samples can be collected via needle biopsy, fine needle aspiration, collection cups or tubes, oral swabs, nasal swabs, vaginal swabs, or cytology smears.

[0163] In some embodiments, the sample is a liquid biopsy sample and may contain, for example, whole blood, plasma, serum, urine, feces, sputum, saliva, or cerebrospinal fluid. In some embodiments, the sample may be a liquid biopsy sample and may contain circulating tumor cells (CTCs). In some embodiments, the sample may be a liquid biopsy sample and may contain cell-free DNA.

[0164] (cfDNA), circulating tumor DNA (ctDNA), or any combination thereof.

[0165] In some embodiments, the disclosed method may further include analyzing a primary control (e.g., a normal blood sample). In some embodiments, the disclosed method may further include determining whether a primary control is available, and if available, isolating a control nucleic acid (e.g., DNA) from the primary control. In some embodiments, if no primary control is available, the sample may contain any normal control. In some embodiments, the method includes evaluating the sample, such as a normal sample, using the methods described herein. In some embodiments, the disclosed method may further include determining that no primary control is available, and labeling the sample for analysis in the absence of a matching control.

[0166] In some embodiments, the nucleic acids extracted from the sample may comprise deoxyribonucleic acid (DNA) molecules. Examples of DNAs suitable for analysis by the disclosed methods include, but are not limited to, genomic DNA or fragments thereof, mitochondrial DNA or fragments thereof, cell-free DNA (cfDNA), and circulating tumor DNA (ctDNA). Cell-free DNA (cfDNA) consists of DNA fragments released from normal cells and / or cancer cells during apoptosis and necrosis and circulating in the bloodstream and / or accumulating in other body fluids. Circulating tumor DNA (ctDNA) consists of DNA fragments released from cancer cells and tumors and circulating in the bloodstream and / or accumulating in other body fluids.

[0167] In some implementations, cell-free or circulating tumor DNA is extracted from the liquid sample. In some implementations, samples with low nucleated cellularity may require more, such as larger volumes, for DNA extraction.

[0168] In some embodiments, the method for determining the topological structure of cell-free DNA further includes obtaining cell-free DNA duplexes from the subject. In some embodiments, the cell-free DNA duplexes are obtained from a liquid biopsy sample. In some embodiments, the sample is a liquid biopsy sample and includes blood, plasma, cerebrospinal fluid, sputum, feces, urine, or saliva. In some embodiments, the cell-free DNA duplexes are circulating tumor DNA (ctDNA) duplexes.

[0169] object

[0170] In some embodiments, the sample is obtained (e.g., collected) from a subject who has a condition or disease (e.g., a hyperproliferative disease (e.g., cancer) or a non-cancer indication) or is suspected of having said condition or disease (e.g., a human patient). In some embodiments, the hyperproliferative disease is cancer. In some embodiments, the cancer is a solid tumor or a metastatic form thereof. In some embodiments, the cancer is a hematologic cancer, such as leukemia or lymphoma.

[0171] In some implementations, the subject has cancer or is at risk of developing cancer. For example, in some implementations, the subject has a genetic predisposition to cancer (e.g., possessing a genetic mutation that increases their baseline risk of developing cancer). In some implementations, the subject has been exposed to environmental disturbances that increase their risk of developing cancer (e.g., radiation or chemicals). In some implementations, monitoring for cancer development is required. In some implementations, monitoring for cancer progression or regression (e.g., after treatment with anticancer therapy). In some implementations, monitoring for cancer recurrence is required. In some implementations, monitoring for minimum residual disease (MRD) is required. In some implementations, the subject has been treated for cancer or is currently being treated for cancer. In some implementations, the subject has not been treated with anticancer therapy.

[0172] In some embodiments, the subject (e.g., a patient) is being treated with one or more anticancer therapies, or has previously been treated with one or more anticancer therapies. In some embodiments, for example, for patients who have previously been treated with targeted anticancer therapies, samples (e.g., specimens) are obtained (e.g., collected) after targeted therapy. In some embodiments, samples after targeted therapy are obtained after the completion of targeted therapy. In some embodiments, one or more anticancer therapies (or anticancer treatments) may include, but are not limited to, surgery (e.g., surgical resection), radiation therapy, or chemotherapy, and combinations thereof.

[0173] In some implementations, the patient has not previously received anticancer treatment. In some implementations, for example, for patients who have not previously received targeted anticancer treatment, the sample contains liquid biopsy material, such as original liquid biopsy material or liquid biopsy material after recurrence.

[0174] In some embodiments, the method for determining the topological structure of cell-free DNA further includes obtaining cell-free DNA duplexes from a subject. In some embodiments, the cell-free DNA duplexes are obtained from a subject suspected of having cancer or confirmed to have cancer. In some embodiments, the cell-free DNA duplexes are obtained from a liquid biopsy sample. In some embodiments, the sample is a liquid biopsy sample and includes blood, plasma, cerebrospinal fluid, sputum, feces, urine, or saliva. In some embodiments, the cell-free DNA duplexes are circulating tumor DNA (ctDNA) duplexes. In some embodiments, the method further includes treating the subject with anticancer therapy.

[0175] Nucleic acid extraction and processing

[0176] DNA can be extracted from liquid biopsy samples (including but not limited to blood, plasma, cerebrospinal fluid, sputum, feces, urine or saliva or other bodily fluid samples) using any of a variety of techniques known to those skilled in the art (see, for example, Example 1 of International Patent Application Publication No. WO 2012 / 092426; Tan, et al. (2009), “DNA, RNA, and Protein Extraction: The Past and The Present”, J. Biomed. Biotech. 2009:574398; Technical documentation for the 16LEV Blood DNA Kit (Promega Corporation, Madison, WI); and the technical manual for the Maxwell 16 buccal swab LEV DNA purification kit (Promega Literature #TM333, January 1, 2011, Promega Corporation, Madison, WI).

[0177] Typical DNA extraction procedures include, for example, (i) collecting a fluid sample from which DNA is to be extracted; (ii) treating the fluid sample with a concentrated salt solution to precipitate proteins, lipids, and RNA, followed by centrifugation to separate the precipitated proteins, lipids, and RNA; and (iii) purifying the DNA from the supernatant to remove detergents, proteins, salts, or other reagents used in the preceding steps. The DNA sample may optionally be further processed using RNases for digesting RNA in the sample.

[0178] Examples of suitable techniques for DNA purification include, but are not limited to, (i) precipitation in ice-cold ethanol or isopropanol followed by centrifugation (DNA precipitation can be enhanced by increasing ionic strength, for example by adding sodium acetate); (ii) phenol-chloroform extraction followed by centrifugation to separate the aqueous phase containing nucleic acids from the organic phase containing denatured proteins; and (iii) solid-phase chromatography, in which nucleic acids are adsorbed onto a solid phase (e.g., silica or others), depending on the pH and salt concentration of the buffer.

[0179] In some cases, cellular proteins and histones bound to DNA can be removed prior to the DNA precipitation step by adding proteases, precipitating the proteins with sodium acetate or ammonium acetate, or by extraction with a phenol-chloroform mixture.

[0180] In some cases, DNA can be extracted using any of a variety of suitable commercial DNA extraction and purification kits. Some examples include, but are not limited to, the QIAamp (for isolating genomic DNA from human samples) and DNAeasy (for isolating genomic DNA from animal or plant samples) kits from Qiagen (Germantown, MD) or the Promega (Madison, WI) kits. and ReliaPrep TM Series of reagent kits.

[0181] In some cases, the disclosed methods may also include determining or acquiring a yield value of nucleic acids extracted from a sample and comparing the determined value with a reference value. For example, if the determined or acquired value is less than the reference value, the nucleic acid can be amplified prior to library construction. In some cases, the disclosed methods may also include determining or acquiring a value of the size (or average size) of nucleic acid fragments in a sample and comparing the determined or acquired value with a reference value, such as a size (or average size) of at least 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 base pairs (bp). In some cases, one or more parameters described herein may be adjusted or selected in response to this determination.

[0182] After separation, nucleic acids are usually dissolved in a weakly alkaline buffer such as Tris-EDTA (TE) buffer, or in ultrapure water.

