Systems and methods for targeted nucleic acid capture

Abstract

To provide systems and methods for targeted indirect, synergistic hybridization capture of a template for amplification and analysis of target sequences.SOLUTION: Provided is a method of sequential enrichment including the steps of: obtaining a sample comprising a plurality of nucleic acid molecules; performing a first target enrichment to enrich for nucleic acid molecules comprising sequences corresponding to a first panel of one or more genome regions, thereby generating a first enriched sample comprising nucleic acids enriched for sequences corresponding to the first panel and a remaining sample comprising nucleic acids depleted for sequences corresponding to the first panel; and performing a second target enrichment upon the remaining sample to enrich for nucleic acid molecules comprising sequences corresponding to a second panel of one or more genome regions, thereby generating a second enriched sample comprising nucleic acids enriched for sequences corresponding to the second panel, wherein the first panel and the second panel are different.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] cross reference This application claims the benefit of U.S. Provisional Application No. 62 / 968,847, filed January 31, 2020, U.S. Provisional Application No. 62 / 987,232, filed March 9, 2020, and U.S. Provisional Application No. 62 / 988,859, filed March 12, 2020, which applications are incorporated herein by reference. This application is related to the following co-pending patent application: International Application No. PCT / US2019 / 062508, filed November 20, 2019, which is incorporated herein by reference. [Background technology]

[0002] background Nucleic acid target capture method can make it possible to enrich specific genes, exons, and other genomic regions of interest, for example, for targeted sequencing.However, target capture-based sequencing methods can involve cumbersome and lengthy protocols and costly processes, and also have low on-target rates for small capture panels (e.g., less than 500 probes).In addition, current nucleic acid target capture methods can be unsuitable for low input and damaged DNA due to low recovery rates. Bisulfite conversion can be a useful technique for studying the methylation patterns of nucleic acid molecules. However, bisulfite conversion can damage nucleic acids, for example, by creating truncations. Treating next-generation sequencing (NGS) DNA libraries with bisulfite can damage a substantial amount of nucleic acid, making it unrecoverable in subsequent amplification steps and resulting in low recovery rates. Furthermore, because bisulfite conversion can result in single-stranded or fragmented DNA and reduced sequence complexity, the converted DNA can be difficult to input into traditional adapter-ligation-based library construction. Given the generally small initial input and low recovery rates (e.g., 5% or less of bisulfite-treated cfDNA), bisulfite-treated cell-free DNA (cfDNA) or circulating tumor cell DNA (ctDNA) may present a greater challenge. Methylation-sensitive enzyme treatment can also be performed to convert methylated cytosines. However, enzyme-based approaches still suffer from loss of methylation status during the long, multistep process, resulting in low recovery rates.

[0003] Methylation analysis in cell-free DNA has great potential for early cancer detection. In the plasma of patients with early-stage cancer, tumor content is estimated to be less than 0.1%, often as low as 0.01% or even lower, thus necessitating highly sensitive assays. Currently, there are two main approaches used for cancer screening: comprehensive approaches, including whole-genome bisulfite sequencing (WGBS), reduced-representation bisulfite sequencing (RRBS), or affinity-based enrichment, and large targeted panels containing 10,000 or more potential methylation markers. Targeted methylation sequencing (TMS) offers the most sensitive and specific analysis of methylation markers. However, the sensitivity and specificity of conventional TMS are compromised by the low efficiency and low recovery of target enrichment and are further hindered by the background noise associated with large panels. A method for thorough analysis using small, focused cancer-specific methylation biomarker panels is needed.

[0004] Therefore, there is a need for more efficient, easy to use, fast, flexible and practical target nucleic acid capture method, and for the improved method of analyzing bisulfite-treated nucleic acid, especially for low-input samples such as cfDNA.The method disclosed herein can be used for the pre-amplification and pre-bisulfite conversion hybridization-based capture for very low DNA input samples. Summary of the Invention [Means for solving the problem]

[0005] overview Disclosed herein is a method comprising the steps of: obtaining a template nucleic acid molecule comprising an adapter at its 5'-end or 3'-end; hybridizing a first target-specific region of a first crosslinking probe to a first target sequence of the template nucleic acid molecule, wherein a first adapter landing sequence of the first crosslinking probe binds to a first crosslinking sequence of the adapter-anchor probe; and hybridizing a second target-specific region of a second crosslinking probe to a second target sequence of the template nucleic acid molecule, wherein a second adapter landing sequence of the second crosslinking probe binds to a second crosslinking sequence of the adapter-anchor probe. The method may further comprise attaching an adapter to the 5'-end or 3'-end of a sample nucleic acid molecule, thereby generating an adapter-containing template nucleic acid molecule. The method may further comprise attaching an adapter to the 5'-end or 3'-end of the sample nucleic acid molecule and attaching an adapter to the 3'-end or 5'-end of the adapter-containing template nucleic acid molecule, respectively, thereby generating a template nucleic acid molecule comprising an adapter at each end. The method may further include hybridizing an adapter primer to the adapter attached to the 3' end of the template nucleic acid molecule hybridized to the first bridge probe and the second bridge probe, and extending the 3' end of the adapter primer, thereby generating an extension product. The method may further include sequencing the extension product.

[0006] Prior to hybridizing to the first target-specific region, the first adapter landing sequence of the first cross-linking probe may be bound to the first cross-linking sequence of the adapter anchor probe. After hybridizing to the first target-specific region, the first adapter landing sequence of the first cross-linking probe may be bound to the first cross-linking sequence of the adapter anchor probe. Prior to hybridizing to the second target-specific region, the second adapter landing sequence of the second cross-linking probe may be bound to the second cross-linking sequence of the adapter anchor probe. After hybridizing to the second target-specific region, the second adapter landing sequence of the second cross-linking probe may be bound to the second cross-linking sequence of the adapter anchor probe.

[0007] The method may further include hybridizing a first landing sequence of the first cross-linking probe to a first cross-linking sequence of the adapter anchor probe. The method may further include hybridizing a second landing sequence of the second cross-linking probe to a second cross-linking sequence of the adapter anchor probe. The adapter anchor probe may further include a spacer located between the first cross-linking sequence and the second cross-linking sequence. The adapter may include a molecular barcode.

[0008] The adaptor-anchor probe can include a binding moiety. The binding moiety can be attached to a support. The support can be a bead. The bead can be a streptavidin bead. The binding moiety can be biotin.

[0009] The first cross-linking probe can include a binding moiety. The binding moiety can be bound to a support. The support can be a bead. The bead can be a streptavidin bead. The binding moiety can be biotin.

[0010] The template nucleic acid molecule may comprise single-stranded DNA. The template nucleic acid molecule may comprise cell-free nucleic acid from a biological sample. The cell-free nucleic acid may comprise cell-free DNA. The cell-free DNA may comprise circulating tumor DNA. The template nucleic acid molecule may comprise damaged DNA.

[0011] Disclosed herein is a method comprising the steps of hybridizing a first target-specific region of a first crosslinking probe to a first target sequence of a template nucleic acid molecule, wherein a first adapter landing sequence of the first crosslinking probe binds to a first crosslinking sequence of an adapter-anchor probe, hybridizing a second target-specific region of a second crosslinking probe to a second target sequence of the template nucleic acid molecule, wherein a second adapter landing sequence of the second crosslinking probe binds to a second crosslinking sequence of the adapter-anchor probe, thereby generating a template nucleic acid molecule hybridized to the first and second crosslinking probes, and treating the template nucleic acid molecule with a methylation assay reagent after the hybridizing of the first target-specific region and the hybridizing of the second target-specific region. The methylation assay reagent can be a disulfide or an enzyme that modifies methylated cytosines. The method may further include hybridizing a third target-specific region of a third cross-linking probe to a third target sequence of the template nucleic acid molecule, wherein a third adapter landing sequence of the third cross-linking probe binds to a third cross-linking sequence of the adapter-anchor probe. The method may further include hybridizing a fourth target-specific region of a fourth cross-linking probe to a fourth target sequence of the template nucleic acid molecule, wherein a fourth adapter landing sequence of the fourth cross-linking probe binds to a fourth cross-linking sequence of the adapter-anchor probe.

[0012] The method may further include attaching an adapter to the 5' or 3' end of the template nucleic acid molecule before hybridizing the first bridge probe and before hybridizing the second bridge probe. The method may further include hybridizing an adapter primer to the adapter attached to the 3' end of the template nucleic acid molecule hybridized to the first bridge probe and the second bridge probe, and extending the 3' end of the adapter primer, thereby generating an extension product. The method may further include sequencing the extension product.

[0013] The adapter primer hybridization step can be performed before bisulfite treatment. The adapter primer hybridization step can be performed after bisulfite treatment. The adapter primer can be designed based on the adapter after bisulfite treatment, and unmethylated cytosines in the adapter can be converted to uracil during the treatment. Prior to hybridizing to the first target-specific region, the first adapter landing sequence of the first cross-linking probe can be bound to the first cross-linking sequence of the adapter anchor probe. After hybridizing to the first target-specific region, the first adapter landing sequence of the first cross-linking probe can be bound to the first cross-linking sequence of the adapter anchor probe. Prior to hybridizing to the second target-specific region, the second adapter landing sequence of the second cross-linking probe can be bound to the second cross-linking sequence of the adapter anchor probe. After hybridizing to the second target-specific region, the second adapter landing sequence of the second cross-linking probe can be bound to the second cross-linking sequence of the adapter anchor probe.

[0014] The method may further include hybridizing a first landing sequence of the first cross-linking probe to a first cross-linking sequence of the adapter anchor probe. The method may further include hybridizing a second landing sequence of the second cross-linking probe to a second cross-linking sequence of the adapter anchor probe. The adapter anchor probe may further include a spacer located between the first cross-linking sequence and the second cross-linking sequence. The adapter may include a molecular barcode.

[0015] The adaptor anchor probe may comprise a binding moiety. The binding moiety may be bound to a support. The support may be a bead. The bead may be a streptavidin bead. The binding moiety may be biotin. The first cross-linking probe may comprise a binding moiety. The binding moiety may be bound to a support. The support may be a bead. The bead may be a streptavidin bead. The binding moiety may be biotin. The template nucleic acid molecule may comprise single-stranded DNA. The template nucleic acid molecule may comprise cell-free nucleic acid from a biological sample. The cell-free nucleic acid may comprise cell-free DNA. The cell-free DNA may comprise circulating tumor DNA. The template nucleic acid molecule may comprise damaged DNA.

[0016] Disclosed herein is a kit that includes a bridge probe comprising a target-specific region configured to hybridize to a target sequence of a template nucleic acid molecule, an adapter anchor probe comprising a bridge binding sequence configured to hybridize to an adapter landing sequence of the bridge probe, and an adapter configured to bind to the 5' or 3' end of the template nucleic acid molecule.

[0017] Disclosed herein is a composition comprising: a template nucleic acid molecule, wherein the 5' or 3' end of the template nucleic acid molecule is linked to an adapter; a first crosslinking probe, wherein a first target-specific region of the first crosslinking probe hybridizes to a first target sequence of the template nucleic acid molecule; a second crosslinking probe, wherein a second target-specific region of the second crosslinking probe hybridizes to a second target sequence of the template nucleic acid molecule; and an adapter anchor probe, wherein a first crosslinking sequence of the adapter anchor probe binds to a first adapter landing sequence of the first crosslinking probe and a second crosslinking sequence of the adapter anchor probe binds to a second adapter landing sequence of the second crosslinking probe.

[0018] Disclosed herein is a nucleic acid complex comprising a template nucleic acid molecule, wherein the 5' or 3' end of the template nucleic acid molecule is bound to an adapter, a first target sequence of the template nucleic acid molecule hybridizes to a first target-specific region of a first bridge probe, a second target sequence of the template nucleic acid molecule hybridizes to a second target-specific region of a second bridge probe, a first adapter landing sequence of the first bridge probe binds to a first cross-linking sequence of the adapter-anchor probe, and a second adapter landing sequence of the second bridge probe binds to a second cross-linking sequence of the adapter-anchor probe. Disclosed herein is a composition comprising the nucleic acid complex.

[0019] Disclosed herein is a method of sequential enrichment comprising: obtaining a sample comprising a plurality of nucleic acid molecules; performing a first targeted enrichment to enrich for nucleic acid molecules comprising sequences corresponding to a first panel of one or more genomic regions, thereby producing a first enriched sample comprising nucleic acids enriched for sequences corresponding to the first panel of one or more genomic regions, and a residual sample comprising nucleic acids depleted for sequences corresponding to the first panel of one or more genomic regions; and performing a second targeted enrichment on the residual sample to enrich for nucleic acid molecules comprising sequences corresponding to a second panel of one or more genomic regions, thereby producing a second enriched sample comprising nucleic acids enriched for sequences corresponding to the second panel of one or more genomic regions, wherein the first panel of one or more genomic regions and the second panel of one or more genomic regions are different.

[0020] The method may further include performing a first analysis of the first enriched sample and a second analysis of the second enriched sample.

[0021] The first analysis can be a sequence analysis and the second analysis can be a methylation analysis.

[0022] In some cases, the first analysis is a first sequence analysis and the second analysis is a second sequence analysis, and the first sequence analysis is performed at a different sequencing depth than the second sequence analysis.

