Methods for generating targeted nucleic acid libraries

Abstract

The present disclosure provides targeted hybridization and / or proximity ligation of probes for target sequence amplification and analysis. Hybridization of the probe to the target sequence can be direct or through indirect association.

Description

[0001] Cross-references

[0002] This application claims priority to U.S. Provisional Application No. 62 / 770,585, filed on November 21, 2018, which is incorporated herein by reference in its entirety. Background Art

[0003] Nucleic acid target capture methods can achieve enrichment of specific genes, exons, and other genomic regions of interest, for example, for targeted sequencing. However, current sequencing methods based on target capture may involve cumbersome and lengthy protocols and expensive processes. Current target capture-based methods may also have low on-target rates for small capture groups (e.g., less than 500 probes), and have low flexibility in target sequencing based on multiplex PCR amplicons due to, for example, primer interactions, especially for PCR primers with molecular barcodes. In addition, due to low conversion rates, current nucleic acid target capture methods may not be suitable for low input and damaged DNA.

[0004] Bisulfite conversion is a useful technique for studying methylation patterns of nucleic acid molecules and can be used to study methylation by sequencing. However, bisulfite conversion can damage nucleic acids by, for example, producing truncations. If a next-generation sequencing (NGS) DNA library is treated with bisulfite, a large amount of nucleic acid may be damaged and cannot be recovered in the subsequent amplification step, resulting in a low conversion rate. In addition, since bisulfite conversion can result in single-stranded or fragmented DNA and reduce sequence complexity, the converted DNA may be difficult to use as input for library construction based on conventional adapter ligation. Given the low conversion rate (e.g., 5% or less for bisulfite-treated cell-free DNA (cfDNA), the initial input of bisulfite-treated cfDNA or circulating tumor cell DNA (ctDNA) is typically small, which may pose a greater challenge. In addition, during bisulfite conversion, any non-methylated "C" can be converted to "U" and then converted to "T" after PCR amplification. Since amplification before bisulfite conversion may not retain methylation information, capture after bisulfite conversion can be more commonly used. The design or production of probes or PCR primers may be based on the sequence after bisulfite conversion, which is a more complex process. If pre-bisulfite conversion capture is used, existing market protocols may require high DNA input (e.g., more than 250 ng). This limitation can result in targeted regions that are difficult to enrich by current probe capture or multiplex PCR methods.

[0005] Therefore, there is a need for more efficient, easy-to-use, rapid, flexible and practical methods for capturing target nucleic acids and improved methods for analyzing bisulfite-treated nucleic acids, particularly for low input samples (e.g., cfDNA). The methods disclosed herein can be used for capture based on pre-amplification and pre-bisulfite conversion hybridization of very low DNA input samples. Summary of the invention

[0006] Disclosed herein is a method comprising: hybridizing a target-specific region of a capture probe with a target sequence of a template nucleic acid molecule; attaching the 3' end of the template nucleic acid molecule to the 5' end of the capture probe, thereby producing a template nucleic acid molecule attached to the capture probe; hybridizing a first adapter primer to the first adapter of the capture probe; extending the first adapter primer, thereby producing a first extension product; and attaching a second adapter to the 3' end of the first extension product, thereby producing a first extension product attached to the second adapter. The capture probe may further include a molecular barcode. The method may further include phosphorylating the 5' end of the template nucleic acid molecule before attaching the second adapter. The second adapter may be a double-stranded adapter, wherein one of the two chains of the second adapter is attached to the template nucleic acid molecule. The method may further include hybridizing a second adapter primer to a second adapter. The method may further include extending the second adapter primer, thereby producing a second extension product. The method may further include amplifying the first extension product and the second extension product attached to the second adapter, thereby producing an amplification product. The method can further include amplifying the first extension product and the second extension product attached to the second adaptor using the first adaptor primer and the second adaptor primer to produce amplification products.

[0007] Disclosed herein is a method comprising: hybridizing a target-specific region of a capture probe with a target sequence of a template nucleic acid molecule; attaching the 3' end of the template nucleic acid molecule to the 5' end of the capture probe, thereby producing a template nucleic acid molecule attached to the capture probe; and treating the template nucleic acid molecule attached to the capture probe with bisulfite. The method may further comprise hybridizing a first adapter primer to a first adapter of the capture probe. The method may comprise extending the first adapter primer, thereby producing a first extension product. The method may further comprise attaching a second adapter to the 3' end of the first extension product, thereby producing a first extension product attached to the second adapter. The capture probe may further comprise a molecular barcode. The method may comprise phosphorylating the 5' end of the template nucleic acid molecule before attaching the second adapter. The second adapter may be a double-stranded adapter, wherein one of the two chains of the second adapter is attached to the template nucleic acid molecule. The method may further comprise hybridizing a second adapter primer to a second adapter. The method may further comprise extending the second adapter primer, thereby producing a second extension product. The method may further include amplifying the first extension product and the second extension product attached to the second adapter, thereby producing an amplified product. The method may further include amplifying the first extension product and the second extension product attached to the second adapter using the first adapter primer and the second adapter primer, thereby producing an amplified product. The method may further include hybridizing the target-specific primer with the first extension product. The method may further include extending the target-specific primer, thereby producing a second extension product. The method may further include amplifying the first extension product and the second extension product attached to the second adapter, thereby producing an amplified product. The method may further include amplifying the first extension product and the second extension product attached to the second adapter using the first adapter primer and the target-specific primer, thereby producing an amplified product.