[0183] System and storage media

[0184] This document also describes systems and computer-readable storage media containing an instructor for causing the system to perform the methods described herein. An exemplary system includes, for example, one or more processors; and a memory communicatively coupled to and configured to store instructions, which, when executed by one or more processors, cause the system to: receive a first sequence readout obtained by: extending the 3' end of the first strand of a cell-free DNA duplex with inosine bases to fill the 5' overhang of the second strand of the cell-free DNA duplex; ligating a sequencing adaptor to the cell-free DNA duplex; and sequencing the first strand of the cell-free DNA duplex to produce the first sequence readout; determining one or more bases in the first sequence readout to be soft-cut; soft-cutting from the first sequence readout the bases corresponding to the 3' inosine extension of the first strand of the cell-free DNA duplex; and determining the length or sequence of the 5' overhang of the second strand of the cell-free DNA duplex based on the soft-cutting of the 3' inosine extension of the first strand of the cell-free DNA duplex.

[0185] In another example, the system includes one or more processors; and a memory communicatively coupled to the one or more processors and configured to store instructions, which, when executed by the one or more processors, cause the system to: receive a first sequence readout obtained by: attaching a sequencing adaptor to the 3' end of the first strand of a cell-free DNA duplex; extending the 3' end of the sequencing adaptor with inosine bases to fill the 3' end of the first strand of the cell-free DNA duplex; and attaching the 3' inosine-extended end of the sequencing adaptor to the 5' end of the second strand of the cell-free DNA duplex, wherein the attached 3' inosine-extended end provides the 5' inosine extension of the second strand of the cell-free DNA duplex; determining one or more bases to be soft-cut in the first sequence readout; soft-cutting the bases corresponding to the 5' inosine extension of the second strand of the cell-free DNA duplex from the first sequence readout; and determining the length or sequence of the 3' end of the first strand of the cell-free DNA duplex based on the soft-cutting of the 5' inosine extension of the second strand of the cell-free DNA duplex.

[0186] Figure 6 An example of a computing device or system according to one embodiment is shown. Device 600 may be a host computer connected to a network. Device 900 may be a client computer or a server. (As in...) Figure 6 As shown, device 600 can be any suitable type of microprocessor-based device, such as a personal computer, workstation, server, or handheld computing device (portable electronic device), such as a telephone or tablet. The device may include, for example, one or more processors 610, input devices 620, output devices 630, memory or storage devices 640, communication devices 660, and nucleic acid sequencers 670. Software 650 residing in memory or storage devices 640 may include, for example, an operating system and software for performing the methods described herein. Input devices 620 and output devices 630 may generally correspond to those described herein and may be connected to or integrated with a computer.

[0187] Input device 620 can be any suitable device that provides input, such as a touchscreen, keyboard or keypad, mouse, or voice recognition device. Output device 630 can be any suitable device that provides output, such as a touchscreen, haptic device, or speaker.

[0188] Memory 640 can be any suitable device providing storage (e.g., electrical memory, magnetic memory, or optical memory, including RAM (volatile and non-volatile), cache, hard disk drive, or removable storage disk). Communication device 660 can include any suitable device capable of sending and receiving signals over a network, such as a network interface chip or device. Computer components can be connected in any suitable manner, such as via wired media (e.g., physical system bus 680, Ethernet connection, or any other wired transmission technology) or wirelessly (e.g., ...). Or any other wireless technology).

[0189] The software module 650, which can be stored as executable instructions in memory 640 and executed by processor 610, may include, for example, an operating system and / or processes embodying the methods of this disclosure (e.g., embodied in the device described herein).

[0190] Software module 650 may also be stored and / or transmitted in any non-transitory computer-readable storage medium for use or in conjunction with an instruction execution system, apparatus, or device (such as those described herein), which may retrieve and execute instructions associated with the software from and execute such instructions. In the context of this disclosure, a computer-readable storage medium may be any medium (e.g., memory 640) that may contain or store processes for use or in conjunction with an instruction execution system, apparatus, or device. Some examples of computer-readable storage media may include memory units such as hard disk drives, flash drives, and distributed modules operating as single functional units. Furthermore, the various processes described herein may be embodied as modules configured to operate according to the above embodiments and techniques. Moreover, while processes may be shown and / or described individually, those skilled in the art will understand that these processes may be routines or modules within other processes.

[0191] Software module 650 can also be propagated in any transmission medium for use by or in conjunction with an instruction execution system, apparatus, or device (such as those described above), which can retrieve and execute instructions associated with the software from and execute such instructions. In the context of this disclosure, the transmission medium can be any medium capable of transmitting, propagating, or transmitting programming for use by or in conjunction with an instruction execution system, apparatus, or device. Transmittable media can include, but are not limited to, wired or wireless transmission media of electronic, magnetic, optical, electromagnetic, or infrared nature.

[0192] Device 600 can connect to a network (e.g., network 704, such as...). Figure 7As shown and / or described below, it can be any suitable type of interconnection communication system. The network can implement any suitable communication scheme and can be protected by any suitable security scheme. The network can contain network links of any suitable arrangement that can implement the transmission and reception of network signals, such as wireless network connections, T1 or T3 lines, wired networks, DSL or telephone lines.

[0193] Device 600 can be implemented using any operating system, such as an operating system suitable for running on a network. Software module 650 can be written in any suitable programming language (e.g., C, C++, Java, or Python). In various embodiments, application software embodying the functionality of this disclosure can be deployed in different configurations, such as client / server deployments or via a web browser, as, for example, web-based applications or web services. In some embodiments, the operating system is executed by one or more processors (e.g., processor 610).

[0194] The device 600 may also include a sequencer 670, which can be any suitable nucleic acid sequencing instrument.

[0195] Figure 7 An example of a computing system according to one embodiment is shown. In system 700, device 600 (e.g., as described above and Figure 6 The device shown is connected to network 704, which is also connected to device 706. In some embodiments, device 706 is a sequencer. Exemplary sequencers may include, but are not limited to, the Roche / 454 Genome Sequencer (GS) FLX system, the Illumina / Solexa Genome Analyzer (GA), and Illumina's... 2500 3000 4000 and 6000 sequencing system, Life / APG Support Oligonucleotide Ligation Detection (SOLiD) system, Polonator G.007 system, Helicos BioSciences HeliScope gene sequencing system, or Pacific Biosciences... RS system.

[0196] Devices 600 and 706 may communicate via network 704 (e.g., a Local Area Network (LAN), Virtual Private Network (VPN), or the Internet) using suitable communication interfaces. In some embodiments, network 704 may be, for example, the Internet, an intranet, a VPN, a cloud network, a wired network, or a wireless network. Devices 600 and 706 may communicate partially or entirely wirelessly or hardwired, such as via Ethernet, IEEE 802.11b wireless, etc. Additionally, devices 600 and 706 may communicate via a second network, such as a mobile / cellular network, using suitable communication interfaces. Communication between devices 600 and 706 may also include or communicate with various servers (e.g., mail servers, mobile servers, media servers, telephone servers, etc.). In some embodiments, devices 600 and 706 may communicate directly (in lieu of or supplement to communication via network 704), for example, via wirelessly or hardwired, such as via Ethernet, IEEE 802.11b wireless, etc. In some implementations, devices 900 and 1006 communicate via communication 708, which may be a direct connection or may occur via a network (e.g., network 704).

[0197] One or both of devices 600 and 706 typically contain logic (e.g., HTTP web server logic) or are programmed to format data, access it from a local or remote database or other data and content source, for providing and / or receiving information via network 704 according to various instances described herein.

[0198] Exemplary Implementation

[0199] The following embodiments are exemplary and are not intended to limit the scope of any invention described herein. Exemplary embodiments include, but are not limited to:

[0200] Implementation Scheme 1. A method for determining the topological structure of cell-free DNA, comprising:

[0201] The 3' end of the first strand of the cell-free DNA duplex is extended with inosine bases to fill the 5' overhang of the second strand of the cell-free DNA duplex.

[0202] The sequencing adaptor is linked to the cell-free DNA duplex;

[0203] The first strand of the cell-free DNA duplex is sequenced to produce a first sequence readout.

[0204] One or more bases to be soft-sheared in the first sequence readout are determined by one or more processors;

[0205] The soft-cleaved bases corresponding to the 3' inosine extension of the first strand of the cell-free DNA duplex are read from the first sequence by one or more processors; and

[0206] The length or sequence of the 5' overhang of the second strand of the cell-free DNA duplex is determined by one or more processors based on soft shearing of the 3' inosine extension of the first strand of the cell-free DNA duplex.

[0207] Implementation Scheme 2. The method of claim 1, further comprising detecting the presence or absence of a disease by means of the one or more processors, based on the length or sequence of the 5' overhang of the second strand of the cell-free DNA duplex.

[0208] Implementation scheme 3. The method of claim 2, wherein the disease is cancer.