[0023] In some cases, the sample is a cfDNA sample.

[0024] In some cases, target enrichment for the genomic regions of the panel of one or more genomic regions comprises target enrichment by hybridization.

[0025] In some cases, target enrichment for a genomic region of the panel of one or more genomic regions comprises hybridizing a first target-specific region of a first cross-linking probe to a first target sequence of a molecule having a sequence corresponding to the genomic region, wherein a first adapter landing sequence of the first cross-linking probe binds to a first cross-linking sequence of the adapter anchor probe; and hybridizing a second target-specific region of a second cross-linking probe to a second target sequence of a molecule having a sequence corresponding to the genomic region, wherein a second adapter landing sequence of the second cross-linking probe binds to a second cross-linking sequence of the adapter anchor probe.

[0026] In some cases, the adaptor anchor probe comprises a binding moiety.

[0027] 74. The method of Claim 73, further comprising the steps of binding the binding moiety to a support and separating the support with the bound binding moiety from unbound nucleic acid.

[0028] In some cases, the first or second panel of genomic regions includes a promoter region.

[0029] In some cases, the first or second panel of genomic regions comprises an intron region.

[0030] 80. The method of Claim 66, 75 or 76, wherein the first or second panel of genomic regions comprises exon regions.

[0031] In some cases, the method further comprises attaching adaptors to the 5' or 3' ends of nucleic acid molecules of the plurality of nucleic acid molecules, thereby generating a library of nucleic acid molecules comprising adaptors.

[0032] In some cases, the second enriched sample is bisulfite treated and subjected to a sequencing reaction.

[0033] In some cases, the number of informative reads of the sequencing reaction is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the number of informative reads that could be obtained from the sample when subjected to single target enrichment to enrich for nucleic acid molecules comprising sequences corresponding to the second panel of one or more genomic regions.

[0034] In some cases, the method further includes performing a third target enrichment on the second remaining sample comprising nucleic acids depleted for sequences corresponding to the first panel of one or more genomic regions and the second panel of one or more genomic regions to enrich for nucleic acid molecules comprising sequences corresponding to the third panel of one or more genomic regions, thereby generating a third enriched sample comprising nucleic acids enriched for sequences corresponding to the third panel of one or more genomic regions, wherein the first panel of one or more genomic regions, the second panel of one or more genomic regions, and the third panel of one or more genomic regions are different.

[0035] In some cases, the method further comprises hybridizing a third target-specific region of a third cross-linking probe to a third target sequence of a molecule having a sequence corresponding to the genomic region, wherein a third adapter landing sequence of the third cross-linking probe binds to a third cross-linking sequence of the adapter anchor probe.

[0036] In some cases, the method further includes hybridizing a fourth target-specific region of a fourth cross-linking probe to a fourth target sequence of a molecule having a sequence corresponding to the genomic region, wherein a fourth adapter landing sequence of the fourth cross-linking probe binds to a fourth cross-linking sequence of the adapter anchor probe.

[0037] INCORPORATION BY REFERENCE All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0038] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which: [Brief explanation of the drawings]

[0039] [Figure 1] 1 illustrates one embodiment of synergistic indirect hybridization capture of template nucleic acid molecules. In this embodiment, a library of template nucleic acid molecules is constructed prior to indirect hybridization.

[0040] [Figure 2] 2A-2B illustrate one embodiment of synergistic indirect hybridization capture of a template nucleic acid molecule for methylation sequencing. Figure 2A shows synergistic indirect hybridization capture of a template nucleic acid molecule, and Figure 2B shows subsequent bisulfite conversion of the captured templated nucleic acid molecule.

[0041] [Figure 3] Figure 3 shows the workflow for synergistic indirect hybridization capture and targeted methylation sequencing (SICON-TMS) of template nucleic acid molecules.

[0042] [Figure 4] FIG. 4 shows a schematic diagram of synergistic indirect hybridization.

[0043] [Figure 5] Figures 5A-5D show schematic diagrams of different hybridization systems. Figure 5A illustrates non-synergistic direct hybridization. Figure 5B illustrates synergistic direct hybridization. Figure 5C illustrates synergistic indirect hybridization. Figure 5D illustrates non-synergistic indirect hybridization.

[0044] [Figure 6] Figures 6A-6B illustrate schematic diagrams of synergistic indirect hybridization using adapter-anchor probes with or without a spacer in the middle of the adapter-anchor probe's cross-linking sequence. Figure 6A shows a schematic diagram of synergistic indirect hybridization using an adapter-anchor probe that includes a spacer. Figure 6B shows synergistic indirect hybridization using an adapter-anchor probe that lacks a spacer.

[0045] [Figure 7] FIG. 7 shows the sequencing coverage of a 15-target panel using the synergistic indirect capture method.

[0046] [Figure 8] Figures 8A-8B show sequencing coverage of a panel of 76 human gene targets (Human ID) using two different hybridization methods. Figure 8A shows coverage by pre-amplification capture with synergistic indirect hybridization. Figure 8B shows coverage by post-amplification capture with direct hybridization.

[0047] [Figure 9] Figure 9 shows the results of a targeted methylation sequencing assay after synergistic indirect capture of cfDNA extracted from non-cancerous individuals.

[0048] [Figure 10] FIG. 10 illustrates the results of a targeted methylation sequencing assay showing a linear relationship between the expected amount of spike-in methylated DNA and the measured value.

[0049] [Figure 11] 11A and 11B show the molecular methylation distribution patterns of DMR1 in the genomic DNA of normal colon tissue and colon cancer tissue, respectively.

[0050] [Figure 12] Figures 12A and 12B show the molecular methylation distribution patterns of DMR2 in the genomic DNA of normal colon tissue and colon cancer tissue, respectively.

[0051] [Figure 13] Figures 13A and 13B show the molecular methylation distribution patterns of DMR1 and DMR2 in plasma cfDNA of healthy individuals and colon cancer patients, respectively.

[0052] [Figure 14] FIG. 14 illustrates a schematic for sequential target enrichment from a sample.

[0053] [Figure 15] FIG. 15 illustrates the mutations identified in the CRC cfDNA samples in Example 11.

[0054] [Figure 16] FIG. 16 illustrates the methylation scores from standalone and dual analysis TMS.

[0055] [Figure 17] FIG. 17 illustrates informative molecule counts from standalone and dual analysis TMS.

[0056] [Figure 18] FIG. 18 illustrates the sensitivity of variant allele detection in personalized panel analysis.

[0057] [Figure 19] FIG. 19 illustrates an implementation of the Point-n-Seq™ technology. DETAILED DESCRIPTION OF THE INVENTION

[0058] Detailed Description Liquid biopsy and mutation analysis based on CfDNA using methylation can be used for early detection and management of cancer.Provided herein is a system and method for combined analysis from limited amount of nucleic acid sample.For example, provided herein is a system and method for combined targeted methylation sequencing (TMS) and mutation analysis from limited DNA sample.These systems and methods can be particularly useful for cfDNA samples that may have a small amount.

[0059] Widespread yet tissue-specific methylation changes in cancer genomes can be used for the sensitive detection of circulating tumor DNA (ctDNA) in plasma from patients with early-stage or recurrent cancer. However, the sensitivity of methylation analysis can be compromised by the low efficiency of methylation marker recovery in the process, and specificity can be further hindered by techniques that include noisy, nonspecific markers that offset the low detection sensitivity. Furthermore, methylation analysis may have advantages for early cancer detection, as actionable mutations can directly provide information to guide treatment selection and further increase assay specificity. The yield of cfDNA from limited clinical blood samples can be small, which can be a major problem for performing multiple analyses from a single sample. Therefore, an assay that can detect both methylation and mutations could offer an improvement for clinical research and diagnostic assays.

[0060] This disclosure provides an improved technology designed for combined analysis of targeted methylation and mutations in cfDNA: Point-n-Seq, which features cytosine conversion and enrichment of target molecules directly from pre-amplification cfDNA. This technology can enable small, focused panels to examine the methylation or mutation status of at least 10, 100, 1,000, or even more than 1,000 markers. A colorectal cancer (CRC) panel designed to cover 100 methylation markers and more than 350 hotspot mutations from 22 genes is presented herein. Point-n-Seq TMS can be used for small, focused combined panel sequencing of methylation and mutations using cfDNA. Point-n-Seq TMS can be used in the development of practical, cost-effective methylation assays for research and clinical use.

[0061] The ultra-efficient pre-conversion / pre-amplification capture Point-n-Seq can be used for disease-focused methylation and mutation panel enrichment. Point-n-Seq TMS enables the analysis of small, focused methylation and mutation panels using cfDNA. Point-n-Seq TMS can be used in practical, cost-effective methylation assays for research and clinical use.

[0062] Also presented herein are systems and methods for synergistic indirect capture of nucleic acids for sequencing (SICON-SEQ, also referred to as Point-n-SEQ). The systems and methods disclosed herein enable efficient capture and enrichment of nucleic acid materials. SICON-SEQ / Point-n-SEQ can be performed for capture enrichment after library construction by binding adapters to template nucleic acid materials. In some embodiments, SICON-SEQ can be performed before library construction. SICON-SEQ can be performed without library construction by adapter binding. The SICON-SEQ method disclosed herein can enable short turnaround times and simple workflows. SICON-SEQ can be used to handle low-input samples, such as cell-free DNA (cfDNA), and therefore may be suitable for methylation sequencing analysis.

[0063] Disclosed herein is a method that involves indirect hybridization of a template nucleic acid molecule with an adapter anchor probe through the hybridization of one or more bridge probes to the template nucleic acid. One or more bridge probes can be designed to hybridize to a specific target sequence within the template nucleic acid molecule, thereby hybridizing to the target template. Next, the adapter anchor probe can be designed to hybridize to one or more bridge probes, thereby creating an assembly of three or more hybridized nucleic acid molecules. Multiplexed hybridization assemblies can act synergistically to provide greater stability to the assembly. The hybridized template nucleic acid molecule can then be treated with bisulfite for methylation sequencing.

[0064] Disclosed herein are kits that include a bridge probe that includes a target-specific region that hybridizes to a target sequence of a template nucleic acid molecule, an adapter anchor probe that includes a bridge binding sequence that hybridizes to the adapter landing sequence of the bridge probe, and an adapter configured to bind to the 5' or 3' end of the template nucleic acid molecule.

[0065] I. Indirect capture by hybridization Target probe hybridization can be facilitated by synergistic interactions between the template nucleic acid and two or more probes to form a hybridization assembly. A multicomplex assembly can stabilize the hybridization interaction between the template and a target probe, such as a bridging probe. The bridging probe can include a target-specific region that hybridizes with the target region of the template and an adapter landing sequence (ALS) that hybridizes to the bridging sequence (BBS) of the adapter anchor probe. Hybridization between the template and the bridging probe and hybridization between the bridging probe and the adapter anchor probe can form a multicomplex assembly.

[0066] The methods disclosed herein can use more than two pre-target regions of the crosslinking probes. For example, at least two, three, four, five, six, seven, eight, nine, ten, twenty-five, fifty, seventy-five, one hundred, or more crosslinking probes can be used to crosslink the template and the adapter-anchor probes. The synergistic indirect capture of nucleic acids for sequencing (SICON-SEQ) method can further include hybridizing a second target-specific region of a second crosslinking probe to a second target sequence of the template nucleic acid molecule, wherein the second adapter landing sequence of the second crosslinking probe can bind to the second crosslinking sequence of the adapter-anchor probe (FIG. 1). In some cases, SICON-SEQ can be performed after the adapter is attached to the template nucleic acid molecule to generate a library (FIG. 1). The library can be a next-generation sequencing (NGS) library.

[0067] The cross-linking probe may further comprise a linker connecting the target-specific region and the adapter landing sequence. The adapter anchor may comprise one or more spacers between the cross-linking sequence. The presence of one or more spacers can improve the efficiency of hybridization capture and increase the specificity of capture.

[0068] Template nucleic acids can be captured and enriched from low-input samples such as cell-free DNA (cfDNA) and circulating tumor DNA (ctDNA). Capture and enrichment can be achieved by indirect association with adapter-anchor probes through hybridization to bridge probes. The bridge probes and / or adapter-anchor probes can contain one or more binding moieties. The binding moiety can be biotin. The binding moiety can be bound to a support. The support can be beads. The beads can be streptavidin beads.

[0069] Disclosed herein are kits that include a bridge probe that includes a target-specific region that hybridizes to a target sequence of a template nucleic acid molecule, an adapter anchor probe that includes a bridge binding sequence that hybridizes to the adapter landing sequence of the bridge probe, and an adapter configured to bind to the 5' or 3' end of the template nucleic acid molecule.

[0070] II. Workflow for methylation analysis A method for nucleic acid methylation analysis is presented herein. Methylation analysis can be performed by bisulfite treatment. Bisulfite-treated nucleic acids can be used to study nucleic acid methylation. Bisulfite treatment can convert unmethylated cytosine to uracil. Methylation of cytosine (e.g., 5'-methylcytosine) can prevent bisulfite-methylated cytosine from being converted to uracil.