[0008] Disclosed herein is a method comprising: hybridizing a target-specific region of a capture probe with a target sequence of a template nucleic acid molecule; attaching the 3' end of the template nucleic acid molecule to the 5' end of the capture probe, thereby producing a template nucleic acid molecule attached to the capture probe; and extending the 3' end of the capture probe, thereby producing a first extension product attached to the template nucleic acid molecule. The method may further include treating the template nucleic acid molecule attached to the capture probe with bisulfite. The capture probe may include a first adapter. The capture probe may further include a molecular barcode. The method may further include phosphorylating the 5' end of the template nucleic acid molecule. The method may further include attaching a second adapter to the 5' end of the template nucleic acid molecule. The method may further include phosphorylating the 5' end of the template nucleic acid molecule before attaching the second adapter. The second adapter may be a double-stranded adapter. The method may further include hybridizing a first adapter primer to the first adapter of the capture probe. The method may further include extending the first adapter primer, thereby producing a second extension product. The method may further include hybridizing a second adapter primer to the second extension product. The method may further include extending the second adapter primer, thereby producing a third extension product. The method may further include amplifying the second extension product and the third extension product, thereby producing an amplified product. The method may further include amplifying the second extension product and the third extension product using the first adapter primer and the second adapter primer, thereby producing an amplified product. The method may further include hybridizing the target-specific primer with the second extension product. The method may further include extending the target-specific primer, thereby producing a third extension product. The method may further include amplifying the second extension product and the third extension product, thereby producing an amplified product. The method may further include amplifying the second extension product and the third extension product using the first adapter primer and the target-specific primer, thereby producing an amplified product.

[0009] Disclosed herein is a method comprising hybridizing a target-specific region of a capture probe with a target sequence of a template nucleic acid molecule; attaching the 3' end of the template nucleic acid molecule to the 5' end of the capture probe, thereby producing a template nucleic acid molecule attached to the capture probe; and isolating the target-specific region. The method may further include treating the template nucleic acid molecule attached to the capture probe with bisulfite before isolating the target-specific region. The capture probe may further include a molecular barcode. The method may further include hybridizing a first adapter primer with a first adapter of the capture probe. The method may further include extending the first adapter primer, thereby producing a first extension product. The method may further include attaching a second adapter to the 3' end of the first extension product, thereby producing a first extension product attached to the second adapter. The method may further include extending the second adapter primer, thereby producing a second extension product. The method may further include amplifying the first extension product and the second extension product attached to the second adapter, thereby producing an amplification product. The method may further include amplifying the first extension product and the second extension product attached to the second adapter using the first adapter primer and the second adapter primer, thereby producing an amplification product. The method may further include hybridizing a target-specific primer to the first extension product. The method may further include extending the target-specific primer, thereby producing a second extension product. The method may further include amplifying the first extension product and the second extension product, thereby producing an amplified product. The method may further include amplifying the first extension product and the second extension product using a first adapter primer and a target-specific primer, thereby producing an amplified product.

[0010] Disclosed herein is a method comprising: hybridizing a first target-specific region of a first bridge probe with a first target sequence of a template nucleic acid molecule, wherein a first adaptor landing sequence of the first bridge probe binds to a first bridge binding sequence of an adaptor anchor probe; and attaching the 3' end of the template nucleic acid molecule to the 5' end of the adaptor anchor probe, thereby producing a template nucleic acid molecule attached to the adaptor anchor probe. The method may further comprise treating the template nucleic acid molecule attached to the adaptor anchor probe with bisulfite. The adaptor anchor probe may further comprise a molecular barcode. The first target-specific region of the first bridge probe may be in the 3' portion of the first bridge probe. The first target-specific region of the first bridge probe may be in the 5' portion of the first bridge probe. The first bridge probe may comprise a first linker between the first target-specific region and the first adaptor landing sequence. The attachment may comprise connecting the 3' end of the template nucleic acid molecule to the 5' end of the adaptor anchor probe using a ligase. The method may further comprise hybridizing a first adaptor primer to a first adaptor of the adaptor anchor probe. The method may further include extending the first adapter primer, thereby producing a first extension product. The method may further include attaching the second adapter to the 3' end of the first extension product, thereby producing a first extension product attached to the second adapter. The method may further include phosphorylating the 5' end of the template nucleic acid molecule before attaching the second adapter. The second adapter may be a double-stranded adapter, wherein one of the two chains of the second adapter is attached to the template nucleic acid molecule. The method may further include hybridizing the second adapter primer with the second adapter in the first extension product attached to the second adapter. The method may further include extending the second adapter primer, thereby producing a second extension product. The method may further include amplifying the first extension product and the second extension product attached to the second adapter, thereby producing an amplified product. The method may further include amplifying the first extension product and the second extension product attached to the second adapter using the first adapter primer and the second adapter primer, thereby producing an amplified product. The method may further include hybridizing the target-specific primer with the first extension product. The method may further include extending the target-specific primer, thereby producing a second extension product. The method may further include amplifying the first extension product and the second extension product, thereby producing an amplified product. The method may further include amplifying the first extension product and the second extension product using a first adapter primer and a target-specific primer, thereby producing an amplified product.