[0209] Implementation Scheme 4. The method according to any one of claims 1 to 3, further comprising:

[0210] The second sequencing adaptor is connected to the 3' overhang of the second strand of the cell-free DNA duplex;

[0211] The 3' end of the second sequencing adaptor is extended with inosine bases to fill the 3' overhang of the second strand of the cell-free DNA duplex;

[0212] The 3' inosine extension of the second sequencing adapter is linked to the 5' end of the first strand of the cell-free DNA duplex, wherein the linked 3' inosine extension provides the 5' inosine extension of the first strand of the cell-free DNA duplex.

[0213] One or more processors are used to determine one or more bases that are connected to the 5' end of the first sequence to be soft-cut;

[0214] The soft-cleaved bases corresponding to the 5' inosine extension of the first strand of the cell-free DNA duplex are read from the first sequence by one or more processors; and

[0215] The length or sequence of the 3' overhang of the second strand of the cell-free DNA duplex is determined by one or more processors based on soft shearing of the 5' inosine extension of the first strand of the cell-free DNA duplex.

[0216] Implementation Scheme 5. The method of claim 4, comprising detecting the presence or absence of a disease by means of the one or more processors, based on the length or sequence of the 3' overhang of the second strand of the cell-free DNA duplex.

[0217] Implementation Scheme 6. The method of claim 4 or 5, wherein linking the second sequencing adaptor to the 3' overhang of the second strand of the cell-free DNA duplex comprises:

[0218] Extending the 3' overhang of the second strand of the cell-free DNA duplex to provide 3' extension, wherein the second sequencing adaptor includes a 3' overhang complementary to the 3' extension of the second strand of the cell-free DNA duplex; and

[0219] The second sequencing adaptor is ligated to the 3' extension of the second strand of the cell-free DNA duplex.

[0220] Implementation Scheme 7. The method of claim 6, wherein the 3' overhang of the second strand of the cell-free DNA duplex is extended using nucleotide bases of the same base type.

[0221] Implementation Scheme 8. The method of any one of claims 1 to 7, wherein determining one or more bases to be soft-cut in the first sequence readout comprises aligning the first sequence readout with a reference sequence and identifying unaligned portions of the first sequence readout.

[0222] Implementation Scheme 9. The method of any one of claims 1 to 8, further comprising:

[0223] Sequencing the second strand of the cell-free DNA duplex to produce a second sequence readout; and

[0224] One or more bases to be soft-cleaved in the second sequence readout are determined by one or more processors.

[0225] Implementation Scheme 10. The method of claim 9, wherein determining one or more bases to be soft-cleaved in the second sequence readout comprises aligning the second sequence with a reference sequence and identifying unaligned portions of the second sequence readout.

[0226] Implementation scheme 11. The method of claim 9 or 10, wherein the first sequence readout and the second sequence readout are associated by a unique molecular identifier (UMI).

[0227] Implementation Scheme 12. The method of claim 11, wherein determining one or more bases to be soft-cleaved in the first sequence readout comprises aligning the first sequence with the second sequence readout and identifying unaligned portions of the first sequence readout.

[0228] Implementation Scheme 13. The method of claim 11 or 12, wherein determining one or more bases to be soft-cleaved in the second sequence readout comprises aligning the second sequence with the first sequence readout and identifying unaligned portions of the second sequence readout.

[0229] Implementation Scheme 14. The method of any one of claims 9 to 13, comprising:

[0230] The 3' end of the second strand of the cell-free DNA duplex is extended with inosine bases to fill the 5' overhang of the first strand of the cell-free DNA duplex.

[0231] The second sequencing adaptor is linked to the cell-free DNA duplex;

[0232] Through one or more processors, the soft-cleaved bases corresponding to the 3' inosine extension of the second strand of the cell-free DNA duplex are read from the second sequence; and

[0233] The length or sequence of the 5' overhang of the first strand of the cell-free DNA duplex is determined by one or more processors based on soft shearing of the 3' inosine extension of the second strand of the cell-free DNA duplex.

[0234] Implementation Scheme 15. The method of claim 14, further comprising detecting the presence or absence of a disease by means of the one or more processors, based on the length or sequence of the 5' overhang of the first strand of the cell-free DNA duplex.

[0235] Implementation Scheme 16. The method of any one of claims 1 to 15, wherein extending the 3' end of the first strand of the cell-free DNA duplex includes forming a single 3' inosine overhang.

[0236] Implementation Scheme 17. The method of claim 16, wherein the sequencing adaptor comprises a 3' cytosine overhang complementary to the 3' inosine overhang.

[0237] Implementation Scheme 18. The method of any one of claims 1 to 17, comprising amplifying the cell-free DNA duplex to link a sample index to the cell-free DNA duplex.

[0238] Implementation Scheme 19. The method of any one of claims 1 to 18, wherein the sequencing adaptor or the second sequencing adaptor comprises a sample index.

[0239] Implementation Scheme 20. The method of any one of claims 11 to 19, wherein the sequencing adaptor or the second sequencing adaptor comprises the UMI.

[0240] Implementation Scheme 21. The method of any one of claims 1 to 20, wherein the sequencing adaptor or the second sequencing adaptor is a Y-shaped sequencing adaptor.

[0241] Implementation Scheme 22. A method for preparing sequencing constructs, comprising:

[0242] The sequencing adaptor is linked to the 3' overhang of the first strand of the cell-free DNA duplex;

[0243] Extend the 3' end of the sequencing adaptor with inosine bases to fill the 3' overhang of the first strand of the cell-free DNA duplex; and

[0244] The 3' inosine extension of the sequencing adapter is linked to the 5' end of the second strand of the cell-free DNA duplex, wherein the linked 3' inosine extension provides the 5' inosine extension of the second strand of the cell-free DNA duplex.

[0245] Implementation Scheme 23. The method of claim 22, wherein linking the sequencing adaptor to the 3' overhang of the first strand of the cell-free DNA duplex comprises:

[0246] Extending the 3' overhang of the first strand of the cell-free DNA duplex to provide 3' extension, wherein the sequencing adaptor includes a 3' overhang complementary to the 3' extension of the first strand of the cell-free DNA duplex; and

[0247] The sequencing adaptor is ligated to the 3' extension of the first strand of the cell-free DNA duplex.

[0248] The method of claim 23 in embodiment 24, wherein the 3' overhang of the first strand of the cell-free DNA duplex is extended using nucleotide bases of the same base type.

[0249] Implementation Scheme 25. A method for determining the topological structure of cell-free DNA, comprising:

[0250] The sequencing construct is prepared according to any one of claims 22 to 24;

[0251] Sequencing the second strand of the cell-free DNA double helix to produce a first sequence readout;

[0252] One or more bases to be soft-sheared in the first sequence readout are determined by one or more processors;

[0253] The soft-cleaved bases corresponding to the 5' inosine extension of the second strand of the cell-free DNA duplex are read from the first sequence by one or more processors; and

[0254] The length or sequence of the 3' overhang of the first strand of the cell-free DNA duplex is determined by one or more processors based on soft shearing of the 5' inosine extension of the second strand of the cell-free DNA duplex.

[0255] Implementation Scheme 26. The method of claim 25, further comprising detecting the presence or absence of a disease by means of the one or more processors, based on the length or sequence of the 3' overhang of the first strand of the cell-free DNA duplex.

[0256] Implementation scheme 27. The method of claim 26, wherein the disease is cancer.

[0257] Implementation Scheme 28. The method of any one of claims 25 to 27, wherein determining one or more bases to be soft-cut in the first sequence readout comprises aligning the first sequence readout with a reference sequence and identifying unaligned portions of the first sequence readout.

[0258] Implementation Scheme 29. The method of any one of claims 25 to 28, further comprising:

[0259] Sequencing the first strand of the cell-free DNA duplex to produce a second sequence readout; and

[0260] One or more bases to be soft-cleaved in the second sequence readout are determined by one or more processors.

[0261] Implementation Scheme 30. The method of claim 29, wherein determining one or more bases to be soft-cleaved in the second sequence readout comprises aligning the second sequence with a reference sequence and identifying unaligned portions of the second sequence readout.

[0262] Implementation scheme 31. The method of claim 29 or 30, wherein the first sequence readout and the second sequence readout are associated by a unique molecular identifier (UMI).

[0263] Implementation Scheme 32. The method of claim 21, wherein determining one or more bases to be soft-cleaved in the first sequence readout comprises aligning the first sequence with the second sequence readout and identifying unaligned portions of the first sequence readout.

[0264] Implementation Scheme 33. The method of claim 11 or 12, wherein determining one or more bases to be soft-cleaved in the second sequence readout comprises aligning the second sequence with the first sequence readout and identifying unaligned portions of the second sequence readout.