[0071] The template nucleic acid molecule can be treated with bisulfite either before or after hybridization capture using a capture probe or a bridge probe / adapter anchor probe. In some cases, the hybridized template nucleic acid molecule can be treated with bisulfite. The formation of a double-stranded sequence (e.g., between the TS of the template and the TSR of the capture probe) can protect cytosines in the hybridized region from conversion to uracil during bisulfite treatment. The double-stranded sequence formed by hybridizing a capture probe to a template or a bridge probe to a template with an adapter anchor probe can protect cytosines in the hybridized region from conversion to uracil by bisulfite. Furthermore, because unmethylated cytosines can be converted to uracil by bisulfite treatment, protecting cytosines from conversion to uracil in the TS region can enable the use of amplification primers designed to anneal to DNA that has not been bisulfite converted. For pre-bisulfite capture, a probe can also be designed against the unconverted sequence. Probes and primers that anneal to unconverted cytosines can be simpler to design and also result in better hybridization. In some cases, enzymatic treatment can be performed for methylation analysis. The enzyme can be a methylation-sensitive enzyme or a methylation-dependent enzyme. The enzyme can be a restriction enzyme. The enzyme can be a methylation-sensitive restriction endonuclease. In other cases, methylation analysis can be performed by using a specific antibody or protein that specifically binds to the methylation site to enrich for methylated nucleic acids.

[0072] a. Methylation treatment or enrichment after hybridization capture of template nucleic acid As described herein, a template nucleic acid (e.g., DNA) can be used for synergistic indirect hybridization and subsequent sequencing (SICON-SEQ) (see, e.g., Figure 3). The template nucleic acid (e.g., DNA) can be, for example, genomic DNA or cfDNA. For example, as described herein, for example, as illustrated in Figures 1 and 2A, the template nucleic acid (e.g., DNA) can be directly hybridized to a capture probe or indirectly bound to an adapter anchor probe (or universal anchor probe) by cross-linking probe hybridization. The hybridization-captured template nucleic acid (e.g., DNA) can be treated with bisulfite, extended, and then amplified, for example, for targeted methylation sequencing (SICON-TMS) (Figure 2B). In some cases, the captured template nucleic acid can be treated with a methylation-sensitive enzyme. In other cases, methylated nucleic acids in the captured template nucleic acid molecule can be enriched by specific binding to an antibody or protein that targets methylated CpG sites within the template nucleic acid molecule. The SICON-TMS is compatible with clinical samples containing a wide range of nucleic acid material amounts. In some cases, the SICON-TMS can be used with sequence samples containing less than 5 ng, less than 4 ng, less than 3 ng, less than 2 ng, or less than 1 ng of nucleic acid molecules.

[0073] The target-specific sequence or target-specific region (TSR) of the capture probe or crosslinking probe can be designed based on the target sequence of the template nucleic acid molecule, and the target sequence of the template nucleic acid molecule can retain unmethylated cytosines after bisulfite treatment.

[0074] In some cases, bisulfite treatment can be performed before the target-specific sequence of the crosslinking probe is removed.Unmethylated cytosines in the TS and TSR sites can be protected from conversion to uracil during bisulfite treatment after the TS and TSR hybridization of the capture probe or crosslinking probe to the template.The hybridized template can then be treated with bisulfite, during which the unmethylated cytosines in the hybridized TSR-TS region are not converted to uracil, while the unmethylated cytosines in the single-stranded region are converted to uracil.Protection of cytosines in the TS region from conversion to uracil can allow the use of probes designed to anneal to DNA that is not bisulfite converted.

[0075] In some cases, bisulfite treatment can be performed after the capture probe or cross-linking probe is removed from the template nucleic acid sequence. One or more cytosine residues in the primer binding site (e.g., in the adapter and / or template) may not be protected from bisulfite conversion. After bisulfite conversion, the primer binding site in the adapter may contain one or more uracils. Primers can be designed to be complementary to adapter sequences containing one or more uracils. Primers can be 100% complementary to adapter sequences containing one or more uracils, or less than 100% complementary to adapter sequences containing one or more uracils.

[0076] The template may contain one or more uracils after bisulfite treatment. A primer annealed to an adapter can use a template containing one or more uracils for strand extension. The extended strand may contain one or more adenines, which are bases that pair with the one or more uracils. The extension product can be denatured from the template. A primer can be annealed to the extension product within the region containing one or more adenines and extended. The primer can be used, for example, to amplify the template using an adapter primer.

[0077] Methylation treatment or enrichment can be applied to the template nucleic acid molecule before the adapter is attached. Methylation treatment or enrichment can be applied to the template nucleic acid molecule after the adapter is attached. Methylation treatment or enrichment can be applied to the template nucleic acid molecule after the first adapter is attached to the template. Methylation treatment or enrichment can be applied to the template nucleic acid molecule after the second adapter is attached to the template.

[0078] b. Methylation treatment or enrichment prior to hybridization capture of template nucleic acid The template nucleic acid molecule can be bisulfite-treated before hybridization to the capture probe or crosslinking probe. DNA can be treated with bisulfite to convert unmethylated cytosines to uracil. The bisulfite-treated DNA can be used as input for synergistic indirect hybridization and subsequent sequencing (SICON-SEQ). The TSR of the probe can be designed to anneal to a template in which existing unmethylated cytosines have been converted to uracil. After hybridization capture, extension can be performed, followed by target amplification. In some cases, the captured template nucleic acid can be treated with a methylation-sensitive enzyme. In other cases, methylated nucleic acids in the captured template nucleic acid molecule can be enriched by specific binding to an antibody or protein that targets methylated CpG sites within the template nucleic acid molecule.

[0079] Before the adapter is attached, the template nucleic acid molecule can be subjected to a methylation treatment or enrichment. After the adapter is attached, the template nucleic acid molecule can be subjected to a methylation treatment or enrichment. After the first adapter is attached to the template, the template nucleic acid molecule can be subjected to a methylation treatment or enrichment. After the second adapter is attached to the template, the template nucleic acid molecule can be subjected to a methylation treatment or enrichment.

[0080] III. Solid phase extraction For example, a method is provided herein for selecting templates hybridized to a bridge probe (or templates to which an adapter anchor probe is associated via a bridge probe) before ligating the adapter anchor probe to the template. This method can employ solid-phase extraction. A method is provided herein for binding a bridge probe or an adapter anchor probe to a solid support. The possibility that an adapter anchor probe may bind (e.g., ligate) to a template independently of the bridge probe can introduce suboptimal specificity. To reduce such nonspecific ligation products and unbound probes, labels (e.g., biotin) and capture moieties (e.g., streptavidin beads) can be utilized.

[0081] The bridge probe or adapter-anchor probe may contain a label. The disclosed method may further include capturing the bridge probe, adapter-anchor probe, or hybridization complex containing the template nucleic acid molecule, the bridge probe, and the adapter-anchor probe with a label. The label may be biotin. The label may be a nucleic acid sequence, such as polyA or polyT, or a specific sequence. The nucleic acid sequence may be approximately 5 to 30 bases in length. The nucleic acid sequence may include DNA and / or RNA. The label may be present at the 3' end of the capture probe, bridge probe, or adapter-anchor probe. The label may be a peptide that can be recognized by an antibody, such as 5-bromouridine and biotin, or a modified nucleic acid. Conjugation of the label to the capture probe, bridge probe, or adapter-anchor probe may be achieved by a reaction such as "click" chemistry. "Click" chemistry may enable the conjugation of a reporter molecule, such as a fluorescent dye, to a biomolecule such as DNA. Click chemistry can be a reaction between an azide and an alkyne that can lead to a covalently linked product (e.g., a 1,5-disubstituted 1,2,3-triazole). Copper can act as a catalyst.

[0082] The label can be captured on a solid support. The solid support can be magnetic. The solid support can include beads, flow cells, glass, plates, devices containing one or more microfluidic channels, or columns. The solid support can be magnetic beads.

[0083] The solid support (e.g., beads) can include (e.g., by coating therewith) one or more capture moieties that can bind to a label. The capture moiety can be streptavidin, which can bind to biotin. The capture moiety can be an antibody. The antibody can bind to a label. The capture moiety can be a nucleic acid, e.g., a nucleic acid comprising DNA and / or RNA. A nucleic acid capture moiety can bind to a sequence on an adapter anchor probe or a bridging probe, for example. In some cases, an anti-RNA / DNA hybrid antibody bound to a solid surface can be used as a capture moiety.

[0084] The label and the capture moiety can be bound through one or more covalent or non-covalent bonds. After the crosslinking probe, adapter-anchor probe, or hybridization complex is captured on the solid support, the solid support can be washed, for example, to remove unbound template from the sample. In some cases, no washing step is performed. The washing can be stringent or gentle. For example, if the label is biotin and the capture moiety is streptavidin, the captured crosslinking probe or adapter-anchor probe that is hybridized to the template nucleic acid molecule can be eluted by adding free biotin to the sample.

[0085] The extension step (e.g., extension of an adapter primer annealed to an adapter) can be performed while the bridge probe or adapter-anchor probe is captured on the solid support, or after elution of the bridge probe (and hybridized template) from the solid support or after elution of the adapter-anchor probe (and indirectly hybridized template) from the solid support.

[0086] After hybridization of the template, bridge probe, and adapter-anchor probe, cleanup can be performed using streptavidin beads, with the 3' end of the adapter-anchor probe being biotinylated. Both the hybridization complex and free adapter-anchor adaptors can be bound to the beads. Unbound template and bridge probe can be washed away. The 5' or 3' end of the first bridge probe and / or the second bridge probe can be biotinylated. Streptavidin beads can be used to remove unhybridized adapter-anchor adaptors and templates, thereby preventing random ligation of the adapter-anchor probe to the template.

[0087] IV. Template nucleic acid molecule The template nucleic acid can be DNA or RNA. The DNA can be genomic DNA (gDNA), mitochondrial DNA, viral DNA, cDNA, cfDNA, or synthetic DNA. The DNA can be double-stranded DNA, single-stranded DNA, fragmented DNA, or damaged DNA. The RNA can be mRNA, tRNA, rRNA, microRNA, snRNA, piRNA, small non-coding RNA, polysomal RNA, intronic RNA, pre-mRNA, viral RNA, or cell-free RNA.

[0088] The template nucleic acid can be a naturally occurring nucleic acid or a synthetic nucleic acid. The template nucleic acid can have a modified heterocyclic base. The modification can be a methylated purine or pyrimidine, an acylated purine or pyrimidine, an alkylated ribose, or other heterocycle. The template nucleic acid can have a modified sugar moiety. The modified sugar moiety can include a peptide nucleic acid. The template nucleic acid can include a peptide nucleic acid. The template nucleic acid can include a threose nucleic acid. The template nucleic acid can include a locked nucleic acid. The template nucleic acid can include a hexitol nucleic acid. The template nucleic acid can be a flexible nucleic acid. The template nucleic acid can include a glycerol nucleic acid.

[0089] Template nucleic acid molecules can be captured and enriched from low-input (e.g., 1 ng of nucleic acid material) samples, such as cell-free DNA (cfDNA) and circulating tumor DNA (ctDNA). A low-input sample can have 1 ng, 2 ng, 3 ng, 4 ng, 5 ng, 6 ng, 7 ng, 8 ng, 9 ng, 10 ng, or more of nucleic acid material. A low-input sample can have less than 10 ng, less than 9 ng, less than 8 ng, less than 7 ng, less than 6 ng, less than 5 ng, less than 4 ng, less than 3 ng, less than 2 ng, less than 1 ng, or less of nucleic acid material. A low-input sample can have 200 pg to 10 ng of nucleic acid material. A low-input sample can have less than 10 ng of nucleic acid material. A low-input sample can have less than 10 ng, less than 5 ng, less than 1 ng, less than 100 pg, less than 50 pg, less than 25 pg, or less of nucleic acid material. In some cases, the input sample may have 1 ng, 10 ng, 20 ng, 30 ng, 40 ng, 50 ng, or more nucleic acid molecules. The input sample may have less than 50 ng, less than 40 ng, less than 30 ng, less than 20 ng, less than 10 ng, less than 1 ng, or less nucleic acid material. Capture and enrichment can be performed by target probe hybridization. The target probe can be a capture probe, a crosslinking probe, and / or an adapter anchor probe. The target probe can include one or more binding moieties. The binding moiety can be biotin. The binding moiety can be attached to a support. The support can be beads. The beads can be streptavidin beads.

[0090] The template nucleic acid may be damaged. The damaged nucleic acid may contain base changes or deletions and / or backbone modifications. The template nucleic acid may be damaged by oxidation, radiation, or random mutation. The template nucleic acid may be damaged by bisulfite treatment.

[0091] With respect to damaged DNA, the present disclosure allows for the elimination of the double-stranded DNA repair step, resulting in higher conversion rates and improved sensitivity due to less DNA loss from fewer steps in the process.

[0092] Damaged dsDNA (nicked) or ssDNA can be used as a template for library construction. For damaged dsDNA, the dsDNA can be denatured, so that at least one undamaged strand can be used as a template. Then, the template can be hybridized and bound to a capture probe and amplified using various primers.