[0011] The method may further include separating the first bridge binding sequence. The method may further include extending the 3' end of the first bridge probe, thereby producing a first extension product. The adapter anchor probe may include a first adapter. The method may further include attaching a second adapter to the 5' end of the template nucleic acid molecule. The method may further include phosphorylating the 5' end of the template nucleic acid molecule before attaching the second adapter. The adapter may be a double-stranded adapter. The method may further include hybridizing a first adapter primer with a first adapter of the adapter anchor probe. The method may further include extending the first adapter primer, thereby producing a second extension product. The method may further include hybridizing a second adapter primer with the second extension product. The method may further include extending the second adapter primer, thereby producing a third extension product. The method may further include amplifying the second extension product and the third extension product, thereby producing an amplification product. The method may further include amplifying the second extension product and the third extension product using the first adapter primer and the second adapter primer, thereby producing an amplification product. The method may further include hybridizing a target-specific primer with the second extension product. The method may further include extending the target-specific primer, thereby producing a third extension product. The method may further include amplifying the second extension product and the third extension product, thereby producing an amplified product. The method may further include amplifying the second extension product and the third extension product using a first adapter primer and a target-specific primer, thereby producing an amplified product. The method may further include hybridizing the second target sequence region of the second bridge probe with the second target sequence of the template nucleic acid molecule, wherein the second adapter landing sequence of the second bridge probe is combined with the second bridge binding sequence of the adapter anchor probe. The second target-specific region of the second bridge probe may be in the 3' portion of the second bridge probe. The second target-specific region of the second bridge probe may be in the 5' portion of the second bridge probe. The second bridge probe may include a second connector between the second target-specific region and the second adapter landing sequence.

[0012] The first target-specific region of the first bridge probe may be in the 3' portion of the first bridge probe and the second target-specific region of the second bridge probe may be in the 3' portion of the second bridge probe; the first target-specific region of the first bridge probe may be in the 5' portion of the first bridge probe and the second target-specific region of the second bridge probe may be in the 5' portion of the second bridge probe; the first target-specific region of the first bridge probe may be in the 3' portion of the first bridge probe and the second target-specific region of the second bridge probe may be in the 5' portion of the second bridge probe; or the first target-specific region of the first bridge probe may be in the 5' portion of the first bridge probe and the second target-specific region of the second bridge probe may be in the 3' portion of the second bridge probe. The first target-specific region of the first bridge probe may be in the 3' portion of the first bridge probe and the second target-specific region of the second bridge probe may be in the 5' portion of the second bridge probe; and the first connector of the first bridge probe and the second connector of the second bridge probe may hybridize to each other.

[0013] The target sequence region of the capture probe or bridge probe can be designed based on the target sequence of the template nucleic acid molecule, and the target sequence of the template nucleic acid can retain non-methylated cytosine after bisulfite treatment. The method can further include treating the nucleic acid molecule with bisulfite to produce a template nucleic acid molecule, wherein the template nucleic acid molecule can be a product of bisulfite treatment. The target sequence region of the capture probe or bridge probe can be designed based on the target sequence of the template nucleic acid molecule after bisulfite treatment, wherein the non-methylated cytosine in the template nucleic acid is converted into uracil during bisulfite treatment. The method can further include dephosphorylating the 5' end of the nucleic acid molecule to produce a template nucleic acid molecule. The capture probe or adapter anchor probe can further include a marker. The method can further include capturing the bridge probe by a marker. The marker can be biotin. The marker can be captured on a solid support. The solid support can be a bead. The bead can include a capture portion of a targeting marker. The capture portion can be streptavidin. The method may further include contacting the nucleic acid molecule with a 3' to 5' exonuclease after attaching the 3' end of the template nucleic acid molecule to the 5' end of the capture probe or the adapter anchor probe, wherein the nucleic acid molecule includes the capture probe and the template nucleic acid molecule. The method may further include sequencing the amplified product. The sequencing may include second generation sequencing. The template nucleic acid molecule may include single-stranded DNA. The template nucleic acid molecule may include RNA. The template nucleic acid molecule may include damaged DNA. The template nucleic acid molecule may be from a formalin-fixed paraffin-embedded (FFPE) sample. The template nucleic acid molecule may include a cell-free nucleic acid from a biological sample. The cell-free nucleic acid may include a cell-free DNA. The cell-free DNA may include circulating tumor DNA. In some cases, the template nucleic acid molecule may not be subjected to an in vitro DNA repair step.

[0014] Disclosed herein is a method comprising: capturing a target sequence using a probe, wherein the probe hybridizes to the target sequence and wherein the probe is attached to a template comprising the target sequence; and treating the target sequence attached to the probe using bisulfite. Disclosed herein is a method comprising capturing a target sequence using a bridge probe, wherein the bridge probe facilitates attachment of an anchor probe to the target sequence. The method may further comprise treating the target sequence attached to the anchor probe using bisulfite.

[0015] Disclosed herein is a kit comprising: a capture probe comprising a target-specific region that hybridizes to a target sequence of a template nucleic acid molecule and a 5' end attached to the 3' end of the template nucleic acid molecule; a first adapter primer that hybridizes to a first adapter of the capture probe, wherein extension of the first adapter primer produces a first extension product; a second adapter that is attached to the first extension product; and a second adapter primer that hybridizes to the second adapter. Disclosed herein is a kit comprising: a bridge probe comprising a target-specific region that hybridizes to a target sequence of a template nucleic acid molecule; an adapter anchor probe comprising a bridge binding sequence that hybridizes to an adapter landing sequence of the bridge probe and a 5' end attached to the 3' end of the template nucleic acid molecule.