[0265] Implementation Scheme 34. The method of any one of claims 22 to 33, comprising:

[0266] The second sequencing adaptor is linked to the 3' overhang of the second strand of the cell-free DNA duplex;

[0267] Extend the 3' end of the second sequencing adaptor with inosine bases to fill the 3' overhang of the second strand of the cell-free DNA duplex; and

[0268] The 3' inosine extension of the second sequencing adapter is joined to the 5' end of the first strand of the cell-free DNA duplex, wherein the 3' inosine extension provides the 5' inosine extension of the first strand of the cell-free DNA duplex.

[0269] Implementation Scheme 35. The method of claim 34, further comprising:

[0270] The first strand of the cell-free DNA duplex is sequenced to produce a second sequence readout.

[0271] One or more processors are used to determine one or more bases that are to be soft-cut and connected to the 5' end of the second sequence read out;

[0272] The soft-cleaved bases corresponding to the 5' inosine extension of the first strand of the cell-free DNA duplex are read from the second sequence by one or more processors; and

[0273] The length or sequence of the 3' overhang of the second strand of the cell-free DNA duplex is determined by one or more processors based on soft shearing of the 5' inosine extension of the first strand of the cell-free DNA duplex.

[0274] Implementation Scheme 36. The method of claim 35, further comprising detecting the presence or absence of a disease by means of the one or more processors, based on the length or sequence of the 3' overhang of the second strand of the cell-free DNA duplex.

[0275] Implementation Scheme 37. The method of any one of claims 34 to 36, wherein linking the second sequencing adaptor to the 3' overhang of the second strand of the cell-free DNA duplex comprises linking a monoinosine to the 3' overhang of the second strand of the cell-free DNA duplex and linking the second sequencing adaptor to the monoinosine.

[0276] Implementation Scheme 38. The method of claim 37, wherein the sequencing adaptor comprises a 3' cytosine overhang complementary to the single inosine and attached to the 3' overhang of the second strand of the cell-free DNA duplex.

[0277] Implementation Scheme 39. The method of any one of claims 35 to 38, wherein determining one or more bases to be soft-cut and connected to the 5' end of the second sequence readout comprises aligning the second sequence readout with a reference sequence and identifying unaligned portions of the second sequence readout.

[0278] Implementation Scheme 40. The method of any one of claims 35 to 39, wherein the first sequence readout and the second sequence readout are associated by a unique molecular identifier (UMI).

[0279] Implementation Scheme 41. The method of claim 40, wherein determining one or more bases to be soft-cut and connected to the 5' end of the second sequence readout comprises aligning the second sequence readout with the first sequence readout and identifying unaligned portions of the second sequence readout.

[0280] Implementation Scheme 42. The method of claim 40 or 41, wherein determining one or more bases to be soft-cut and connected to the 5' end of the first sequence readout comprises aligning the first sequence readout with the second sequence readout and identifying unaligned portions of the first sequence readout.

[0281] Implementation Scheme 43. The method of any one of claims 22 to 42, comprising amplifying the cell-free DNA duplex to link a sample index to the cell-free DNA duplex.

[0282] Implementation Scheme 44. The method of any one of claims 22 to 42, wherein the sequencing adaptor or the second sequencing adaptor comprises a sample index.

[0283] Implementation Scheme 45. The method of any one of claims 40 to 44, wherein the sequencing adaptor or the second sequencing adaptor comprises the UMI.

[0284] Implementation Scheme 46. The method of any one of claims 22 to 45, wherein the sequencing adaptor or the second sequencing adaptor is a Y-shaped sequencing adaptor.

[0285] Implementation Scheme 47. The method of any one of claims 1 to 46, wherein the cell-free DNA duplex is obtained from a subject suspected of having cancer or confirmed to have cancer.

[0286] Implementation scheme 48. The method of claim 47, further comprising treating the subject with anticancer therapy.

[0287] Implementation Scheme 49. The method of any one of claims 1 to 48, further comprising obtaining the cell-free DNA duplex from the object.

[0288] Implementation Scheme 50. The method of any one of claims 1 to 49, wherein the cell-free DNA duplex is obtained from a liquid biopsy sample.

[0289] Implementation Scheme 51. The method of claim 50, wherein the sample is a liquid biopsy sample and comprises blood, plasma, cerebrospinal fluid, sputum, feces, urine or saliva.

[0290] Implementation Scheme 52. The method of any one of claims 1 to 51, wherein the cell-free DNA double strand is a circulating tumor DNA (ctDNA) double strand.

[0291] Implementation Scheme 53. The method of any one of claims 1 to 52, wherein the sequencing adaptor or the second sequencing adaptor comprises an amplification primer binding site, a flow cell adaptor sequence, or a substrate adaptor sequence.

[0292] Implementation Scheme 54. The method of any one of claims 1 to 53, further comprising amplifying the first and second strands of the cell-free DNA duplex.

[0293] Implementation Scheme 55. The method of claim 54, wherein the amplification includes polymerase chain reaction (PCR) amplification, non-PCR amplification, or isothermal amplification.

[0294] Implementation Scheme 56. The method of any one of claims 1 to 55, wherein the sequencing comprises using massively parallel sequencing (MPS), whole genome sequencing (WGS), whole exome sequencing, targeted sequencing, direct sequencing, or Sanger sequencing.

[0295] Implementation Scheme 57. The method of claim 56, wherein the sequencing comprises massively parallel sequencing, and the massively parallel sequencing technology comprises next-generation sequencing (NGS).

[0296] Implementation scheme 58. The method of any one of claims 1 to 57, wherein the sequencing is performed using a next-generation sequencer.

[0297] Implementation Scheme 59. The method of any one of claims 1 to 58, further comprising generating a report by one or more processors, the report indicating the lengths of: the 3' protrusion of the first strand of the cell-free DNA duplex, the 3' protrusion of the second strand of the cell-free DNA duplex, the 5' protrusion of the first strand of the cell-free DNA duplex, and / or the 5' protrusion of the second strand of the cell-free DNA duplex.

[0298] Implementation scheme 60. The method of claim 59, further comprising transmitting the report to a healthcare provider.

[0299] Implementation Scheme 61. The method of claim 60, wherein the report is transmitted via a computer network or peer-to-peer connection.

[0300] Implementation Scheme 62. The method of any one of claims 1 to 61, further comprising generating a genomic profile of the object, the genomic profile comprising the following lengths: a 3' overhang of the first strand of the cell-free DNA duplex, a 3' overhang of the second strand of the cell-free DNA duplex, a 5' overhang of the first strand of the cell-free DNA duplex, and / or a 5' overhang of the second strand of the cell-free DNA duplex.

[0301] Implementation Scheme 63. The method of claim 62, wherein the genomic profile of the object further comprises results from: comprehensive genome profiling (CGP) test, gene expression profiling test, cancer hotspot group test, DNA methylation test, DNA fragmentation test, RNA fragmentation test, or any combination thereof.

[0302] Implementation Scheme 64. The method of claim 62 or 63, wherein the genomic profile of the object further comprises results from nucleic acid sequencing-based tests.

[0303] Implementation plan 65. System, which includes:

[0304] One or more processors; and

[0305] A memory, communicatively coupled to the one or more processors and configured to store instructions, which, when executed by the one or more processors, cause the system to:

[0306] Receive a first sequence readout obtained by: extending the 3' end of the first strand of the cell-free DNA duplex with inosine bases to fill the 5' overhang of the second strand of the cell-free DNA duplex; connecting a sequencing adaptor to the cell-free DNA duplex; and sequencing the first strand of the cell-free DNA duplex to produce the first sequence readout;

[0307] Identify one or more bases in the first sequence readout that are to be soft-cleaved;

[0308] Read from the first sequence the soft cut bases corresponding to the 3' inosine extension of the first strand of the cell-free DNA double helix; and

[0309] The length or sequence of the 5' overhang of the second strand of the cell-free DNA duplex is determined by soft shearing of the 3' inosine extension of the first strand.

[0310] Implementation Scheme 66. The system of claim 65, wherein the instructions further cause the system to detect the presence or absence of a disease based on the length or sequence of the 5' overhang of the second strand of the cell-free DNA duplex.

[0311] Implementation Scheme 67. The system of claim 66, wherein the disease is cancer.

[0312] Implementation Scheme 68. The system of any one of claims 65 to 67, wherein the first sequence readout is further obtained by: linking a second sequencing adaptor to the 3' overhang of the second strand of the cell-free DNA duplex; extending the 3' end of the second sequencing adaptor with inosine bases to fill the 3' overhang of the second strand of the cell-free DNA duplex; and linking the 3' inosine-extended end of the second sequencing adaptor to the 5' end of the first strand of the cell-free DNA duplex, wherein the linked 3' inosine-extended end provides a 5' inosine extension of the first strand of the cell-free DNA duplex.