[0093] The template can be derived from cell-free DNA (cfDNA) or circulating tumor DNA (ctDNA). cfDNA can be from a fetus or tumor. The template can be derived from a liquid biopsy, solid biopsy, or fixed tissue of a subject. The template can be cDNA and can be generated by reverse transcription. The template nucleic acid can be derived from a body fluid sample, including, but not limited to, plasma, serum, sputum, saliva, urine, or sweat. The body fluid sample can be bisulfite-treated to investigate the methylation pattern of the template nucleic acid and / or to determine the tissue origin of the template nucleic acid. The template nucleic acid can be derived from the liver, esophagus, kidney, heart, lung, spleen, bladder, colon, or brain. The template nucleic acid can be bisulfite-treated to analyze the methylation pattern in the organ from which the template nucleic acid is derived. The subject can suffer from a methylation-related disease, such as an autoimmune disease, cardiovascular disease, atherosclerosis, neurological disorders, and cancer.

[0094] The template nucleic acid can be from a male or female subject. The subject can be an infant. The subject can be a teenager. The subject can be a young adult. The subject can be an elderly person.

[0095] The template nucleic acid may originate from a human, rat, mouse, other animal, or a particular plant, bacterium, algae, virus, etc. The template nucleic acid may originate from a primate. The primate may be a chimpanzee or a gorilla. The other animal may be a rhesus monkey. The template may also be derived from a mixture of genomes from different species, including host-pathogen, bacterial populations, etc. The template may be cDNA made from RNA expressed from the genomes of two or more species.

[0096] The template nucleic acid may comprise a target sequence. The target sequence may be an exon. The target sequence may be an intron. The target sequence may comprise a promoter. The target sequence may be previously known. The target sequence may be previously partially known. The target sequence may be previously unknown. The target sequence may comprise a chromosome, a chromosome arm, or a gene. The gene may be a gene associated with a condition, e.g., cancer. The template nucleic acid molecule may be dephosphorylated prior to hybridization, e.g., to reduce the rate of self-ligation.

[0097] V. Cross-linking Probes A bridge probe can be used to hybridize an adapter anchor probe with a template nucleic acid molecule having a target sequence. The bridge probe also allows for indirect association of the adapter anchor probe with the template, thereby facilitating their binding. The ligation rate of a free adapter anchor probe with a template can be very low due to the randomness of the interaction. However, a hybridized bridge probe can increase the probability of ligation of the template with the adapter anchor probe compared to the probability of ligation with a free adapter anchor probe. The bridge probe can include DNA. The bridge probe can include RNA. The bridge probe can include uracil and methylated cytosine. The bridge probe cannot include uracil.

[0098] The bridge probe may include a target-specific region (TSR) that hybridizes to the target sequence. The bridge probe may include an adapter landing sequence (ALS) that hybridizes to the bridge-binding sequence of the adapter-anchor probe. The bridge probe may include a linker connecting the TSR and ALS. The TSR may be located at the 3' portion of the bridge probe. The TSR may be located at the 5' portion of the bridge probe.

[0099] The cross-linking probe may comprise one or more molecular barcodes. The cross-linking probe may comprise one or more binding moieties. The binding moiety may be biotin. The binding moiety may be bound to a support. The support may be a bead. The bead may be a streptavidin bead.

[0100] The bridge probe may comprise about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 120 nucleotides, about 100 nucleotides, about 90 nucleotides, about 80 nucleotides, about 70 nucleotides, about 50 nucleotides, about 40 nucleotides, about 30 nucleotides, about 20 nucleotides, or about 10 nucleotides.

[0101] Multiple crosslinking probes can be used to anneal to multiple target sequences in a sample. The crosslinking probes can be designed to have similar melting temperatures. The melting temperatures of a set of crosslinking probes can be within about 15°C, about 10°C, about 5°C, or about 2°C. The melting temperature of one or more crosslinking probes can be about 75°C, about 70°C, about 65°C, about 60°C, about 55°C, about 50°C, about 45°C, or about 40°C. The melting temperature of a crosslinking probe can be about 40°C to about 75°C, about 45°C to about 70°C, 45°C to about 60°C, or about 52°C to about 58°C.

[0102] The use of an adapter anchor probe together with one or more surrounding cross-linking probes can help stabilize the hybridization of a specific cross-linking probe to its target sequence through a synergistic effect. The hybridization temperature for forming a multiple cross-linking probe assembly can be higher than the melting temperature of a single cross-linking probe. Higher temperatures can result in better capture specificity by reducing nonspecific hybridization that can occur at lower temperatures. The hybridization temperature can be about 5°C, about 10°C, about 15°C, or about 20°C higher than the melting temperature of an individual cross-linking probe. The hybridization temperature can be about 5°C to about 20°C higher than the melting temperature of the cross-linking probe, or about 5°C to about 20°C higher than the average melting temperature of multiple cross-linking probes.

[0103] The hybridization temperature of multiple cross-linking probes can be about 75°C, about 70°C, about 65°C, about 60°C, about 55°C, or about 50°C. The hybridization temperature of multiple cross-linking probes can be about 50°C to about 75°C, 55°C to about 75°C, 60°C to about 75°C, or 65°C to about 75°C.

[0104] The cross-linking probe may further comprise a label. The label may be fluorescent. The fluorescent label may be an organic fluorescent dye, a metal chelate, a carbon nanotube, a quantum dot, a gold particle, or a fluorescent inorganic substance. The label may be radioactive. The label may be biotin. The cross-linking probe may bind to a labeled nucleic acid binding molecule. The nucleic acid binding molecule may be an antibody, an antibiotic, a histone, an antibody, or a nuclease.

[0105] The crosslinking probe may comprise a linker. The linker may comprise about 30 nucleotides, about 25 nucleotides, about 20 nucleotides, about 15 nucleotides, about 10 nucleotides, or about 5 nucleotides. The linker may comprise about 5 to about 20 nucleotides.

[0106] The linker can comprise a non-nucleic acid polymer (e.g., a stretch of carbon). The linker non-nucleotide polymer can comprise about 30 units, about 25 units, about 20 units, about 15 units, about 10 units, or about 5 units.

[0107] The bridge probe can be blocked at the 3' and / or 5' end. The bridge probe can lack a 5' phosphate. The bridge probe can lack a 3' OH. The bridge probe can contain a 3' ddC, a 3' inverted dT, a 3' C3 spacer, a 3' amino, or a 3' phosphorylation.

[0108] VI. Adapter anchor probe An adapter anchor probe or universal anchor probe may contain one or more bridge sequences that hybridize to the adapter landing sequences of one or more bridge probes.

[0109] The adapter-anchor probe may include a spacer in the middle of the BBS. The presence of one or more spacers can improve the efficiency of hybridization capture and increase the specificity of the capture.

[0110] The adapter anchor probe may include a molecular barcode (MB). The adapter anchor probe may include a bridging sequence (BBS) to which one or more bridging probes can be hybridized. The adapter anchor probe may include 1 to 100 BBSs. The adapter anchor probe may include an index for identifying the sample. The molecular barcode or index may be located 5' of the adapter sequence and 5' of the BBS.

[0111] The adaptor-anchor probe can comprise about 400 nucleotides, about 200 nucleotides, about 120 nucleotides, about 100 nucleotides, about 90 nucleotides, about 80 nucleotides, about 70 nucleotides, about 50 nucleotides, about 40 nucleotides, about 30 nucleotides, about 20 nucleotides, or about 10 nucleotides. The adaptor-anchor probe can be about 20 to about 70 nucleotides.

[0112] The melting temperature of the adapter-anchored probe relative to the bridge probe can be about 65°C, about 60°C, about 55°C, about 50°C, about 45°C, or between about 45°C and about 70°C.

[0113] The adaptor anchor probe may include a label. The label may be fluorescent. The fluorescent label may be an organic fluorescent dye, a metal chelate, a carbon nanotube, a quantum dot, a gold particle, or a fluorescent inorganic substance. The label may be radioactive. The label may be biotin. The adaptor anchor probe may bind to a labeled nucleic acid binding molecule. The nucleic acid binding molecule may be an antibody, an antibiotic, a histone, an antibody, or a nuclease.

[0114] VII. Adapter / Adapter Primer For library construction, one or more adapters can be attached to multiple template nucleic acids. The library can be a next-generation sequencing (NGS) library. One adapter can be attached to the 5' or 3' end of the template nucleic acid molecule. Two adapters can be attached to the 5' and 3' ends of the template nucleic acid molecule. One or more adapters can be attached to the template nucleic acid by ligation. Attachment of one or more adapters can be performed before hybridization of the template nucleic acid and the target probe. In some cases, adapters can be added to the captured template nucleic acid after hybridization. One or more adapters can include a molecular barcode (MB).

[0115] One or more adapter primers can hybridize to one or more adapters attached to the template nucleic acid molecule. In some cases, the adapter is incorporated into an adapter anchor probe or a capture probe. In certain cases, the attached, added, or incorporated adapter can provide a site for primer hybridization for amplification. A first adapter (AD1) can be attached to the template via a capture probe or an adapter anchor probe. A primer to AD1 can be used to synthesize a strand complementary to the template. A second adapter (AD2) can be attached to the 5' end of the template and / or the 3' end of the complementary strand for further amplification of the template. A library can be constructed using AD1 primers and AD2 primers. Selective amplification can be performed using an AD1 primer and a primer to the TSR or its adjacent region.

[0116] The adaptor can be a single-stranded nucleic acid. The adaptor can be a double-stranded nucleic acid. The adaptor can be partially duplex, having a short strand and a longer strand, or having two strands of equal length.

[0117] VIII. Enzymes Examples of DNA polymerases that can be used in the methods and kits described herein include Klenow polymerase, Bst DNA polymerase, Bca polymerase, phi29 DNA polymerase, Vent polymerase, Deep Vent polymerase, Taq polymerase, T4 polymerase, T7 polymerase, or E. coli DNA polymerase 1.

[0118] Examples of ligases that can be used in the methods and kits described herein include CircLigase, CircLigase II, E. coli DNA ligase, T3 DNA ligase, T4 DNA ligase, T7 DNA ligase, DNA ligase I, DNA ligase II, DNA ligase III, DNA ligase IV, Taq DNA ligase, or Tth DNA ligase.

[0119] Examples of methylation-sensitive or methylation-dependent restriction enzymes that can be used in the methods and kits described herein include Aat II, Acc II, Aor13H I, Aor51H I, BspT104 I, BssH II, Cfr10 I, Cla I, Cpo I, Eco52 I, Hae II, Hap II, Hha I, Mlu I, Nae I, Not I, Nru I, Nsb I, PmaC I, Psp1406 I, Pvu I, Sac II, Sal I, Sma I, and SnaB I.

[0120] IX. Downstream Analysis of Amplification Products Amplification products generated using the methods described herein can be further analyzed using a variety of methods, including Southern blotting, polymerase chain reaction (PCR) (e.g., real-time PCR (RT-PCR), digital PCR (dPCR), droplet digital PCR (ddPCR), quantitative PCR (Q-PCR), nCounter analysis (Nanostring technology), gel electrophoresis, DNA microarrays, mass spectrometry (e.g., tandem mass spectrometry, matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS)), chain termination sequencing (Sanger sequencing), or next-generation sequencing.

[0121] Next generation sequencing includes 454 sequencing (ROCHE) (using pyrosequencing), sequencing using reversible terminator dyes (ILLUMINA sequencing), semiconductor sequencing (THERMOFISHER ION TORRENT), single molecule real-time (SMRT) sequencing (PACIFIC BIOSCIENCES), nanopore sequencing (e.g., using technology from OXFORD NANOPORE or GENIA), microdroplet single molecule sequencing using pyrophosphate dissolution (BASE4), single molecule electronic detection sequencing, e.g., measuring the tunneling current through a nanoelectrode as nucleic acid (DNA / RNA) passes through a nanogap and calculating the current difference (QUANTUM SEQUENCING from QUANTUM BIOSYSTEMS), GenapSys Gene Electronic Nano-Integrated Ultra-Sensitive (GENIUS) technology (GENAPYS), QIAGEN's GENEREADER, and partially random oligonucleotides identified by specific fluorophores and centrally determined bases. The sequencing may include sequencing using sequential hybridization and ligation with a base (or base pairs) (SOLiD sequencing). The sequencing may be paired-end sequencing.

[0122] The number of target sequences from a sample that can be sequenced using the methods described herein can be about 5, 10, 15, 25, 50, 100, 1000, 10,000, 100,000, or 1,000,000, or about 5 to about 100, about 100 to about 1000, about 1000 to about 10,000, about 10,000 to about 100,000, or about 100,000 to about 1,000,000.

[0123] Nucleic acid libraries generated using the methods described herein can be generated from more than one sample. Each library can have a different index associated with the sample. For example, capture probes or adapter-anchor probes can include an index that can be used to identify nucleic acids as originating from the same sample (e.g., a first set of capture probes or adapter-anchor probes containing the same first index can be used to generate a first library from a first sample from a first subject, a second set of capture probes or adapter-anchor probes containing the same second index can be used to generate a second library from a second sample from a second subject, the first and second libraries can be pooled and sequenced, and the index can be used to distinguish the sample from which the sequenced nucleic acid originated). Amplification products generated using the methods described herein can be used to generate libraries from at least 2, 5, 10, 25, 50, 100, 1000, or 10,000 samples, each with a different index, and the libraries can be pooled and sequenced, for example, using next-generation sequencing technology.