[0016] Disclosed herein is a method comprising hybridizing a target-specific region of a capture probe with a target sequence of a template nucleic acid molecule, wherein the capture probe further comprises a first adapter located at the 5' end of the target-specific region; after hybridizing with the target-specific region, contacting the template nucleic acid molecule with a 3' to 5' exonuclease; and extending the 3' end of the template nucleic acid molecule using the first adapter as a template, thereby producing a first extension product. The method may further include extending the 3' end of the capture probe, thereby producing a second extension product. The method may further include hybridizing a primer comprising the first adapter with the first extension product; and extending the 3' end of the primer comprising the first adapter, thereby producing a second extension product. The method may further include attaching a second adapter to the 5' end of the first extension product. The method may further include phosphorylating the 5' end of the first extension product before attaching the second adapter. The second adapter may be a double-stranded adapter, wherein one of the two chains of the second adapter is attached to the second extension product. The method may further include amplifying the first extension product and the second extension product, thereby producing an amplification product.

[0017] The method may further include hybridizing a target-specific primer to the second extension product, wherein the target-specific primer is attached to a second adaptor; and extending the target-specific primer to produce a third extension product. The method may further include amplifying the second extension product and the third extension product to produce an amplification product.

[0018] The capture probe may further include a molecular barcode. The capture probe may further include a binding moiety. The binding moiety may be attached to a support. The support may be a bead. The bead may be a streptavidin bead. The binding moiety may be biotin.

[0019] Disclosed herein is a method comprising hybridizing a first target-specific region of a first bridge probe to a first target sequence of a template nucleic acid molecule, wherein a first adapter landing sequence of the first bridge probe binds to a first bridge binding sequence of an adapter anchor probe; hybridizing a second target-specific region of a second bridge probe to a second target sequence of a template nucleic acid molecule, wherein a second adapter landing sequence of the second bridge probe binds to a second bridge binding sequence of the adapter anchor probe; and extending the 3' end of the first bridge probe to produce a first extension product. The first bridge probe may include a first adapter. The method may further include attaching a second adapter to the 3' end of the first extension product. The second adapter may be a double-stranded adapter, wherein one of the two chains of the second adapter is attached to a template nucleic acid molecule. The method may further include phosphorylating the 5' end of the template nucleic acid molecule before attaching the second adapter.

[0020] The method may further include hybridizing a target-specific primer to the first extension product; and extending the target-specific primer, thereby producing a second extension product. The target-specific primer may be attached to a second adaptor. The method may further include hybridizing a primer including the first adaptor to the second extension product; and extending the 3' end of the primer including the first adaptor, thereby producing a third extension product. The method may further include amplifying the second extension product and the third extension product, thereby producing an amplification product.

[0021] The first bridge probe may further include a molecular barcode. The first bridge probe may further include a binding moiety. The binding moiety may be attached to a support. The support may be a bead. The bead may be a streptavidin bead. The binding moiety may be biotin.

[0022] Disclosed herein is a method comprising hybridizing a first target-specific region of a first bridge probe to a first target sequence of a template nucleic acid molecule, wherein a first adapter landing sequence of the first bridge probe binds to a first bridge binding sequence of an adapter anchor probe, wherein the first bridge probe further comprises a first adapter located at the 5' end of the target-specific region; and after hybridizing with the first target-specific region, contacting the template nucleic acid molecule with a 3' to 5' exonuclease; and extending the 3' end of the template nucleic acid molecule using the first adapter as a template, thereby generating a first extension product. The method may further comprise hybridizing a primer comprising the first adapter to the first extension product; and extending the 3' end of the primer comprising the first adapter, thereby generating a second extension product. The method may further comprise attaching a second adapter to the 5' end of the first extension product. The method may further comprise phosphorylating the 5' end of the first extension product before attaching the second adapter. The second adapter may be a double-stranded adapter, wherein one of the two chains of the second adapter is attached to the second extension product. The method may further comprise hybridizing a target-specific primer to the second extension product; and extending the target-specific primer, thereby generating a third extension product. The target-specific primer may be attached to a second adaptor. The method may further include amplifying the second extension product and the third extension product, thereby generating an amplification product. The contacting of the template nucleic acid molecule with the 3' to 5' exonuclease may cleave one or more nucleotides at the 3' end of the template nucleic acid molecule.

[0023] The first bridge probe may further include a molecular barcode. The first bridge probe may further include a binding moiety. The binding moiety may be attached to a support. The support may be a bead. The bead may be a streptavidin bead. The binding moiety may be biotin.

[0024] Incorporation by reference

[0025] 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The novel features of the present disclosure are particularly set forth in the appended claims. The features and advantages of the present disclosure may be better understood by reference to the following detailed description of illustrative embodiments that employ the principles of the present disclosure and the accompanying drawings, in which:

[0027] Figure 1A-Figure 1C One embodiment of direct hybridization of the capture probe to the target sequence (TS) in the template and subsequent amplification is shown. Figure 1A Hybridization of the capture probe to TS and proximity ligation of the capture probe to the template are shown. Figure 1B Synthesis of the complementary strand of the template using the AD1 primer is shown. Figure 1C The second adaptor (AD2) is shown Figure 1B The extension product and the template are connected, and the primer is annealed to the second adapter to further amplify the TS using the AD2 primer and the AD1 primer.

[0028] Figure 2A-2C One embodiment of direct hybridization of capture probes to target sequences (TS) in templates and different amplification methods is shown. Figure 2A Hybridization of the capture probe to the target sequence and proximity ligation of the capture probe to the template are shown. Figure 2B Synthesis of the complementary strand of the template using the AD1 primer is shown. Figure 2C Shown is the use of target specific region (TSR) primers for further amplification of the target sequence with the AD1 primer.