[0313] Implementation Scheme 69. The system of claim 68, wherein the instructions, when executed by the one or more processors, further cause the system to:

[0314] Read from the first sequence the soft-cleaved bases corresponding to the 5' inosine extension of the first strand of the cell-free DNA double helix; and

[0315] The length or sequence of the 3' overhang of the second strand of the cell-free DNA duplex is determined by soft shearing of the 5' inosine extension of the first strand.

[0316] Implementation Scheme 70. The system of claim 69, wherein the instructions further cause the system to detect the presence or absence of a disease based on the length or sequence of the 3' overhang of the second strand of the cell-free DNA duplex.

[0317] Implementation Scheme 71. The system of any one of claims 68 to 70, wherein linking the second sequencing adaptor to the 3' overhang of the second strand of the cell-free DNA duplex comprises:

[0318] Extending the 3' overhang of the second strand of the cell-free DNA duplex to provide 3' extension, wherein the second sequencing adaptor includes a 3' overhang complementary to the 3' extension of the second strand of the cell-free DNA duplex; and

[0319] The second sequencing adaptor is ligated to the 3' extension of the second strand of the cell-free DNA duplex.

[0320] Implementation Scheme 72. The system of claim 71, wherein the 3' overhang of the second strand of the cell-free DNA duplex is extended using nucleotide bases of the same base type.

[0321] Implementation Scheme 73. The system of any one of claims 65 to 72, wherein one or more bases in the first sequence readout are determined to be soft-cut by a method comprising aligning the first sequence readout with a reference sequence and identifying unaligned portions of the first sequence readout.

[0322] Implementation Scheme 74. The system of any one of claims 65 to 73, wherein the instructions, when executed by the one or more processors, further cause the system to:

[0323] Receive a second sequence readout obtained by sequencing the second strand of the cell-free DNA duplex, and

[0324] Identify one or more bases in the second sequence readout that are to be soft-cleaved.

[0325] Implementation Scheme 75. The system of claim 74, wherein one or more bases read from the second sequence are determined to be soft-cut according to a method comprising aligning the second sequence with a reference sequence and identifying unaligned portions of the second sequence read.

[0326] Implementation scheme 76. The system of claim 74 or 75, wherein the first sequence readout and the second sequence readout are associated by a unique molecular identifier (UMI).

[0327] Implementation Scheme 77. The system of claim 76, wherein one or more bases of the first sequence readout are determined to be soft-cut according to a method comprising aligning the first sequence with the second sequence readout and identifying unaligned portions of the first sequence readout.

[0328] Implementation Scheme 78. The system of claim 76 or 77, wherein one or more bases of the second sequence readout are determined to be soft-cut according to a method comprising aligning the second sequence with the first sequence readout and identifying unaligned portions of the second sequence readout.

[0329] Implementation Scheme 79. The system of any one of claims 74 to 78, wherein the second sequence readout is further obtained by: extending the 3' end of the second strand of the cell-free DNA duplex with inosine bases to fill the 5' overhang of the first strand of the cell-free DNA duplex; and linking the second sequencing adaptor to the cell-free DNA duplex.

[0330] Implementation Scheme 80. The system of claim 79, wherein the instructions, when executed by the one or more processors, further cause the system to:

[0331] Read from the second sequence the soft-cleaved bases corresponding to the 3' inosine extension of the second strand of the cell-free DNA double helix; and

[0332] The length or sequence of the 5' overhang of the first strand of the cell-free DNA duplex is determined by soft shearing of the 3' inosine extension of the second strand of the cell-free DNA duplex.

[0333] Implementation Scheme 81. The system of any one of claims 65 to 80, wherein the 3' end of the first strand extending the cell-free DNA duplex includes forming a single 3' inosine overhang.

[0334] Implementation Scheme 82. The system of claim 81, wherein the sequencing adaptor comprises a 3' cytosine overhang complementary to the 3' inosine overhang.

[0335] Implementation plan 83. System, which includes:

[0336] One or more processors; and

[0337] A memory, communicatively coupled to the one or more processors and configured to store instructions, which, when executed by the one or more processors, cause the system to:

[0338] Receive a first sequence readout obtained by: linking a sequencing adaptor to the 3' overhang of the first strand of a cell-free DNA duplex; extending the 3' end of the sequencing adaptor with inosine bases to fill the 3' overhang of the first strand of the cell-free DNA duplex; and linking the 3' inosine-extended end of the sequencing adaptor to the 5' end of the second strand of the cell-free DNA duplex, wherein the linked 3' inosine-extended end provides a 5' inosine extension of the second strand of the cell-free DNA duplex;

[0339] Identify one or more bases in the first sequence readout that are to be soft-cleaved;

[0340] Read from the first sequence the soft-cleaved bases corresponding to the 5' inosine extension of the second strand of the cell-free DNA double helix; and

[0341] The length or sequence of the 3' overhang of the first strand of the cell-free DNA duplex is determined by soft shearing of the 5' inosine extension of the second strand of the cell-free DNA duplex.

[0342] Implementation Scheme 84. The system of claim 83, wherein the instructions further cause the system to detect the presence or absence of a disease based on the length or sequence of the 3' overhang of the first strand of the cell-free DNA duplex.

[0343] Implementation scheme 85. The system of claim 84, wherein the disease is cancer.

[0344] Implementation Scheme 86. The system of any one of claims 83 to 85, wherein linking the second sequencing adaptor to the 3' overhang of the second strand of the cell-free DNA duplex comprises:

[0345] Extending the 3' overhang of the second strand of the cell-free DNA duplex to provide 3' extension, wherein the second sequencing adaptor includes a 3' overhang complementary to the 3' extension of the second strand of the cell-free DNA duplex; and

[0346] The second sequencing adaptor is ligated to the 3' extension of the second strand of the cell-free DNA duplex.

[0347] Implementation Scheme 87. The system of claim 86, wherein the 3' overhang of the second strand of the cell-free DNA duplex is extended using nucleotide bases of the same base type.

[0348] Implementation Scheme 88. The system of any one of claims 83 to 87, wherein one or more bases in the first sequence readout are determined to be soft-cut by a method comprising aligning the first sequence readout with a reference sequence and identifying unaligned portions of the first sequence readout.

[0349] Implementation Scheme 89. The system of any one of claims 83 to 88, wherein the instructions, when executed by the one or more processors, further cause the system to:

[0350] Receive a second sequence readout obtained by sequencing the second strand of the cell-free DNA duplex, and

[0351] Identify one or more bases in the second sequence readout that are to be soft-cleaved.

[0352] Implementation Scheme 90. The system of claim 89, wherein one or more bases read from the second sequence are determined to be soft-cut according to a method comprising aligning the second sequence with a reference sequence and identifying unaligned portions of the second sequence read.

[0353] Implementation scheme 91. The system of claim 89 or 90, wherein the first sequence readout and the second sequence readout are associated by a unique molecular identifier (UMI).

[0354] Implementation Scheme 92. The system of claim 91, wherein one or more bases in the first sequence readout are determined to be soft-cut according to a method comprising aligning the first sequence with the second sequence readout and identifying unaligned portions of the first sequence readout.

[0355] Implementation Scheme 93. The system of claim 91 or 92, wherein one or more bases of the second sequence readout are determined to be soft-cut according to a method comprising aligning the second sequence with the first sequence readout and identifying unaligned portions of the second sequence readout.

[0356] Implementation Scheme 94. The system of any one of claims 89 to 93, wherein the second sequence readout is further obtained by:

[0357] The second sequencing adaptor is linked to the 3' overhang of the second strand of the cell-free DNA duplex;

[0358] The 3' end of the second sequencing adaptor is extended with inosine bases to fill the 3' overhang of the second strand of the cell-free DNA duplex;

[0359] The 3' inosine extension of the second sequencing adapter is joined to the 5' end of the first strand of the cell-free DNA duplex, wherein the 3' inosine extension provides the 5' inosine extension of the first strand of the cell-free DNA duplex.

[0360] Implementation Scheme 95. The system of claim 94, wherein the instructions, when executed by the one or more processors, further cause the system to:

[0361] Read from the second sequence the soft-cleaved bases corresponding to the 3' inosine extension of the first strand of the cell-free DNA double helix; and

[0362] The length or sequence of the 5' overhang of the second strand of the cell-free DNA duplex is determined by soft shearing of the 3' inosine extension of the first strand.