[0124] Sequencing can generate at least 100, 1000, 5000, 10,000, 100,000, 1,000,000, or 10,000,000 sequence reads. Sequencing can generate between about 100 and about 1000 sequence reads, between about 1000 and about 10,000 sequence reads, between about 10,000 and about 100,000 sequence reads, between about 100,000 and about 1,000,000 sequence reads, or between about 1,000,000 and about 10,000,000 sequence reads.

[0125] The sequencing depth can be about 1×, 5×, 10×, 50×, 100×, 1000×, or 10,000×. The sequencing depth can be between about 1× and about 10×, between about 10× and about 100×, between about 100× and about 1000×, or between about 1000× and about 10,000×. X. Bioinformatics Analysis Presented herein are methods for bioinformatic analysis of sequencing data, such as methods to exclude incompletely bisulfite converted molecules and methods to analyze methylation patterns in samples with very low disease molecule content.

[0126] a. Elimination of incompletely bisulfite-converted molecules Filtering techniques to eliminate molecules with incomplete C>T conversion are used to enhance the robustness of the molecule count and methylation percentage data.

[0127] Using the nucleotide positions of the start and end of the read within the genome and unique molecular identifier information, sequencing reads mapped to each differentially methylated region (DMR) can be de-duped. De-duplication can also be performed with lower precision using only the start and end position information.

[0128] The de-duplicated reads are filtered according to the number of unconverted Cs in the CH environment, where C represents cytosine and H represents one of the three nucleotides: C (cytosine), A (adenine), or T (thymine). The presence of Cs in the CH environment that are not converted to T indicates a high likelihood of incomplete bisulfite or enzymatic processing of the molecule. If the number of unconverted Cs in the CH environment is greater than a preset threshold, the read is discarded. In some cases, the threshold number of unconverted Cs in the CH environment is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some cases, a read may be discarded if the percentage of unconverted Cs in the CH environment (as a percentage of the total number of Cs in the CH environment) is greater than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 14%, 16%, 18%, 20%, 25%, 30%, 35%, 40%, or 50%. b.SICON TMS analysis

[0129] Current methods for analyzing methylation sequencing data can involve calculating either or both of two metrics for downstream analysis: (1) the methylation percentage of individual CpG sites, or (2) the methylation density of a genomic region of interest. For (1), the number of methylated Cs at a CpG site can be divided by the total number of molecules covering the CpG site. For (2), the average overall methylation percentage of CpG sites within a defined genomic region can be calculated. As a slight modification to the above concept, methylation haplotype load (MHL) can be introduced to take into account differences in methylation patterns among molecules in the region. Essentially, MHL represents an average measure across a mixture of molecules, weighted to take into account the length of the block. These methods take an average measure across all DNA molecules in the sequenced molecules, including both disease-derived and healthy-derived materials.

[0130] In tissue sequencing data, averaging across all molecules is usually a reasonable and necessary technique. For example, in the case of tumor biopsy tissue, the tumor content may be moderately high (e.g., 20% or higher). Significant differences in methylation levels between tumor and normal tissue may reflect the average of mixed tumor-normal tissue and pure normal tissue. Because most bisulfite sequencing data have low complexity in each genomic region, averaging is often performed out of necessity. For example, 30x may be considered deep coverage in whole-genome bisulfite sequencing, while many studies have much lower coverage. Averaging across many CpG sites within a region can smooth out variability due to low coverage and enhance the robustness of the measurement. In the context of samples with very low disease molecule content, such as liquid biopsies using plasma cfDNA from tumor patients, the tumor content is often below 0.1%, and averaging across a mixture of healthy, normal, and disease-derived molecules may result in a predominance of normal molecules. In other words, tumor-derived methylation information is overwhelmed by normal-derived molecules in an act of averaging.

[0131] The method for analyzing methylation sequencing data is referred to herein as "SICON TMS analysis." Briefly, the number of CpG sites on each sequenced molecule is counted, and the methylation percentage of these sites is calculated. A data pair consisting of CpG count and methylation percentage represents one data point in the downstream classification model. Compared to average-based methods, the methylation information from disease-derived molecules and normal-derived molecules is not averaged. Therefore, the methylation profiles of disease-derived molecules and normal cell-derived molecules can be kept separate. Each resulting read can contain CpG methylation information from the unique DNA molecules captured by the assay. Two metrics are collected from each read: 1) N: total number of CpGs in the read; 2) M: Number of methylated CpGs in the read. From 1) and 2), the third metric is 3) f = M / N, the fraction of CpGs that are methylated within the current read It is calculated as:

[0132] Data pairs (N, f) are collected for each molecule at every DMR in the assay. A scatter plot showing f (y-axis) versus N (x-axis) can be generated for the DMR, with all reads in the DMR represented as dots in the plot. For example, Figure 11 shows the molecular methylation distribution pattern of DMR1 in the genomic DNA of normal colon tissue (Figure 11A) and colon cancer tissue (Figure 11B). This demonstrates a DMR with no hypermethylated DNA molecules in normal colon tissue and a large amount of hypermethylated molecules in colon cancer tissue. Figures 12A and 12B show the molecular methylation distribution pattern of DMR2 in the genomic DNA of normal colon tissue and colon cancer tissue, respectively. This demonstrates a DMR with some hypermethylated DNA molecules in normal colon tissue (Figure 12A) and a large amount of hypermethylated molecules in colon cancer tissue (Figure 12B). Figure 13 shows the molecular methylation distribution patterns of DMR1 and DMR2 in plasma cfDNA from healthy individuals (Figure 13A) and colon cancer patients (Figure 13B). The counts of hypermethylated molecules illustrated in the upper part of Figure 13B from each DMR are the criterion for disease detection from liquid biopsies.

[0133] Several further analyses can be performed. For example, a filter can be applied to count hypermethylated molecules. Filter for hypermethylated molecules: A threshold f0 can be selected to count all molecules with f>f0 (i.e., the upper part of the scatter plot). These reads are hypermethylated reads that are indicative of diseased tissue (such as colon cancer). The hypermethylation filter threshold (f0) can be set to 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, or 0.9. In some cases, the hypermethylation filter threshold (f0) can be set based on an analysis of methylation in samples from normal tissue or healthy subjects. For example, the hypermethylation filter threshold (f0) can be set as 0.5, 1, 1.5, 2, 2.5, or 3 standard deviations from the average methylation fraction in normal tissue samples or samples from healthy subjects.

[0134] Molecules can also be filtered for robust signals. Filter for molecules with robust signals: An additional threshold, N0, can be selected to retain only reads with N>N0, enhancing the robustness of molecule counting. The threshold, N0, can be set to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 30.

[0135] Filtering for hypermethylated molecules and robust signals can ensure that only robustly hypermethylated molecules are counted for each DMR, which may improve the quality and / or sensitivity of the analysis.

[0136] In some cases, the thresholds f0 and N0 are the same across all DMRs. In some cases, the thresholds f0 and N0 may be customized for each individual DMR. In some cases, the threshold f0 may be the same across all DMRs, and the threshold N0 may be customized for each individual DMR. In some cases, the threshold N0 may be the same across all DMRs, and the threshold f0 may be customized for each individual DMR. In some cases, both the thresholds f0 and N0 may be customized for each individual DMR.

[0137] The robust hypermethylated molecule counts across all DMRs in the assay can be fed into a model to determine the disease status of the sample using machine learning classifiers. XI. Sequential target enrichment The present disclosure provides a method of sequential hybridization-based enrichment that can be used to enrich for two or more panels of sequences from the same undivided DNA input. Figure 14 illustrates a method for performing sequential enrichment. In some cases, the method of sequential enrichment may include obtaining a sample containing a plurality of nucleic acid molecules and performing a first target enrichment to enrich for nucleic acid molecules containing sequences corresponding to a first panel of one or more genomic regions, thereby generating a first enriched sample containing nucleic acids enriched for sequences corresponding to the first panel of one or more genomic regions. The first target enrichment may also generate a residual sample (or first residual sample) containing nucleic acids depleted for sequences corresponding to the first panel of one or more genomic regions. This residual sample can be used to perform a second target enrichment on the residual sample to enrich for nucleic acid molecules containing sequences corresponding to a second panel of one or more genomic regions, thereby generating a second enriched sample containing nucleic acids enriched for sequences corresponding to the second panel of one or more genomic regions. The first panel of one or more genomic regions and the second panel of one or more genomic regions are usually different. In some cases, a third, fourth, or further round of target enrichment can be performed using a third, fourth, or further panel of genomic regions.

[0138] For example, the panel of one or more genomic regions may include a panel of 1 to 50,000, 5 to 10,000, or 5 to 5,000 genomic regions associated with mutation hotspots, oncogenes, tumor suppressor genes, oncogene exons, tumor suppressor exons, or regulatory regions. In another example, the panel of one or more genomic regions may include a panel of 5 to 5,000 genomic regions associated with differentially methylated regions, epigenetic modifications, introns, promoters, or other regulatory sequences. In some examples, the panel includes 50 to 500 genomic regions associated with hypermethylation in cancer.

[0139] Because Point-n-Seq is a pre-amplification and pre-conversion enrichment technique, the enriched sample can be analyzed by sequencing or can be bisulfide-treated (or enzyme-treated) before sequencing to evaluate methylation. In some cases, the first enriched sample can be analyzed by sequencing to evaluate mutations, while the second enriched sample can be bisulfide-treated (or enzyme-treated) before sequencing to evaluate methylation. In some cases, both the first enriched sample and the second enriched sample can be evaluated by simple sequencing to evaluate genomic alterations, but the samples can be sequenced at different depths. In some cases, the analysis of the first enriched sample can be performed before performing the second target enrichment step. The results of the analysis of the first enriched sample can be used to select a second panel for the second enrichment step.

[0140] Target enrichment may include any method disclosed herein or known in the art. In some cases, target enrichment includes hybridizing a first target-specific region of a first cross-linking probe to a first target sequence of a molecule having a sequence corresponding to a genomic region, wherein a first adapter landing sequence of the first cross-linking probe binds to a first cross-linking sequence of an adapter anchor probe, and hybridizing a second target-specific region of a second cross-linking probe to a second target sequence of a molecule having a sequence corresponding to a genomic region, wherein a second adapter landing sequence of the second cross-linking probe binds to a second cross-linking sequence of the adapter anchor probe. As described herein, the anchor probe may include a binding moiety. The method typically includes attaching an adapter to the 5' or 3' end of a plurality of nucleic acid molecules, thereby generating a library of nucleic acid molecules containing adapters.

[0141] The sequential target enrichment described herein can be highly efficient.For example, when the second enriched sample is subjected to bisulfite treatment and subjected to sequencing reaction, the number of informative reads of sequencing reaction can be at least 60%, 65%, 70%, 75%, 80% or 85% of the number of informative reads that can be obtained from the sample when subjected to single target enrichment to enrich for the nucleic acid molecules that comprise the sequence corresponding to the second panel of one or more genomic regions.

[0142] The sequential target enrichment methods described herein can be generalized to any nucleic acid sample. The methods can be particularly useful for the analysis of limited nucleic acid samples.

[0143] XII. Application a. Detection of nucleic acid features Amplified nucleic acid products generated using the methods and kits described herein can be analyzed for one or more nucleic acid features. The one or more nucleic acid features can be one or more methylation events. The methylation can be methylation of a cytosine within a CpG dinucleotide. The methylated base can be 5-methylcytosine. Cytosines that are not in a CpG state can also be methylated. The methylated or unmethylated cytosine can be within a CpG island. A CpG island can be a region of the genome with a high frequency of CpG sites. A CpG island can be at least 200 bp, or about 300 to about 3000 bp. The CpG dinucleotide content of a CpG island can be at least 60%. A CpG island can be within the promoter region of a gene. The methylation can be 5-hmC (5-hydroxymethylcytosine), 5-fC (5-formylcytosine), or 5-caC (5-carboxylcytosine). The methods and kits described herein can be used to detect methylation patterns of DNA from, for example, solid tissue or from, for example, biological fluids containing cell-free DNA, such as plasma, serum, urine, or saliva.

[0144] The one or more nucleic acid features can be novel mutations, nonsense mutations, missense mutations, silent mutations, frameshift mutations, insertions, substitutions, point mutations, single nucleotide polymorphisms (SNPs), single nucleotide variants (SNVs), novel single nucleotide variants, deletions, rearrangements, amplifications, chromosomal translocations, interstitial deletions, chromosomal inversions, loss of heterozygosity, loss of function, gain of function, dominant negative, or lethal mutations. The amplified nucleic acid products can be analyzed to detect germline or somatic mutations. The one or more nucleic acid features can be associated with a condition, such as cancer, an autoimmune disease, a neurological disease, an infection (e.g., a viral infection), or a metabolic disease.