[0029] Figure 3A-3C An embodiment of direct hybridization of capture probes to target sequences (TS) and another amplification method is shown. Figure 3A Hybridization of the capture probe to the target sequence and proximity ligation of the capture probe to the template are shown. Figure 3B Phosphorylation of the 5' end of the template and synthesis of the complementary strand of the template using the target specific region (TSR) of the capture probe (3' end of the capture probe) as a primer are shown. Figure 3C The AD2 adapter is shown Figure 3B The products were ligated and primers annealed with AD1 and AD2 for further amplification of the target sequence.

[0030] Figure 4A-4C Removal of the target specific region (TSR) of the capture probe is shown to facilitate subsequent amplification of the target sequence. Figure 4A Hybridization of the capture probe to the target sequence of the template and proximity ligation of the capture probe to the template are shown. Figure 4B Cleavage of the capture probe by TSR is shown. Figure 4C Extension of the AD1 primer annealing to the AD1 sequence in the capture probe and amplification of the target sequence are shown.

[0031] Figure 5A-5C Indirect association of the adapter anchor probe to the template is shown. Figure 5A The association of the adapter anchor probe with the template due to hybridization of the bridge probe to both the adapter anchor probe and the template and the subsequent ligation of the adapter anchor probe to the template is shown.The target specific region (TSR) is located at the 3' portion of the bridge probe. Figure 5B Synthesis of the complementary strand of the template using the AD1 primer is shown. Figure 5C The AD2 adapter is shown Figure 5BThe products were ligated and further amplified using primers AD1 and AD2.

[0032] Figure 6A-6C Indirect association of adapter anchor probes and different amplification methods are shown. Fig. 6A The association of the adapter anchor probe with the template due to hybridization of the bridge probe to both the adapter anchor probe and the template and the subsequent ligation of the adapter anchor probe to the template is shown.The target specific region (TSR) is located at the 3' portion of the bridge probe. Figure 6B Synthesis of the complementary strand of the template using the AD1 primer is shown. Figure 6C Further amplification using primers annealing to the complement of the target sequence and the AD1 primer is shown.

[0033] Figure 7A-7C Indirect association of the adapter anchor probe to the template using a bridge probe is shown, wherein the target specific region (TSR) is located at the 5' portion of the bridge probe. Fig. 7A The association of the adapter anchor probe with the template due to hybridization of the bridge probe to both the adapter anchor probe and the template and the subsequent ligation of the adapter anchor probe to the template is shown. Figure 7B The synthesis of the complementary strand of the template using the AD1 primer is shown. Prior to annealing the primer to the AD1 sequence, the bonds between the bridge probe, template and adapter anchor probe can be denatured. Figure 7C The AD2 adapter is shown Figure 7B The products were ligated and further amplified using primers AD1 and AD2.

[0034] Figure 8A-8C Indirect association of the adapter anchor probe to the template via the bridge probe and extension of the target specific region (TSR) of the bridge probe is shown. Fig. 8A The association of the adapter anchor probe with the template due to hybridization of the bridge probe to both the adapter anchor probe and the template and the subsequent ligation of the adapter anchor probe to the template is shown. Figure 8B The synthesis of the complementary strand of the template using TSR as a primer (3' end of the bridge probe) is shown. Figure 8C The AD2 adapter is shown Figure 8B The products were ligated and further amplified using primers AD1 and AD2.

[0035] Fig. 9 Hybridization of multiple bridge probes to the template and the adapter anchor probe is shown, resulting in proximity ligation of the adapter anchor probe to the template. Both bridge probes include a target specific region (TSR) at the 3' portion.

[0036] Fig.10 Hybridization of another set of bridge probes to the template and the adapter anchor probes, and subsequent ligation of the adapter anchor probes to the template are shown.Both bridge probes include a target specific region (TSR) in the 5' portion.

[0037] Fig.11 Hybridization of different bridge probes to the template and ligation of the adapter anchor probe to the template are shown.The first bridge probe includes a target specific region (TSR) at the 3' portion and the second bridge probe includes a TSR at the 5' portion, resulting in adapter hybridization and a stable assembly of the two bridge probes.

[0038] Fig.12 Hybridization of the bridge probe to the template and ligation of the adapter anchor probe to the template are shown.The first bridge probe includes a TSR at the 5' portion and the second bridge probe includes a TSR at the 3' portion.

[0039] Fig.13A - Panel B shows two embodiments of target amplification in which different bisulfite treatment sequences are employed for ligation of the probe to the template comprising the target. Fig.13A Bisulfite treatment of DNA input prior to hybridization to capture probes or bridge probes is shown. Fig. 13B Bisulfite treatment after hybridization of the DNA input and proximity ligation to the capture probe or bridge probe is shown.

[0040] Fig.14 The effect of bisulfite treatment on ligated template DNA is shown. Bisulfite conversion can leave the double-stranded portion of the template DNA intact, allowing the use of TSR PCR primers designed to anneal to the complementary sequence of the non-bisulfite converted TS of the template DNA.

[0041] Figure 15A-15B A scheme for selective removal of off-target ligation products using exonucleases is shown.

[0042] Figure 16A-16B A solid phase capture method for selecting correctly ligated target template and adapter anchor probes is shown.

[0043] Figure 17A-17C Library construction methods using ssDNA or damaged dsDNA as starting material are shown.