[0363] Implementation Scheme 96. The system of claim 94 or 95, wherein linking the second sequencing adaptor to the 3' overhang of the second strand of the cell-free DNA duplex comprises linking a monoinosine to the 3' overhang of the second strand of the cell-free DNA duplex and linking the second sequencing adaptor to the monoinosine.

[0364] Implementation Scheme 97. The system of claim 96, wherein the sequencing adaptor comprises a 3' cytosine overhang complementary to the single inosine and connected to the 3' overhang of the second strand of the cell-free DNA duplex.

[0365] Implementation scheme 98. The system of any one of claims 65 to 97, wherein the sequencing adaptor or the second sequencing adaptor comprises the UMI.

[0366] Implementation Scheme 99. The system of any one of claims 65 to 98, wherein the sequencing adaptor or the second sequencing adaptor is a Y-shaped sequencing adaptor.

[0367] Implementation Scheme 100. The system of any one of claims 65 to 99, further comprising a nucleic acid amplification instrument configured to amplify the cell-free DNA duplex to link a sample index to the cell-free DNA duplex.

[0368] Implementation Scheme 101. The system of claim 100, wherein the nucleic acid amplification instrument is a thermal cycler.

[0369] Implementation scheme 102. The system of any one of claims 65 to 101, wherein the cell-free DNA duplex is obtained from a subject suspected of having cancer or confirmed to have cancer.

[0370] Implementation scheme 103. The system of any one of claims 65 to 102, wherein the cell-free DNA duplex is obtained from the object.

[0371] Implementation scheme 104. The system of any one of claims 65 to 103, wherein the cell-free DNA duplex is obtained from a liquid biopsy sample.

[0372] Implementation Scheme 105. The system of claim 104, wherein the sample is a liquid biopsy sample and comprises blood, plasma, cerebrospinal fluid, sputum, feces, urine or saliva.

[0373] Implementation Scheme 106. The system of any one of claims 65 to 105, wherein the cell-free DNA duplex is a circulating tumor DNA (ctDNA) duplex.

[0374] Implementation Scheme 107. The system of any one of claims 65 to 106, wherein the sequencing adaptor or the second sequencing adaptor comprises an amplification primer binding site, a flow cell adaptor sequence, or a substrate adaptor sequence.

[0375] Implementation Scheme 108. The system of any one of claims 65 to 107, further comprising a nucleic acid amplification instrument configured to amplify the first and second strands of the cell-free DNA duplex.

[0376] Implementation Scheme 109. The system of claim 108, wherein the amplification includes polymerase chain reaction (PCR) amplification, non-PCR amplification, or isothermal amplification.

[0377] Implementation Scheme 110. The system of any one of claims 65 to 109, further comprising a sequencer configured to sequence the first strand and / or the second strand of the cell-free DNA duplex.

[0378] Implementation Scheme 111. The system of any one of claims 65 to 110, wherein the sequencing comprises using massively parallel sequencing (MPS), whole genome sequencing (WGS), whole exome sequencing, targeted sequencing, direct sequencing, or Sanger sequencing.

[0379] Implementation Scheme 112. The system of claim 111, wherein the sequencing comprises massively parallel sequencing, and the massively parallel sequencing technology comprises next-generation sequencing (NGS).

[0380] Implementation scheme 113. The system of any one of claims 65 to 112, wherein the sequencing is performed using a next-generation sequencer.

[0381] Implementation Scheme 114. The system of any one of claims 65 to 113, wherein the instructions, when executed by the one or more processors, further cause the system to generate a report indicating the lengths of: the 3' overhang of the first strand of the cell-free DNA duplex, the 3' overhang of the second strand of the cell-free DNA duplex, the 5' overhang of the first strand of the cell-free DNA duplex, and / or the 5' overhang of the second strand of the cell-free DNA duplex.

[0382] Implementation scheme 115. The system of claim 114, wherein the instructions, when executed by the one or more processors, further cause the system to transmit the report to a healthcare provider.

[0383] Implementation scheme 116. The system of claim 114 or 115, wherein the report is transmitted via a computer network or peer-to-peer connection.

[0384] Implementation Scheme 117. The system of any one of claims 65 to 116, wherein the instructions, when executed by the one or more processors, further cause the system to generate a genomic spectrum of the object by the one or more processors, the genomic spectrum comprising the following lengths: a 3' overhang of the first strand of the cell-free DNA duplex, a 3' overhang of the second strand of the cell-free DNA duplex, a 5' overhang of the first strand of the cell-free DNA duplex, and / or a 5' overhang of the second strand of the cell-free DNA duplex.

[0385] Implementation Scheme 118. The system of claim 117, wherein the genomic profile of the object further comprises results from: comprehensive genome profiling (CGP) test, gene expression profiling test, cancer hotspot group test, DNA methylation test, DNA fragmentation test, RNA fragmentation test, or any combination thereof.

[0386] Implementation scheme 119. The system of claim 117 or 118, wherein the genomic profile of the object further comprises results from a nucleic acid sequencing-based test.

[0387] Implementation Scheme 120. A non-transitory computer-readable storage medium storing one or more programs, said one or more programs comprising instructions that, when executed by one or more processors of a system, cause the system to:

[0388] Receive a first sequence readout obtained by: extending the 3' end of the first strand of the cell-free DNA duplex with inosine bases to fill the 5' overhang of the second strand of the cell-free DNA duplex; connecting a sequencing adaptor to the cell-free DNA duplex; and sequencing the first strand of the cell-free DNA duplex to produce the first sequence readout;

[0389] Identify one or more bases in the first sequence readout that are to be soft-cleaved;

[0390] Read from the first sequence the soft cut bases corresponding to the 3' inosine extension of the first strand of the cell-free DNA double helix; and

[0391] The length or sequence of the 5' overhang of the second strand of the cell-free DNA duplex is determined by soft shearing of the 3' inosine extension of the first strand.

[0392] Implementation scheme 121. The storage medium of claim 120, wherein the instructions further cause the system to detect the presence or absence of a disease based on the length or sequence of the 5' overhang of the second strand of the cell-free DNA duplex.

[0393] Implementation scheme 122. The storage medium of claim 121, wherein the disease is cancer.

[0394] Implementation Scheme 123. The storage medium of any one of claims 120 to 122, wherein the first sequence readout is further obtained by: connecting a second sequencing adaptor to the 3' overhang of the second strand of the cell-free DNA duplex; extending the 3' end of the second sequencing adaptor with inosine bases to fill the 3' overhang of the second strand of the cell-free DNA duplex; and connecting the 3' inosine-extended end of the second sequencing adaptor to the 5' end of the first strand of the cell-free DNA duplex, wherein the connected 3' inosine-extended end provides a 5' inosine extension of the first strand of the cell-free DNA duplex.

[0395] Implementation Scheme 124. The storage medium of claim 123, wherein the instructions, when executed by the one or more processors, further cause the system to:

[0396] Read from the first sequence the soft-cleaved bases corresponding to the 5' inosine extension of the first strand of the cell-free DNA double helix; and

[0397] The length or sequence of the 3' overhang of the second strand of the cell-free DNA duplex is determined by soft shearing of the 5' inosine extension of the first strand of the cell-free DNA duplex.

[0398] Implementation scheme 125. The storage medium of claim 124, wherein the instructions further cause the system to detect the presence or absence of a disease based on the length or sequence of the 3' overhang of the second strand of the cell-free DNA duplex.

[0399] Implementation Scheme 126. The storage medium of any one of claims 123 to 125, wherein linking the second sequencing adaptor to the 3' overhang of the second strand of the cell-free DNA duplex comprises:

[0400] Extending the 3' overhang of the second strand of the cell-free DNA duplex to provide 3' extension, wherein the second sequencing adaptor includes a 3' overhang complementary to the 3' extension of the second strand of the cell-free DNA duplex; and

[0401] The second sequencing adaptor is ligated to the 3' extension of the second strand of the cell-free DNA duplex.

[0402] Implementation Scheme 127. The storage medium of claim 126, wherein the 3' overhang of the second strand of the cell-free DNA duplex is extended using nucleotide bases of the same base type.

[0403] Implementation Scheme 128. The storage medium of any one of claims 120 to 127, wherein one or more bases of the first sequence readout are determined to be soft-cut by a method comprising aligning the first sequence readout with a reference sequence and identifying unaligned portions of the first sequence readout.

[0404] Implementation Scheme 129. The storage medium of any one of claims 120 to 128, wherein the instructions, when executed by the one or more processors, further cause the system to:

[0405] Receive a second sequence readout obtained by sequencing the second strand of the cell-free DNA duplex, and

[0406] Identify one or more bases in the second sequence readout that are to be soft-cleaved.