[0145] b. Diagnosis / detection / monitoring The disclosed methods and kits can also be used to diagnose or detect a disease or condition. The disease or condition can be associated with aberrant methylation. The condition can be a psychological disorder. The condition can be aging. The condition can be a disease. The condition (e.g., disease) can be cancer, a neurological disease (e.g., Alzheimer's disease, autism spectrum disorder, Rett syndrome, schizophrenia), an immune deficiency, a skin disease, an autoimmune disease (e.g., ocular Behçet's disease, systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), multiple sclerosis), an infection (e.g., a viral infection), or a metabolic disease (e.g., hyperglycemia, hyperlipidemia, type 2 diabetes). The cancer can be, for example, colon cancer, breast cancer, liver cancer, bladder cancer, Wilms' cancer, ovarian cancer, esophageal cancer, prostate cancer, bone cancer, or hepatocellular carcinoma, glioblastoma, breast cancer, lung squamous cell carcinoma, thyroid cancer, or leukemia (see, e.g., Jin and Liu (2018) DNA methylation in human disease. Genes & Diseases, 5:1-8). The condition can be Beckwith-Wiedemann syndrome, Prader-Willi syndrome, or Angelman syndrome.

[0146] The methylation pattern of cell-free DNA generated using the methods and kits provided herein can be used as a marker for cancer (see, e.g., Hao et al., DNA methylation markers for diagnosis and prognosis of common cancers. Proc. Natl. Acad. Sci. 2017; International PCT Application Publication No. WO2015116837). The methylation pattern of cell-free DNA can be used to determine the tissue of origin of DNA (see, e.g., International PCT Application Publication No. WO2005019477). The methods and kits described herein can be used to determine methylation haplotype information and can be used to determine the tissue or cellular origin of cell-free DNA (see, for example, Seioighe et al. (2018) DNA methylation haplotypes as cancer markers. Nature Genetics 50, 1062-1063; International PCT Publication No. WO2015116837; US Patent Application Publication No. 20170121767). The methods and kits described herein can be used to detect, for example, the methylation level of cell-free DNA in subjects with and without cancer (see, for example, Vidal et al. A DNA methylation map of human cancer at single base-pair resolution. Oncogenomics 36, 5648-5657; International PCT Publication No. WO2014043763). The methods and kits described herein can be used to determine methylation levels or to determine the fractional contributions of different tissues to a cell-free DNA mixture (see, e.g., International PCT Application Publication No. WO2016008451).The methods and kits described herein can be used, for example, based on comparing the patterns and abundance of methylation haplotypes with respect to the tissue of origin of cell-free DNA in plasma (see, for example, Tang et al., (2018) Tumor origin detection with tissue-specific miRNA and DNA methylation markers. Bioinformatics 34, 398-406; International PCT Application Publication No. WO2018119216). The methods and kits described herein can be used to distinguish cancer cells from normal cells and to classify different cancer types according to their tissue of origin (see, for example, U.S. Patent Application Publication No. 20170175205 A1). The methods and kits provided herein can be used to detect fetal DNA or fetal abnormalities using maternal samples (see, e.g., Poon et al. (2002) Differential DNA Methylation between Fetus and Mother as a Strategy for Detecting Fetal DNA in Maternal Plasma. Clinical Chemistry, 48: 35-41).

[0147] The disclosed methods can be used to monitor a condition. The condition can be a disease. The disease can be cancer, a neurological disease (e.g., Alzheimer's disease), an immunodeficiency, a skin disease, an autoimmune disease (e.g., ocular Behcet's disease), an infection (e.g., a viral infection), or a metabolic disease. The cancer can be in remission. Because the disclosed methods can detect low-level abnormalities using cfDNA and ctDNA, the present disclosure can provide a relatively non-invasive method for monitoring disease. The disclosed methods can be used to monitor treatment or therapy. The treatment or therapy can be used for a condition, such as a disease, such as cancer, or for any condition disclosed herein. The methods described herein may allow for the enrichment of target molecules directly from cfDNA prior to bisulfite conversion and amplification. The methods may also allow for the development of small, focused panels that examine the methylation status of 1 to approximately 1,000 markers for a given disease. In some cases, kits may be created for panels that examine the methylation status of 1 to approximately 10,000 differentially methylated regions for a given disease. [Example]

[0148] Example 1 Synergistic indirect hybridization capture Synergistic indirect capture of nucleic acids for sequencing (SICON-SEQ) experiments were performed using two bridge probes with different sequences and an adapter anchor probe / universal anchor probe (UP, SEQ ID NO: 1). Two bridge probes (EGFR-BP2, SEQ ID NO: 2, and EGFR-BP3, SEQ ID NO: 3) were designed to target the EGFR genomic sequence. Each bridge probe contained an approximately 25-bp targeting sequence (TS1 or TS2) region, a linker containing at least 15 thymines, and a 20-bp landing sequence (LS1 or LS1, in italics) designed to be complementary to the bridge sequence on the adapter anchor probe. The adapter anchor probe contained two bridge sequences (BBS1 or BBS2) designed to hybridize to either of the bridge probe's landing sequences. The adapter anchor probe was further biotinylated at the 5' end of the nucleic acid sequence. Figure 4 presents a schematic diagram of synergistic indirect hybridization. [Table 1]

[0149] For hybridization capture, 20 ng of fragmented (peak size 160 bp) gDNA was mixed with two bridging probes for EGFR (1 fmole each) and one universal anchor probe (200 fmole) in a final solution volume of 20 μl. The DNA input and hybridization probes were denatured in hybridization buffer at 95°C for 30 min and gradually cooled to 65°C. The hybridization complex was incubated at 65°C for 1 h in a thermocycler. The final hybridization buffer contained 100 ng / μl blocking DNA, 1 μg / μl bovine serum albumin (BSA), 1 μg / μl Ficoll, 1 μg / μl polyvinylpyrrolidone (PVP), 0.075 M sodium citrate, 0.75 M NaCl, 5x SSC, and 1x Denhardt's solution.

[0150] For capture / cleanup, the hybridization assemblies were incubated with streptavidin beads (Thermo Fisher Dynabeads M270 Streptavidin) for 10 min at room temperature. Cleanup was performed using three washes (Wash 1: 5x SSPE, 1% SDS; Wash 2: 2x SSPE, 0.1% SDS; Wash 3: 0.1x SSPE, 0.01% Triton).

[0151] The enriched DNA was evaluated by qPCR using primers for the EGFR targeting sequence (SEQ ID NOs: 4 and 5). qPCR results for the captured EGFR DNA were compared to the same portion of gDNA without capture enrichment. Over 65% to 90% of the EGFR was recovered.

[0152] Example 2 Capture by different hybridization schemes To determine the capture performance of various hybridization systems, four hybridization schemes were tested: non-synergistic hybridization, direct (Figure 5A), synergistic direct hybridization (Figure 5B), synergistic indirect hybridization (Figure 5C), and non-synergistic indirect hybridization (Figure 5D).

[0153] The non-synergistic direct method involved hybridization of a biotinylated capture probe (120 bp, SEQ ID NO: 6) containing a target-specific sequence (hatched, Figure 5A). The synergistic direct method involved hybridization of four short biotinylated capture probes (SEQ ID NOs: 7-10), each containing a 25 bp target-specific sequence (hatched, Figure 5B). The synergistic indirect method utilized four short bridging probes (SEQ ID NOs: 12-15) without biotin (Figure 5C), each containing the same target-specific sequence as one of the capture probes used in the synergistic direct method. Each of the bridging probes (BP) contained one of two different landing sequences (dotted and vertically hatched) designed to be complementary to one of the bridging sequences in the universal anchor probe (SEQ ID NO: 11). A non-synergistic, but indirect, approach (Figure 5D) was tested by using a short bridge probe (SEQ ID NO: 16) paired with the same universal anchor probe used for the synergistic direct hybridization. The capture probe or universal anchor probe (UP) used in the experiment was biotinylated at the 5' end. [Table 2-1] [Table 2-2]

[0154] Before the hybridization reaction, 10 ng of cfDNA was used to construct an NGS library using the NEBNext Ultra II DNA Library Preparation Kit by following the steps in the accompanying protocol. After library construction, hybridization-based capture was performed directly with the ligation mix without bead purification to enrich the library. The enriched library was then subjected to qPCR analysis.

[0155] Capture efficiency was assessed by comparing the percentage of EGFR present before and after capture. The post-capture ct was compared to 2.5 ng of human gDNA library (an appropriate fraction of the capture input). Capture efficacy PCR was performed using a primer designed for EGFR (SEQ ID NO: 17) and the NGS adapter P7 sequence (SEQ ID NO: 18). The background (total DNA present) was assessed by qPCR using primers (SEQ ID NOs: 18, 19) capable of amplifying all DNA libraries. All background delta ct were normalized to the average CT obtained from the "C" probe design.

[0156] Indirect synergistic hybridization capture demonstrated superior hybridization sensitivity and specificity to either the non-synergistic or direct methods (Table 3). The synergistic indirect probe design demonstrated the highest capture efficiency (average approximately 91%) and the lowest background noise. Non-synergistic direct hybridization showed recovery of 0-14.87% at much higher (300x) bridging probe concentrations, but a greater than 200-fold increase over background. Lowering the hybridization temperature did not benefit capture efficiency, but instead dramatically increased background noise. For the synergistic, but not indirect, design, neither increasing bridging probe concentration nor decreasing hybridization benefited capture efficiency. No capture enrichment was detected for the indirect, non-synergistic method. [Table 3]

[0157] Example 3 Indirect capture by universal anchor probes with or without spacers A study was performed to determine whether the presence of a spacer between two or more bridging sequences on a universal anchor probe (UP) affects the capture performance of indirect synergistic hybridization capture. The same bridging probe was used in both cases.

[0158] Table 4 lists the sequences of the cross-linking probes and UPs used. Figure 6A shows a schematic of synergistic indirect hybridization using a UP with a spacer. Figure 6B shows synergistic indirect hybridization using a UP without a spacer. [Table 4-1] [Table 4-2]

[0159] The capture efficiency and background noise were determined for either hybridization capture. The background noise was calculated by normalizing the qPCR results to the average background signal. The capture efficiency was not significantly affected by the presence of a spacer, but the background noise of the capture hybridization without a spacer was approximately 100-fold higher than that of the capture with a spacer (Table 5). This suggests that the spacer in the universal anchor probe played an important role in enabling highly specific (low background) capture. [Table 5]

[0160] Example 4 Determining NGS metrics using synergistic indirect capture methods Next-generation sequencing (NGS) metrics were determined using 3-, 15-, and 76-target panels. The mapping rate was calculated as the percentage of sequencing reads aligned to the human genome. The mapping rates for the 3-, 15-, and 76-target panels were 97%, 94%, and 95%, respectively (Table 6). The on-target rate was calculated using de-duplicated mapped reads across the region covered by the capture probe and 100-bp flanking. For small panels such as the 3-, 15-, and 76-target panels, traditional hybridization-based DNA enrichment was not feasible. However, the study demonstrated comparable on-target rates of 83.6% and 85.3% for the 15- and 76-target panels compared with standard target panels with over 50 kb.

[0161] Furthermore, uniformity across the panel was high (>99% of positions had reads higher than 0.2x average coverage, and over 95% had 0.5x coverage). 0.2x or 0.5x coverage was not suitable for the three-target micropanel. The high uniformity of the 15-target panel also reflected uniform coverage of regions with high GC content (Figure 7). Coverage of regions with 80% GC content was higher than 0.5x average coverage. [Table 6]

[0162] Example 5 Determination of NGS metrics for human SNPs using a synergistic indirect capture method. A synergistic indirect hybridization assay was performed covering 76 human ID single nucleotide polymorphisms (SNPs). Pre-amplification hybridization was performed on 20 ng of human cell-free DNA (cfDNA). Results were compared with post-amplification hybridization results using the commercially available IDT xGen Hybridization and Cleanup Kit. The xGen Human ID Research Panel V1.0, which covers the same 76 ID SNPs, was used for capture. Hybridization-based capture was performed using the xGen Human ID Panel on an NGS library constructed using 20 ng of cfDNA as the original input according to the commercial protocol.

[0163] Next-generation sequencing (NGS) metrics were determined using a 76-target panel (Table 7). The post-amplification capture target rate was low, with an on-target rate of 30.7%. In contrast, the SICON-MAS panel covering the same genomic region had an on-target rate of 88%. [Table 7]

[0164] Example 6 Comparison of SICON-SEQ and post-amplification methods Synergistic indirect capture of nucleic acids for sequencing (SICON-SEQ) was performed on a panel of 76 human gene targets, providing an on-target rate of >80% for 1M reads from 10 ng of cfDNA input with only 1 hour of pre-amplification capture. Post-amplification capture using the company "I" kit was used on the same panel, yielding only 6-30% on-target rate for 1M reads from twice the amount of input (20 ng of cfDNA) with 16 hours of post-amplification capture. Pre-amplification capture using the company "I" kit was also performed, but yielded no results.

[0165] Figures 8A-8B show coverage of regions of different percentages of GC content by the SICON-SEQ and IDT xGEN hybridization and wash kits. Coverage from regions with low GC content (<30%) to high GC content (>50%) was highly uniform for the SICON-SEQ assay (Figure 8A). For the capture protocol using the IDT xGEN kit, which did not result in library enrichment (Figure 8B), coverage of regions with different GC content was systematically biased.

[0166] Example 7 Methylation assay by SICON-TMS The SICON targeted methylation sequencing (SICON-TMS) assay was performed as illustrated in Figures 2A and 2B. Sample cfDNA was extracted from 3-5 ml of plasma from different non-cancer individuals and interrogated for 120 different differentially methylated regions (DMRs). The readout showed a nearly linear relationship with input (R ) even with a cfDNA input as low as 1 ng. 2 =0.9474) relationship was shown (Figure 9).