[0044] Fig.18A Schematic diagram showing the attachment of an adapter anchor probe to a template with (1) or without (2) a bridge probe. Fig.18B An agarose gel image is shown with ligated template and adapter anchor probe products where the bridge probe (1) setup was used; ligation products are absent where the bridge probe (2) setup was not used.

[0045] Figure 19A-Figure 19C Different methods of adding adaptors after direct hybridization of capture probes to template nucleic acids are shown.

[0046] Figures 20A-20D Different methods of adding adaptors after the template nucleic acid has been indirectly associated with the adaptor anchor probe through interaction with the bridge probe are shown.

[0047] Figure 21A-Figure 21E Various configurations of indirect association of the template nucleic acid and the adapter anchor probe through interaction with the bridge probe are shown.

[0048] Figure 22A-22G The steps of adding adaptors by direct hybridization of the template nucleic acid to the target probe and digestion of the template nucleic acid by a 3' to 5' exonuclease are shown.

[0049] Figure 23A-23F The addition of adaptors and the extension steps of the bridge probe by indirect association of the target probe and template nucleic acid are shown.

[0050] Figure 24A-Figure 24F The steps of adding adaptors through indirect association of target probe and template nucleic acid and digestion of the template nucleic acid by 3' to 5' exonuclease are shown.

[0051] Figure 25A-Figure 25D The steps of adding adaptors by using a polynucleotide primer with adaptors are shown.

[0052] Fig.26 Shown is a schematic diagram of an experiment using a synthetic DNA template, addition of adaptors by direct hybridization of capture probes, and 3' to 5' exonuclease digestion of the template nucleic acid.

[0053] Fig. 27 Shown is a schematic diagram of another experiment using fragmented genomic DNA, addition of adaptors by direct hybridization of capture probes, and 3' to 5' exonuclease digestion of the template nucleic acid. DETAILED DESCRIPTION

[0054] I. Overview

[0055] Provided herein are methods and kits for targeted adapter proximity ligation and enrichment (TALE) and subsequent sequencing (TALE-SEQ). TALE can complete target enrichment and library construction in one step. TALE can also use single-stranded DNA as input and enrich target sequences before amplification. In addition, the methods and kits disclosed herein can use damaged DNA, while the use of second-generation sequencing (NGS) may have low conversion rates. The methods and kits provided herein can provide higher conversion rates and improved sensitivity because there is less DNA loss associated with fewer steps in the process. In addition, the use of multiple bridge probe-adapter anchors can improve capture specificity and efficiency. Therefore, the methods and kits provided herein can provide fast, simple and flexible designs and low-cost solutions for target enrichment in NGS.

[0056] Methods disclosed herein include targeted hybridization of capture probes with templates and proximity connection of the ends of capture probes with the ends of templates, and subsequent amplification of target sequences in templates using, for example, attached adapters. Compared with multiple polymerase chain reaction (PCR) second generation sequencing analysis, these methods can improve the flexibility and practicality of nucleic acid combination (panel) design, and can save a lot of time and cost. The method can also maintain the high complexity of nucleic acid samples, and, for example, when using damaged DNA as input, can have the characteristics of low bias display due to high conversion rate.

[0057] In addition, the local target capture method disclosed herein can eliminate double-stranded DNA repair steps and cumbersome probe hybridization / enrichment. The disclosed method can be used for single-stranded DNA, damaged DNA, fragmented DNA (e.g., from formalin-fixed paraffin-embedded (FFPE) samples), bisulfite-treated DNA, and RNA.

[0058] a. Overview of adding targeted probes by direct hybridization

[0059] The method disclosed herein includes selectively attaching a capture probe to a template nucleic acid molecule having a target sequence by direct hybridization and attachment. The capture probe can be designed to include a target-specific region that can hybridize with a specific target sequence in a template nucleic acid molecule and can thus hybridize with a target template. The proximity brought by hybridization can promote the 5' end of the capture probe to attach to the 3' end of the template nucleic acid molecule. The 5' end of the capture probe can be attached to the 3' end of the template by connecting using a ligase. The 5' end of the capture probe can be phosphorylated to promote its connection to the template nucleic acid molecule. The 5' end of the template nucleic acid molecule can be dephosphorylated to reduce self-connection, for example, before the target-specific region of the capture probe hybridizes with the target sequence in the template.

[0060] The capture probe may include an adaptor that can serve as a primer annealing site for amplification. The capture probe may further include a molecular barcode (MB) to uniquely identify the attached template nucleic acid molecule for subsequent sequencing, detection, identification, measurement and / or analysis of the template nucleic acid molecule.

[0061] b. Overview of adding targeting probes by indirect association

[0062] The disclosed method further includes certain embodiments, which include selectively attaching the adapter anchor probe to a template nucleic acid molecule having a target sequence by indirect association and attachment. The bridge probe can be designed to hybridize with a specific target sequence in the template nucleic acid molecule, thereby hybridizing with the target template. The adapter anchor probe can be designed to hybridize with the bridge probe and can be hybridized with the bridge probe to produce an assembly of three hybrid nucleic acid molecules. The proximity brought about by the hybridization between the three nucleic acid molecules can promote the 5' end of the adapter anchor probe to attach to the 3' end of the template nucleic acid molecule. The 5' end of the adapter anchor probe can be attached to the 3' end of the template by connecting using a ligase. The 5' end of the adapter anchor probe can be phosphorylated to facilitate its connection to the template. The 5' end of the template nucleic acid molecule can be dephosphorylated to reduce self-connection.