[0407] Implementation Scheme 130. The storage medium of claim 129, wherein one or more bases read from the second sequence are determined to be soft-cut according to a method comprising aligning the second sequence with a reference sequence and identifying unaligned portions of the second sequence read.

[0408] Implementation scheme 131. The storage medium of claim 129 or 130, wherein the first sequence readout and the second sequence readout are associated by a unique molecular identifier (UMI).

[0409] Implementation Scheme 132. The storage medium of claim 131, wherein one or more bases of the first sequence readout are determined to be soft-cut according to a method comprising comparing the first sequence with the second sequence readout and identifying unaligned portions of the first sequence readout.

[0410] Implementation Scheme 133. The storage medium of claim 131 or 132, wherein one or more bases of the second sequence readout are determined to be soft-cut according to a method comprising comparing the second sequence with the first sequence readout and identifying unaligned portions of the second sequence readout.

[0411] Implementation Scheme 134. The storage medium of any one of claims 129 to 133, wherein the second sequence readout is further obtained by: extending the 3' end of the second strand of the cell-free DNA duplex with inosine bases to fill the 5' overhang of the first strand of the cell-free DNA duplex; and connecting the second sequencing adaptor to the cell-free DNA duplex.

[0412] Implementation Scheme 135. The storage medium of claim 134, wherein the instructions, when executed by the one or more processors, further cause the system to:

[0413] Read from the second sequence the soft-cleaved bases corresponding to the 3' inosine extension of the second strand of the cell-free DNA double helix; and

[0414] The length or sequence of the 5' overhang of the first strand of the cell-free DNA duplex is determined by soft shearing of the 3' inosine extension of the second strand of the cell-free DNA duplex.

[0415] Implementation Scheme 136. The storage medium of any one of claims 120 to 135, wherein the 3' end of the first strand extending the cell-free DNA duplex includes a 3' single inosine overhang.

[0416] Implementation Scheme 137. The storage medium of claim 136, wherein the sequencing adaptor comprises a 3' cytosine overhang complementary to the single 3' inosine overhang.

[0417] Implementation Scheme 138. A non-transitory computer-readable storage medium storing one or more programs, said one or more programs comprising instructions that, when executed by one or more processors of a system, cause the system to:

[0418] Receive a first sequence readout obtained by: linking a sequencing adaptor to the 3' overhang of the first strand of a cell-free DNA duplex; extending the 3' end of the sequencing adaptor with inosine bases to fill the 3' overhang of the first strand of the cell-free DNA duplex; and linking the 3' inosine-extended end of the sequencing adaptor to the 5' end of the second strand of the cell-free DNA duplex, wherein the linked 3' inosine-extended end provides a 5' inosine extension of the second strand of the cell-free DNA duplex;

[0419] Identify one or more bases in the first sequence readout that are to be soft-cleaved;

[0420] Read from the first sequence the soft cut bases corresponding to the 5' inosine extension of the second strand of the cell-free DNA double helix; and

[0421] The length or sequence of the 3' overhang of the first strand of the cell-free DNA duplex is determined by soft shearing of the 5' inosine extension of the second strand of the cell-free DNA duplex.

[0422] Implementation scheme 139. The storage medium of claim 138, wherein the instructions further cause the system to detect the presence or absence of a disease based on the length or sequence of the 3' overhang of the first strand of the cell-free DNA duplex.

[0423] Implementation scheme 140. The storage medium of claim 139, wherein the disease is cancer.

[0424] Implementation Scheme 141. The storage medium of any one of claims 138 to 140, wherein linking the second sequencing adaptor to the 3' overhang of the second strand of the cell-free DNA duplex comprises:

[0425] Extending the 3' overhang of the second strand of the cell-free DNA duplex to provide 3' extension, wherein the second sequencing adaptor includes a 3' overhang complementary to the 3' extension of the second strand of the cell-free DNA duplex; and

[0426] The second sequencing adaptor is ligated to the 3' extension of the second strand of the cell-free DNA duplex.

[0427] Implementation Scheme 142. The storage medium of claim 141, wherein the 3' overhang of the second strand of the cell-free DNA duplex is extended using nucleotide bases of the same base type.

[0428] Implementation Scheme 143. The storage medium of any one of claims 138 to 142, wherein one or more bases of the first sequence readout are determined to be soft-cut by a method comprising aligning the first sequence readout with a reference sequence and identifying unaligned portions of the first sequence readout.

[0429] Implementation Scheme 144. The storage medium of any one of claims 138 to 143, wherein the instructions, when executed by the one or more processors, further cause the system to:

[0430] Receive a second sequence readout obtained by sequencing the second strand of the cell-free DNA duplex, and

[0431] Identify one or more bases in the second sequence readout that are to be soft-cleaved.

[0432] Implementation Scheme 145. The storage medium of claim 144, wherein one or more bases read from the second sequence are determined to be soft-cut according to a method comprising aligning the second sequence with a reference sequence and identifying unaligned portions of the second sequence read.

[0433] Implementation scheme 146. The storage medium of claim 144 or 145, wherein the first sequence readout and the second sequence readout are associated by a unique molecular identifier (UMI).

[0434] Implementation Scheme 147. The storage medium of claim 146, wherein one or more bases of the first sequence readout are determined to be soft-cut according to a method comprising comparing the first sequence with the second sequence readout and identifying unaligned portions of the first sequence readout.

[0435] Implementation Scheme 148. The storage medium of claim 146 or 147, wherein one or more bases of the second sequence readout are determined to be soft-cut according to a method comprising comparing the second sequence with the first sequence readout and identifying unaligned portions of the second sequence readout.

[0436] Implementation Scheme 149. The storage medium of any one of claims 144 to 148, wherein the second sequence readout is further obtained by:

[0437] The second sequencing adaptor is linked to the 3' overhang of the second strand of the cell-free DNA duplex;

[0438] Extend the 3' end of the second sequencing adaptor with inosine bases to fill the 3' overhang of the second strand of the cell-free DNA duplex; and

[0439] The 3' inosine extension of the second sequencing adapter is joined to the 5' end of the first strand of the cell-free DNA duplex, wherein the 3' inosine extension provides the 5' inosine extension of the first strand of the cell-free DNA duplex.

[0440] Implementation Scheme 150. The storage medium of claim 149, wherein the instructions, when executed by the one or more processors, further cause the system to:

[0441] Read from the second sequence the soft-cleaved bases corresponding to the 3' inosine extension of the first strand of the cell-free DNA double helix; and

[0442] The length or sequence of the 5' overhang of the second strand of the cell-free DNA duplex is determined by soft shearing of the 3' inosine extension of the first strand.

[0443] Implementation Scheme 151. The storage medium of claim 149 or 150, wherein linking the second sequencing adaptor to the 3' overhang of the second strand of the cell-free DNA duplex comprises linking a monoinosine to the 3' overhang of the second strand of the cell-free DNA duplex and linking the second sequencing adaptor to the monoinosine.

[0444] Implementation Scheme 152. The storage medium of claim 151, wherein the sequencing adaptor comprises a single inosine-complementary 3' cytosine overhang connected to the 3' overhang of the second strand of the cell-free DNA duplex.

[0445] Implementation Scheme 153. The storage medium of any one of claims 120 to 152, wherein the sequencing adapter or the second sequencing adapter comprises the UMI.

[0446] Implementation Scheme 154. The storage medium of any one of claims 120 to 153, wherein the sequencing adaptor or the second sequencing adaptor is a Y-shaped sequencing adaptor.

[0447] Implementation Scheme 155. The storage medium of any one of claims 120 to 154, wherein the cell-free DNA double strand is obtained from a subject suspected of having cancer or confirmed to have cancer.

[0448] Implementation Scheme 156. The storage medium of any one of claims 120 to 155, wherein the cell-free DNA duplex is obtained from the object.

[0449] Implementation Scheme 157. The storage medium of any one of claims 120 to 156, wherein the cell-free DNA duplex is obtained from a liquid biopsy sample.

[0450] Implementation scheme 158. The storage medium of claim 157, wherein the sample is a liquid biopsy sample and comprises blood, plasma, cerebrospinal fluid, sputum, feces, urine or saliva.

[0451] Implementation Scheme 159. The storage medium of any one of claims 120 to 158, wherein the cell-free DNA duplex is a circulating tumor DNA (ctDNA) duplex.

[0452] Implementation Scheme 160. The storage medium of any one of claims 120 to 159, wherein the sequencing adaptor or the second sequencing adaptor comprises an amplification primer binding site, a flow cell adaptor sequence, or a substrate adaptor sequence.