[0167] Example 8 Detection of methylated DNA in cfDNA by SICON-TMS SICON-TMS assays were performed to examine 60 different differentially methylated regions (DMRs).

[0168] Next-generation sequencing (NGS) libraries were first constructed using cfDNA by following the NEBNext Ultra II kit manual. Library DNA (cfDNA with spike-in methylated DNA at 0.01%, 0.1%, 1%, 10%, or 100%) was input for hybridization capture. 20 ng of unamplified DNA was mixed with the probe, and the library / probe mixture was denatured in hybridization buffer at 95°C for 30 minutes. The mixture was gradually cooled to 60°C. The hybridization mixture was incubated at 60°C for 1 hour in a thermocycler. The final hybridization buffer contained 100 ng / μl salmon sperm DNA, 1 μg / μl bovine serum albumin (BSA), 1 μg / μl Ficoll, 1 μg / μl polyvinylpyrrolidone (PVP), 0.075 M sodium citrate, 0.75 M NaCl, 5× SSC, and 1× Denhardt's solution.

[0169] For cleanup, the captured assemblies were incubated with streptavidin beads (Thermo Fisher Dynabeads M270 Streptavidin) for 10 min at room temperature, followed by three washes (Wash 1: 5x SSPE, 1% SDS; Wash 2: 2x SSPE, 0.1%; Wash 3: 0.1x SSPE, 0.01% Triton). The cleaned assemblies were treated with bisulfite for methylation analysis.

[0170] Figure 10 shows the relationship between predicted spike-in and measured values. The SICON-TMS assay demonstrated analytical sensitivity and linearity down to 0.01% methylation. Methylation percentage is expressed as R 2 was 0.99 and highly correlated with the predicted value, indicating high accuracy of the assay.

[0171] Example 9 Detection of cancer methylation patterns in cfDNA by SICON-TMS Samples from normal colon tissue and colon cancer tissue, as well as plasma cfDNA samples from healthy individuals and colon cancer patients, were bisulfite treated and sequenced. The sequencing reads were mapped to each differentially methylated region (DMR) that was de-duplicated. Each resulting read contained CpG methylation information from the unique DNA molecule captured by the assay. Two metrics were then calculated for each read: 1) N: total number of CpGs in the read; 2) M: Number of methylated CpGs in the read. From 1) and 2), the third metric is 3) f = M / N, the fraction of CpGs that are methylated within the current read It was calculated as:

[0172] The results are shown as a scatter plot of f (y-axis) versus N (x-axis) for each DMR, with all reads of the DMR shown as dots in the plot. Figure 11 shows the molecular methylation distribution pattern of DMR1 in the genomic DNA of normal colon tissue (Figure 11A) and colon cancer tissue (Figure 11B). This demonstrates the absence of hypermethylated DNA molecules in normal colon tissue and the presence of a large amount of hypermethylated molecules in colon cancer tissue.

[0173] Figures 12A and 12B show the molecular methylation distribution patterns of DMR2 in the genomic DNA of normal colon tissue and colon cancer tissue, respectively. These figures demonstrate the presence of some hypermethylated DNA molecules in normal colon tissue and a large amount of hypermethylated molecules in colon cancer tissue.

[0174] Figures 13A and 13B show the molecular methylation distribution patterns of DMR1 and DMR2 in plasma cfDNA of healthy individuals and colon cancer patients, respectively. The counts of hypermethylated molecules illustrated in the upper part of Figure 13B from each DMR can be used as a criterion for disease detection from liquid biopsies.

[0175] Example 10 Detection of cancer methylation patterns in cfDNA by SICON-TMS We designed a Point-n-Seq colorectal cancer (CRC) panel covering 100 methylation markers in three steps. First, approximately 1,000 CRC-specific markers were identified from public databases. Second, markers with high background signal in baseline cfDNA from healthy populations were filtered out. Finally, the list was refined to include markers that best discriminated between patient and healthy cfDNA. The SICON CRC panel capture was highly efficient, yielding high uniformity (94% >0.5×, 100% >0.2×) and on-target rates (>80%). For a 20 ng cfDNA input, an average of >1,000 de-duplicated informative reads were obtained for each marker, despite the high GC content (>80%). The informative read output was linear with respect to cfDNA input in the range of 1 ng to 40 ng. In a dose-finding study, 0.6 pg (0.2 × genome equivalent) of methylated DNA (0.003%) in 20 ng of cfDNA was reliably detected above the cfDNA background. In a pilot clinical study using plasma samples from patients with colorectal adenocarcinoma—early stage I (n=7; II (n=7)), late stage III (n=11; IV (n=3))—and control individuals (n=105), the mean percentage of methylation signals was 0.0034%, 0.013%, 0.09%, 0.17%, and 0.29% for controls, stages I, II, III, and IV, respectively. The methylation percentage in stage I samples was significantly different from the control group (P<0.001). Using a simple cutoff using the methylation percentage, the Point-n-Seq CRC Panel achieved a sensitivity of 86% for stage I and 100% for stages II-IV, with a specificity of 91% and an area under the curve (AUC) of 0.96.

[0176] Example 11 Point-n-Seq SNV+Methyl Dual Capture Analysis in CRC Plasma Samples Genetic and epigenetic alterations were detected by an integrated Point-n-Seq assay in plasma samples (1 ml) from patients with advanced CRC. A Point-n-Seq colorectal cancer (CRC) panel covering methylation markers and over 350 hotspot mutations from 22 genes was designed.

[0177] Two sequential rounds of target enrichment were performed using a methylation marker panel and a mutation hotspot panel by synergistic indirect hybridization capture as described herein. Briefly, 20 μL of each cfDNA sample was added to a PCR tube. For DNA volumes less than 20 μL, IDTE or buffer EB was added to a final volume of 20 μL. For each sample, 2.8 μL of final preparation buffer and 1.2 μL of final preparation enzyme were added. The tubes were gently vortexed to mix thoroughly, and then briefly centrifuged. The tubes were run in a thermal cycler with a heated lid at 20 ° C for 30 minutes, followed by 65 ° C for 30 minutes. 2.5 μL of adapter solution was added, and 13 μL of ligation mix was added, and the mixture was incubated at 20 ° C for 30 minutes.

[0178] The sample-bound beads were equilibrated at room temperature for at least 15 minutes and vortexed to resuspend. 48 μL (approximately 1.2x volume) of library-bound beads were added to 39.5 μL of ligation reaction mixture. These were thoroughly mixed by pipetting at least 10 times and briefly centrifuged. The mixture was incubated at room temperature for 10 minutes and placed on a magnet for at least 2 minutes or until the solution was clear. The supernatant was removed and discarded. While on the magnet, 150 μL of sample wash buffer was added to the beads without disturbing them, incubated for 2 minutes, and the supernatant was discarded.

[0179] For target capture, the hybridization mixture containing the mutation capture panel and probe binding mixture was added and mixed thoroughly by gentle vortexing or flicking. The mixture was heated to 98°C for 2 minutes, then ramped down to 60°C at a rate of 2.5°C / second and incubated at 60°C for 60 minutes. After 60 minutes of hybridization, the sample was placed on a magnet for 30 seconds, and the supernatant was carefully transferred to a labeled tube and saved for the second hybridization step. The beads were washed three times, resuspended, and DNA was amplified on the beads.

[0180] The pooled supernatant from above was mixed with a hybridization mixture containing the TMS capture panel, and capture hybridization was performed with the mutation capture panel. The captured TMS DNA was disulfide treated, repaired, and eluted from the beads, followed by index PCR. Both amplified DNA samples were prepared for sequencing and sequenced on an Illumina platform.

[0181] Figure 14 illustrates sequential target enrichment. Table 8 lists the DNA input amount and the percentage of methylation and mutant signals for each patient sample. Details of the detected mutations are shown in Figure 15. As shown by Table 8, the Point-n-Seq CRC Mutation Capture and Methylation Panel yielded highly efficient detection of hypermethylation and mutations from a wide range of starting DNA amounts. Furthermore, combined methylation and mutation analysis using plasma cfDNA from CRC patients demonstrated concordant tumor content estimates from methylation status and driver mutation allele frequencies.

[0182] [Table 8]

[0183] Example 12 Methylation signals from dual analysis are comparable to standalone methylation (TMS) analysis To evaluate the methylation signal derived from the sequential target enrichment method, a dose-finding experiment was performed using gDNA from the cell line HCT116 spiked into control cfDNA. HCT116 gDNA was spiked at concentrations ranging from 0.001% to 10%. As outlined in Figure 14, the same DNA input was subjected to TMS analysis alone or sequential SICON mutation-TMS dual analysis, in which the enrichment step for mutation analysis was performed first and the enrichment step for TMS analysis was performed second. As shown in Figure 16, the methylation scores from the standalone and dual analyses were comparable, indicating that the sensitivity of the methylation assay was not compromised with the second capture in sequential capture dual analysis. Figure 17 shows that the second capture TMS recovery (informative molecule counts from sequencing per differentially methylated region (DMR)) was approximately 85% of the first capture TMS.

[0184] Example 13 Tumor-informed personalized panel analysis CRC tumor gDNA was subjected to full exon sequencing, and 114 single base variants were selected to create personalized panel.CRC tumor gRNA was added to control cfDNA in dose-finding experiments at concentrations of 0.001%, 0.003%, 0.01%, 0.03% and 0.1%.As shown in Figure 18, the sample added at 0.003% can be distinguished from 0%, suggesting a detection limit of 0.003% for certain personalized hybridization-based assays.It is expected that a larger panel will result in a lower detection limit.

[0185] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous modifications, changes, and substitutions will readily occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein can be employed in practicing the invention. It is intended that the following claims define the scope of the invention, and that methods and structures that fall within the scope of these claims and their equivalents be covered thereby.