[0063] The adapter anchor probe can act as an adapter for a primer annealing site for amplification. The adapter anchor probe can further include a molecular barcode to uniquely identify the attached template for subsequent sequencing, detection, identification, measurement and / or analysis of the template nucleic acid molecule.

[0064] c. Overview of adapter addition by direct hybridization

[0065] The method disclosed herein includes selective hybridization of a capture probe with a template nucleic acid molecule having a target sequence by direct hybridization. The disclosed method includes certain embodiments, which include adding or incorporating one or more adapters of the target probe into the extension product without attaching or connecting the target probe to the template nucleic acid. The capture probe can be designed to include a target-specific region that can hybridize with a specific target sequence in a template nucleic acid molecule and can thus hybridize with a target template. The capture probe can further include an adapter that can serve as a primer annealing site for amplification. The adapter can be located at the 5' end of the target-specific region.

[0066] After hybridization with the capture probe, the template nucleic acid molecule can be contacted with a 3' to 5' exonuclease to digest any 3' overhangs of the template nucleic acid.After digestion, the 3' end of the template nucleic acid molecule can be extended to generate an extension product including a sequence complementary to the adaptor.

[0067] In some cases, following hybridization of the capture probe and template nucleic acid, the 3' end of the capture probe can be extended to generate an extension product that includes an adaptor.

[0068] The capture probe may further include a molecular barcode (MB) to uniquely identify the attached template nucleic acid molecule for subsequent sequencing, detection, identification, measurement and / or analysis of the template nucleic acid molecule.

[0069] d. Overview of Adaptor Addition by Indirect Ligation

[0070] The disclosed method further includes certain embodiments, which include selectively hybridizing the bridge probe to the template nucleic acid. The disclosed method includes certain embodiments, which include adding or incorporating one or more adapters of the bridge probe into the extension product without attaching or connecting the targeting probe to the template nucleic acid. The bridge probe can be designed to hybridize with a specific target sequence in the template nucleic acid molecule, so that it can hybridize with the target template. The adapter anchor probe can be designed to hybridize with the bridge probe and it can be hybridized with the bridge probe to produce an assembly of three hybrid nucleic acid molecules. The multiple hybridization assembly can synergistically provide higher stability for the complex. The bridge probe can further include an adapter that can be used as a primer annealing site for amplification. The adapter can be located at the 5' end of the target-specific region.

[0071] After hybridization with the capture probe, the template nucleic acid molecule can be contacted with a 3' to 5' exonuclease to digest any 3' overhangs of the template nucleic acid. After digestion, the 3' end of the template nucleic acid molecule can be extended to produce an extension product including a sequence complementary to the adaptor. The 3' end of the bridge probe can also be extended when the bridge probe and the template hybridize to produce an extension product including the adaptor.

[0072] The bridge probe may further include a molecular barcode (MB) to uniquely identify the attached template nucleic acid molecule for subsequent sequencing, detection, identification, measurement and / or analysis of the template nucleic acid molecule.

[0073] II. Addition of probes by direct hybridization

[0074] The methods disclosed herein may include direct hybridization and ligation of a capture probe to a template. The capture probe may include an adapter to which a primer may hybridize. Amplification may be performed using primers for sequencing and / or library construction.

[0075] a. Targeted Probe Proximity Ligation and Amplification Using Adaptors

[0076] Disclosed herein is a method comprising hybridizing a target-specific region of a capture probe with a target sequence of a template nucleic acid molecule; attaching the 3' end of the template nucleic acid molecule to the 5' end of the capture probe, thereby producing a template nucleic acid molecule attached to the capture probe; hybridizing a first linker primer with a first linker of the capture probe; extending the first linker primer, thereby producing a first extension product; and attaching a second linker to the 3' end of the first extension product, thereby producing a first extension product attached to the second linker.

[0077] The capture probe can include a molecular barcode (MB) with a unique recognition sequence to uniquely identify the template and / or ensure the specificity of the capture probe to the template nucleic acid molecule. The template nucleic acid molecule can be phosphorylated at its 5' end to promote the connection of one chain of the adapter (e.g., a double-stranded second adapter) to the template nucleic acid molecule. In order to amplify the template nucleic acid molecule, a second adapter primer that hybridizes with the second adapter can be used together with the first adapter primer. A primer that hybridizes with the target sequence of the template nucleic acid molecule can be used together with the first adapter primer to amplify the template nucleic acid molecule.

[0078] Figure 1A Methods for direct hybridization of a capture probe to a template nucleic acid molecule and ligation of the capture probe to the template are shown. A nucleic acid, such as a double-stranded (ds) DNA containing a target sequence (hash tag, TS) can be used as a template. One of the strands may be damaged (e.g., nicked). The capture probe may include a target specific region (TSR) complementary to the TS of the template and a first adapter (AD1). The 5' end of the capture probe may be phosphorylated. The template may be dephosphorylated at its 5' end. The TSR of the capture probe may hybridize to the TS of the template, and the phosphorylated 5' end of the capture probe may be ligated to the 3' end of the template because they are very close after hybridization. The first adapter primer (AD1 primer) may hybridize to the first adapter (AD1) of the capture probe and extend, for example, using a chain displacement mechanism to synthesize such as Figure 1B The complementary strand is shown. Figure 1C A second adapter (AD2) connected is shown for further amplification of TS. Double-stranded AD2 can be connected to the template and its extension product, and PCR can be performed using primers that anneal to AD1 and AD2 at each end to amplify TS and generate a library. In some cases, a single-stranded AD2 adapter is connected to the 3' of the extension product. The capture probe can include a unique molecular barcode (MB) to uniquely identify the template in subsequent analysis.