[0453] Implementation Scheme 161. The storage medium of any one of claims 120 to 160, wherein the sequencing comprises using massively parallel sequencing (MPS), whole genome sequencing (WGS), whole exome sequencing, targeted sequencing, direct sequencing, or Sanger sequencing.

[0454] Implementation Scheme 162. The storage medium of claim 161, wherein the sequencing comprises massively parallel sequencing, and the massively parallel sequencing technology comprises next-generation sequencing (NGS).

[0455] Implementation Scheme 163. The storage medium of any one of claims 120 to 162, wherein the sequencing is performed using a next-generation sequencer.

[0456] Implementation Scheme 164. The storage medium of any one of claims 120 to 163, wherein the instructions, when executed by the one or more processors, further cause the system to generate a report indicating the lengths of: the 3' protrusion of the first strand of the cell-free DNA duplex, the 3' protrusion of the second strand of the cell-free DNA duplex, the 5' protrusion of the first strand of the cell-free DNA duplex, and / or the 5' protrusion of the second strand of the cell-free DNA duplex.

[0457] Implementation scheme 165. The storage medium of claim 164, wherein the instructions, when executed by the one or more processors, further cause the system to transmit the report to a healthcare provider.

[0458] Implementation scheme 166. The storage medium of claim 164 or 165, wherein the report is transmitted via a computer network or peer-to-peer connection.

[0459] Implementation Scheme 167. The storage medium of any one of claims 120 to 166, wherein the instructions, when executed by the one or more processors, further cause the system to generate a genomic spectrum of the object by the one or more processors, the genomic spectrum comprising the following lengths: a 3' overhang of the first strand of the cell-free DNA duplex, a 3' overhang of the second strand of the cell-free DNA duplex, a 5' overhang of the first strand of the cell-free DNA duplex, and / or a 5' overhang of the second strand of the cell-free DNA duplex.

[0460] Implementation Scheme 168. The storage medium of claim 167, wherein the genomic profile of the object further comprises results from: comprehensive genome profiling (CGP) test, gene expression profiling test, cancer hotspot group test, DNA

[0461] Methylation test, DNA fragmentation test, RNA fragmentation test, or any combination thereof.

[0462] Implementation scheme 169. The storage medium of claim 167 or 168, wherein the genomic spectrum of the object further comprises results from a nucleic acid sequencing-based test.

[0463] It should be understood from the foregoing that while specific embodiments of the disclosed methods and systems have been shown and described, various modifications are possible and such modifications are contemplated herein. The invention is not intended to be limited by the specific examples provided in the specification. Although the invention has been described with reference to the foregoing specification, the description and illustrations of preferred embodiments herein are not intended to be limiting. Furthermore, it should be understood that all aspects of the invention are not limited to the specific descriptions, configurations, or relative proportions set forth herein, and depend on a variety of conditions and variables. Various modifications in form and detail of embodiments of the invention will be apparent to those skilled in the art. Therefore, it is contemplated that the invention should also cover any such modifications, variations, or equivalents.

Claims

1. A method for determining the topological structure of cell-free DNA, comprising: The 3' end of the first strand of the cell-free DNA duplex is extended with inosine bases to fill the 5' overhang of the second strand of the cell-free DNA duplex. The sequencing adaptor is linked to the cell-free DNA duplex; The first strand of the cell-free DNA duplex is sequenced to produce a first sequence readout. One or more bases to be soft-sheared in the first sequence readout are determined by one or more processors; The soft shearing of the bases corresponding to the 3' inosine extension of the first strand of the cell-free DNA duplex is performed by one or more processors from the first sequence; as well as The length or sequence of the 5' overhang of the second strand of the cell-free DNA duplex is determined by one or more processors based on soft shearing of the 3' inosine extension of the first strand of the cell-free DNA duplex.

2. The method of claim 1, further comprising: The second sequencing adaptor is connected to the 3' overhang of the second strand of the cell-free DNA duplex; The 3' end of the second sequencing adaptor is extended with inosine bases to fill the 3' overhang of the second strand of the cell-free DNA duplex; The 3' inosine extension of the second sequencing adapter is linked to the 5' end of the first strand of the cell-free DNA duplex, wherein the linked 3' inosine extension provides the 5' inosine extension of the first strand of the cell-free DNA duplex. One or more processors are used to determine one or more bases that are connected to the 5' end of the first sequence to be soft-cut; The soft shearing of the bases corresponding to the 5' inosine extension of the first strand of the cell-free DNA duplex is performed by one or more processors from the first sequence; as well as The length or sequence of the 3' overhang of the second strand of the cell-free DNA duplex is determined by one or more processors based on soft shearing of the 5' inosine extension of the first strand of the cell-free DNA duplex.

3. The method of claim 2, wherein linking the second sequencing adaptor to the 3' overhang of the second strand of the cell-free DNA duplex comprises: Extending the 3' overhang of the second strand of the cell-free DNA duplex to provide 3' extension, wherein the second sequencing adaptor includes a 3' overhang complementary to the 3' extension of the second strand of the cell-free DNA duplex; and The second sequencing adaptor is ligated to the 3' extension of the second strand of the cell-free DNA duplex.

4. The method of claim 1, wherein determining one or more bases to be soft-cleaved in the first sequence readout comprises aligning the first sequence readout with a reference sequence and identifying unaligned portions of the first sequence readout.

5. The method of claim 1, further comprising: The second strand of the cell-free DNA duplex is sequenced to produce a second sequence readout. as well as One or more bases to be soft-cleaved in the second sequence readout are determined by one or more processors.

6. The method of claim 5, wherein determining one or more bases to be soft-cleaved in the second sequence readout comprises aligning the second sequence with a reference sequence and identifying unaligned portions of the second sequence readout.

7. The method of claim 5, wherein the first sequence readout and the second sequence readout are associated by a unique molecular identifier (UMI).

8. The method of claim 7, wherein: Determining one or more bases to be soft-cleaved in the first sequence readout includes comparing the first sequence with the second sequence readout and identifying the unaligned portions of the first sequence readout; or Identifying one or more bases to be soft-cleaved in the second sequence readout includes aligning the second sequence with the first sequence readout and identifying any unaligned portions of the second sequence readout.

9. The method of claim 5, comprising: The 3' end of the second strand of the cell-free DNA duplex is extended with inosine bases to fill the 5' overhang of the first strand of the cell-free DNA duplex. The second sequencing adaptor is linked to the cell-free DNA duplex; Through one or more processors, the soft cleavage of the bases corresponding to the 3' inosine extension of the second strand of the cell-free DNA duplex is read from the second sequence; as well as The length or sequence of the 5' overhang of the first strand of the cell-free DNA duplex is determined by one or more processors based on soft shearing of the 3' inosine extension of the second strand of the cell-free DNA duplex.

10. The method of claim 1, wherein extending the 3' end of the first strand of the cell-free DNA duplex includes forming a single 3' inosine overhang.

11. The method of claim 10, wherein the sequencing adaptor comprises a 3' cytosine overhang complementary to the 3' inosine overhang.

12. A method for preparing sequencing constructs, comprising: The sequencing adaptor is linked to the 3' overhang of the first strand of the cell-free DNA duplex; The 3' end of the sequencing adaptor is extended with inosine bases to fill the 3' overhang of the first strand of the cell-free DNA duplex; as well as The 3' inosine extension of the sequencing adapter is linked to the 5' end of the second strand of the cell-free DNA duplex, wherein the linked 3' inosine extension provides the 5' inosine extension of the second strand of the cell-free DNA duplex.

13. The method of claim 12, wherein linking the sequencing adaptor to the 3' overhang of the first strand of the cell-free DNA duplex comprises: Extending the 3' overhang of the first strand of the cell-free DNA duplex to provide 3' extension, wherein the sequencing adaptor includes a 3' overhang complementary to the 3' extension of the first strand of the cell-free DNA duplex; and The sequencing adaptor is ligated to the 3' extension of the first strand of the cell-free DNA duplex.

14. A method for determining the topological structure of cell-free DNA, comprising: The sequencing construct is prepared by the method according to any one of claims 12 to 13; Sequencing the second strand of the cell-free DNA double helix to produce a first sequence readout; One or more bases to be soft-sheared in the first sequence readout are determined by one or more processors; The first sequence is read from by one or more processors, where the bases corresponding to the 5' inosine extension of the second strand of the cell-free DNA duplex are softly cleaved. as well as The length or sequence of the 3' overhang of the first strand of the cell-free DNA duplex is determined by one or more processors based on soft shearing of the 5' inosine extension of the second strand of the cell-free DNA duplex.

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