[0186] The spirit and scope of the present invention lie in the appended claims, but are also implicit in the following [preliminary claims], which existed as claims in the parent application and have been partially amended to eliminate them, the subject matter of which is incorporated herein by reference. [Preliminary Claims] [Preliminary Claim 1] obtaining a template nucleic acid molecule comprising an adaptor at the 5' or 3' end of the template nucleic acid molecule; hybridizing a first target-specific region of a first cross-linking probe to a first target sequence of the template nucleic acid molecule, wherein a first adaptor landing sequence of the first cross-linking probe binds to a first cross-linking sequence of an adaptor-anchor probe; hybridizing a second target-specific region of a second cross-linking probe to a second target sequence of the template nucleic acid molecule, wherein a second adaptor landing sequence of the second cross-linking probe binds to a second cross-linking sequence of the adaptor-anchor probe; A method comprising: [Preliminary Claim 2] The method of claim 1, further comprising the step of attaching the adaptor to the 5' end or 3' end of a sample nucleic acid molecule, thereby generating a template nucleic acid molecule comprising the adaptor. [Preliminary Claim 3] 3. The method of claim 2, comprising attaching the adaptor to the 5' end or the 3' end of a sample nucleic acid molecule, and attaching an adaptor to the 3' end or the 5' end of the template nucleic acid molecule comprising the adaptor, respectively, thereby generating a template nucleic acid molecule comprising an adaptor at each end. [Preliminary Claim 4] 4. The method of claim 2 or 3, further comprising hybridizing an adapter primer to the adapter attached to the 3' end of the template nucleic acid molecule hybridized to the first bridge probe and the second bridge probe, and extending the 3' end of the adapter primer, thereby generating an extension product. [Preliminary Claim 5] 5. The method of claim 4, further comprising sequencing the extension products. [Preliminary Claim 6] 6. The method of claim 1, wherein prior to the hybridizing step to the first target-specific region, the first adapter landing sequence of the first cross-linking probe binds to the first cross-linking sequence of the adapter anchor probe. [Preliminary Claim 7] 6. The method of claim 1, wherein after the hybridizing step to the first target-specific region, the first adapter landing sequence of the first cross-linking probe binds to the first cross-linking sequence of the adapter anchor probe. [Preliminary Claim 8] 8. The method of claim 1, wherein prior to the hybridizing step to the second target-specific region, the second adapter landing sequence of the second bridging probe binds to the second bridging sequence of the adapter anchor probe. [Preliminary Claim 9] 8. The method of claim 1, wherein after the hybridizing step to the second target-specific region, the second adapter landing sequence of the second bridging probe binds to the second bridging sequence of the adapter anchor probe. [Preliminary Claim 10] 10. The method of claim 1, further comprising hybridizing the first landing sequence of the first cross-linking probe to the first cross-linking sequence of the adaptor anchor probe. [Preliminary Claim 11] 11. The method of claim 1, further comprising hybridizing the second landing sequence of the second cross-linking probe to the second cross-linking sequence of the adaptor anchor probe. [Preliminary Claim 12] 12. The method of claim 1, wherein the adaptor anchor probe further comprises a spacer located between the first cross-linking sequence and the second cross-linking sequence. [Preliminary Claim 13] The method of any one of claims 1 to 12, wherein the adapter comprises a molecular barcode. [Preliminary Claim 14] The method of claim 1 , wherein the adaptor anchor probe comprises a binding moiety. [Preliminary Claim 15] The method of claim 14 , wherein the binding moiety is bound to a support. [Preliminary Claim 16] The method of claim 15, wherein the support is a bead. [Preliminary Claim 17] 17. The method of claim 16, wherein the beads are streptavidin beads. [Preliminary Claim 18] 18. The method of claim 14, wherein the binding moiety is biotin. [Preliminary Claim 19] The method of any one of claims 1 to 18, wherein the first cross-linking probe comprises a binding moiety. [Preliminary Claim 20] The method of claim 19 , wherein the binding moiety is bound to a support. [Preliminary Claim 21] 21. The method of claim 20, wherein the support is a bead. [Preliminary Claim 22] 22. The method of claim 21, wherein the beads are streptavidin beads. [Preliminary Claim 23] 23. The method of any one of claims 19 to 22, wherein the binding moiety is biotin. [Preliminary Claim 24] The method according to any one of claims 1 to 23, wherein the template nucleic acid molecule comprises single-stranded DNA. [Preliminary Claim 25] 24. The method according to claim 1, wherein the template nucleic acid molecule comprises cell-free nucleic acid derived from a biological sample. [Preliminary Claim 26] 26. The method of claim 25, wherein the cell-free nucleic acid comprises cell-free DNA. [Preliminary Claim 27] 27. The method of claim 26, wherein the cell-free DNA comprises circulating tumor DNA. [Preliminary Claim 28] The method according to any one of claims 1 to 27, wherein the template nucleic acid molecule comprises damaged DNA. [Preliminary Claim 29] hybridizing a first target-specific region of a first cross-linking probe to a first target sequence of a template nucleic acid molecule, wherein a first adaptor landing sequence of the first cross-linking probe binds to a first cross-linking sequence of an adaptor-anchor probe; hybridizing a second target-specific region of a second crosslinking probe to a second target sequence of the template nucleic acid molecule, wherein a second adaptor landing sequence of the second crosslinking probe binds to a second crosslinking sequence of the adaptor-anchor probe, thereby generating a template nucleic acid molecule hybridized to the first crosslinking probe and the second crosslinking probe; treating the template nucleic acid molecule with a methylation assay reagent after said hybridizing step of the first target-specific region and said hybridizing step of the second target-specific region; A method comprising: [Auxiliary Claim 30] 30. The method of claim 29, wherein the methylation assay reagent is a disulfide or an enzyme that modifies methylated cytosine. [Preliminary Claim 31] 30. The method of claim 29, further comprising hybridizing a third target-specific region of a third cross-linking probe to a third target sequence of the template nucleic acid molecule, wherein a third adapter landing sequence of the third cross-linking probe binds to a third cross-linking sequence of an adapter-anchor probe. [Preliminary Claim 32] 32. The method of Claim 31, further comprising hybridizing a fourth target-specific region of a fourth bridge probe to a fourth target sequence of the template nucleic acid molecule, wherein a fourth adapter landing sequence of the fourth bridge probe binds to a fourth bridge binding sequence of an adapter anchor probe. [Preliminary Claim 33] 30. The method of Claim 29, further comprising attaching an adaptor to the 5' end or the 3' end of the template nucleic acid molecule prior to the hybridizing step of the first bridge probe and prior to the hybridizing step of the second bridge probe. [Preliminary Claim 34] 34. The method of Claim 33, further comprising hybridizing an adapter primer to the adapter attached to the 3' end of the template nucleic acid molecule hybridized to the first bridge probe and the second bridge probe, and extending the 3' end of the adapter primer, thereby generating an extension product. [Preliminary Claim 35] 35. The method of claim 34, further comprising sequencing the extension products. [Preliminary Claim 36] 35. The method of claim 34, wherein the hybridizing step of the adapter primer is performed before treatment with bisulfite. [Preliminary Claim 37] 35. The method of claim 34, wherein the hybridizing step of the adapter primer is performed after treatment with bisulfite. [Preliminary Claim 38] 35. The method of claim 34, wherein the adapter primer is designed based on the adapter after treatment with bisulfite, and unmethylated cytosines in the adapter are converted to uracils during the treatment. [Preliminary Claim 39] 39. The method of any one of claims 29 to 38, wherein prior to the hybridizing step to the first target-specific region, the first adapter landing sequence of the first cross-linking probe binds to the first cross-linking sequence of the adapter anchor probe. [Auxiliary Claim 40] 39. The method of any one of claims 29 to 38, wherein after the hybridizing step to the first target-specific region, the first adapter landing sequence of the first bridging probe binds to the first bridging sequence of the adapter anchor probe. [Preliminary Claim 41] 41. The method of claim 29, wherein prior to the hybridizing step to the second target-specific region, the second adapter landing sequence of the second bridging probe binds to the second bridging sequence of the adapter anchor probe. [Preliminary Claim 42] 41. The method of claim 29, wherein after the hybridizing step to the second target-specific region, the second adapter landing sequence of the second bridging probe binds to the second bridging sequence of the adapter anchor probe. [Preliminary Claim 43] 43. The method of any one of claims 29 to 42, further comprising hybridizing the first landing sequence of the first cross-linking probe to the first cross-linking sequence of the adaptor anchor probe. [Preliminary Claim 44] 44. The method of any one of claims 29 to 43, further comprising hybridizing the second landing sequence of the second cross-linking probe to the second cross-linking sequence of the adaptor anchor probe. [Preliminary Claim 45] 45. The method of any one of claims 1 to 44, wherein the adaptor-anchored probe further comprises a spacer positioned between the first cross-linking sequence and the second cross-linking sequence. [Preliminary Claim 46] 46. ​​The method of any one of claims 30 to 45, wherein the adapter comprises a molecular barcode. [Preliminary Claim 47] 47. The method of any one of claims 29 to 46, wherein the adaptor anchor probe comprises a binding moiety. [Auxiliary Claim 48] The method of claim 47, wherein the binding moiety is bound to a support. [Preliminary Claim 49] 49. The method of claim 48, wherein the support is a bead. [Preliminary Claim 50] 50. The method of claim 49, wherein the beads are streptavidin beads. [Preliminary Claim 51] 51. The method of any one of claims 47 to 50, wherein the binding moiety is biotin. [Preliminary Claim 52] 51. The method of any one of claims 29 to 50, wherein the first cross-linking probe comprises a binding moiety. [Preliminary Claim 53] 53. The method of claim 52, wherein the binding moiety is bound to a support. [Preliminary Claim 54] 54. The method of claim 53, wherein the support is a bead. [Preliminary Claim 55] 55. The method of claim 54, wherein the beads are streptavidin beads. [Preliminary Claim 56] 56. The method of any one of claims 52 to 55, wherein the binding moiety is biotin. [Preliminary Claim 57] 57. The method of claim 29, wherein the template nucleic acid molecule comprises single-stranded DNA. [Preliminary Claim 58] 57. The method according to claim 29, wherein the template nucleic acid molecule comprises cell-free nucleic acid derived from a biological sample. [Preliminary Claim 59] 58. The method of Claim 57, wherein said cell-free nucleic acid comprises cell-free DNA. [Preliminary Claim 60] 59. The method of Claim 58, wherein the cell-free DNA comprises circulating tumor DNA. [Preliminary Claim 61] 61. The method of claim 29, wherein the template nucleic acid molecule comprises damaged DNA. [Preliminary Claim 62] a cross-linking probe comprising a target-specific region configured to hybridize to a target sequence of a template nucleic acid molecule; an adapter anchor probe comprising a bridge binding sequence configured to hybridize to the adapter landing sequence of the bridge probe; and an adaptor configured to bind to the 5' end or the 3' end of the template nucleic acid molecule. [Preliminary Claim 63] a template nucleic acid molecule, wherein the 5' or 3' end of the template nucleic acid molecule is linked to an adaptor; a first cross-linking probe, wherein a first target-specific region of the first cross-linking probe hybridizes to a first target sequence of the template nucleic acid molecule; a second cross-linking probe, wherein a second target-specific region of the second cross-linking probe hybridizes to a second target sequence of the template nucleic acid molecule; an adapter anchor probe, wherein a first cross-linking sequence of the adapter anchor probe binds to a first adapter landing sequence of the first cross-linking probe and a second cross-linking sequence of the adapter anchor probe binds to a second adapter landing sequence of the second cross-linking probe. [Preliminary Claim 64] A nucleic acid complex comprising a template nucleic acid molecule, wherein the 5' or 3' end of the template nucleic acid molecule is bound to an adapter, a first target sequence of the template nucleic acid molecule hybridizes to a first target-specific region of a first crosslinking probe, a second target sequence of the template nucleic acid molecule hybridizes to a second target-specific region of a second crosslinking probe, a first adapter landing sequence of the first crosslinking probe binds to a first crosslinking sequence of an adapter-anchor probe, and a second adapter landing sequence of the second crosslinking probe binds to a second crosslinking sequence of the adapter-anchor probe. [Preliminary Claim 65] 65. A composition comprising the nucleic acid complex of claim 64.

Claims

1. 1. A method of sequential enrichment comprising: obtaining a sample comprising a plurality of nucleic acid molecules; performing a first target enrichment to enrich for nucleic acid molecules comprising sequences corresponding to a first panel of one or more genomic regions, thereby generating a first enriched sample comprising nucleic acids enriched for sequences corresponding to said first panel of one or more genomic regions, and a residual sample comprising nucleic acids depleted for sequences corresponding to said first panel of one or more genomic regions; performing a second target enrichment on the remaining sample to enrich for nucleic acid molecules comprising sequences corresponding to a second panel of one or more genomic regions, thereby generating a second enriched sample comprising nucleic acids enriched for sequences corresponding to the second panel of one or more genomic regions, wherein the first panel of one or more genomic regions and the second panel of one or more genomic regions are different; A method comprising:

2. 10. The method of claim 1, further comprising the steps of performing a first analysis of the first enriched sample and a second analysis of the second enriched sample.

3. 3. The method of claim 2, wherein the first analysis is a sequence analysis and the second analysis is a methylation analysis.

4. 3. The method of claim 2, wherein the first analysis is a first sequence analysis and the second analysis is a second sequence analysis, and the first sequence analysis is performed at a different sequencing depth than the second sequence analysis.

5. The method of any one of claims 1 to 4, wherein the sample is a cfDNA sample.

6. 5. The method of any one of claims 1 to 4, wherein target enrichment for the genomic regions of said panel of one or more genomic regions comprises target enrichment by hybridization.

7. Target enrichment for a genomic region of the panel of one or more genomic regions comprises hybridizing a first target-specific region of a first cross-linking probe to a first target sequence of a molecule having a sequence corresponding to said genomic region, wherein a first adapter landing sequence of said first cross-linking probe binds to a first cross-linking sequence of an adapter anchor probe; 5. The method of claim 1, wherein a second target-specific region of a second cross-linking probe is hybridized to a second target sequence of the molecule having a sequence corresponding to the genomic region, wherein a second adapter landing sequence of the second cross-linking probe binds to a second cross-linking sequence of the adapter anchor probe.

8. The method of claim 7 , wherein the adaptor anchor probe comprises a binding moiety.

9. 9. The method of claim 8, further comprising the steps of binding the binding moiety to a support and separating the support with the bound binding moiety from unbound nucleic acid.

10. 10. The method of claim 1, wherein the first or second panel of genomic regions comprises a promoter region.

11. 11. The method of claim 1 or 10, wherein the first or second panel of genomic regions comprises an intron region.

12. 12. The method of claim 1, 10, or 11, wherein the first or second panel of genomic regions comprises exon regions.

13. 13. The method of any one of claims 1 to 12, further comprising attaching adaptors to the 5' or 3' ends of nucleic acid molecules of the plurality of nucleic acid molecules, thereby generating a library of adaptor-containing nucleic acid molecules.

14. The method of any one of claims 1 to 13, wherein the second enriched sample is bisulfite treated and subjected to a sequencing reaction.

15. 15. The method of Claim 14, wherein the number of informative reads of the sequencing reaction is at least 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% of the number of informative reads that could be obtained from the sample when subjected to single target enrichment to enrich for nucleic acid molecules comprising sequences corresponding to a second panel of one or more genomic regions.

16. 2. The method of claim 1, further comprising: performing a third target enrichment on a second remaining sample comprising nucleic acids depleted for sequences corresponding to the first and second panels of one or more genomic regions to enrich for nucleic acid molecules comprising sequences corresponding to the third panel of one or more genomic regions, thereby generating a third enriched sample comprising nucleic acids enriched for sequences corresponding to the third panel of one or more genomic regions, wherein the first panel of one or more genomic regions, the second panel of one or more genomic regions, and the third panel of one or more genomic regions are different.

17. 8. The method of claim 7, further comprising hybridizing a third target-specific region of a third cross-linking probe to a third target sequence of the molecule having a sequence corresponding to the genomic region, wherein a third adapter landing sequence of the third cross-linking probe binds to a third cross-linking sequence of the adapter anchor probe.

18. 8. The method of claim 7, further comprising hybridizing a fourth target-specific region of a fourth cross-linking probe to a fourth target sequence of the molecule having a sequence corresponding to the genomic region, wherein a fourth adapter landing sequence of the fourth cross-linking probe binds to a fourth cross-linking sequence of the adapter anchor probe.

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