[0079] Once the capture probe is attached to the template, amplification can be performed using different primers. Figure 2C As shown, the target sequence (TS) can be amplified using primers that hybridize to the complementary sequences of AD1 and TS. In another embodiment, the primers can be designed to anneal to the region flanking the complementary sequence of TS (eg, 5' or 3' of the complementary sequence of TS).

[0080] After the capture probe is attached to the template, the TSR can be separated or cleaved from the capture probe. The cleavage can remove the hairpin loop. The original template strand may not be an ideal template for the PCR reaction because the hairpin loop formed by the hybridization of the TSR and TS and the ligation of the capture probe to the template may interfere with the annealing and subsequent extension of the primer to AD1. After the proximity ligation of the capture probe to the template, the TSR can be cleaved, for example, between the 5' end of the TSR and the 3' end of AD1 by, for example, a restriction enzyme, such as Figure 4A and Figure 4B The cleaved TSR can be removed from the template, and the AD1 primer can be used in an extension reaction to amplify the template after removal of the TSR (see, e.g., Figure 4C ).

[0081] b. Targeted Probe Proximity Ligation and Amplification Using TSR

[0082] The 3' end of the capture probe can serve as an extension primer to synthesize the complementary strand of the template nucleic acid molecule without the need for a separate extension primer. Figure 3A-3C The possible use of the TSR region of the capture probe for amplifying the template is shown after the capture probe is adjacently ligated to the template. Once the 5' end of the hybridized capture probe is ligated to the 3' end of the template, the hybridized capture probe can form a hairpin loop (see, e.g., Figure 3A and Figure 3B ). The TSR (at the 3' end of the capture probe) can be extended toward the 5' end of the template like a primer to form a complementary sequence that hybridizes with the template (see, e.g., Figure 3B-Figure 3C The double-stranded AD2 can be ligated to the end of the circularized and extended template (see, e.g., Figure 3C ). In some cases, a single-stranded AD2 adapter can be ligated to the 5' end of the template. The 5' end of the template can be phosphorylated to ensure that the AD2 strand is ligated to the template. The template strand can then be further amplified using primers that anneal to the AD1 and AD2 sequences (see, e.g., Figure 3C ).

[0083] III. Addition of probes by indirect association

[0084] The probe can be indirectly associated with the template and connected adjacent to the template. This method can provide flexibility for the design of the capture probe combination and can allow the TSR to be separated from the template after connection. The bridge probe can include a TSR hybridized with the TS of the template and an adapter landing sequence (ALS) hybridized with the bridge binding sequence (BBS) of the adapter anchor probe. The hybridization between the template and the bridge probe and between the bridge probe and the adapter anchor probe can bring the adapter anchor probe close to the template, promoting the connection of the adapter anchor probe to the template. The adapter anchor probe can further include AD1, a unique identifier, MB and 5' phosphate.

[0085] a. Targeted Probe Proximity Ligation and Amplification Using Adaptors

[0086] Disclosed herein is a method comprising hybridizing a first target specific region (TSR) of a first bridge probe to a first target sequence (TS) of a template nucleic acid molecule, wherein a first adapter landing sequence (ALS) of the first bridge probe binds to a first bridge binding sequence (BBS) of an adapter anchor probe; and attaching (e.g., ligating) the 3' end of the template nucleic acid molecule to the 5' end of the adapter anchor probe, thereby generating a template nucleic acid molecule attached to the adapter anchor probe (see, e.g., Figure 5A and Fig. 6A The first bridge probe may further include a linker connecting the target specific region (TSR) and the ALS (see, e.g., Figure 5A and Fig. 6A ).

[0087] The adapter anchor probe may include a MB with a unique recognition sequence to uniquely identify the template in subsequent analysis (see, e.g., Figure 5A and Fig. 6A ) and / or to ensure the specificity of the capture probe to the template nucleic acid molecule. The template nucleic acid molecule can be phosphorylated at its 5' end to facilitate the attachment of one strand of the second adaptor (eg, a single-stranded or double-stranded adaptor) to the template nucleic acid molecule (see, e.g., Figure 5A and Fig. 6A ). To amplify the template nucleic acid molecule, a second adapter primer that hybridizes to the second adapter can be used together with the first adapter primer. In some cases, a primer that hybridizes to the complement of the target sequence (TS) of the template nucleic acid molecule can be used together with the first adapter primer to amplify the template nucleic acid molecule (see, e.g., Figure 6C ). The first target-specific region (TSR) of the first bridge probe can be in the 3' portion of the first bridge probe (see, e.g., Figure 5A and Fig. 6A ). The first target-specific region (TSR) of the first bridge probe can be in the 5' portion of the first bridge probe (see, e.g., Fig. 7A ). The adapter anchor probe can be associated with the template through the bridge probe, and the phosphorylated 5' end of the adapter anchor probe can be ligated to the 3' end of the template, thereby attaching AD1 and MB of the adapter anchor probe to the template (see, e.g., Figure 5A , Fig. 6A , Fig. 7A and Fig. 8A ). Primers that anneal to AD1 and AD2 can be used (see, e.g., Figure 5C and Figure 7C ) or a primer that anneals to the complementary sequence of AD1 and TS (see, e.g., Figure 6C) to amplify the ligated template. The adapter anchor probe may lack TSR; when the adapter anchor probe lacks TSR, it will not form a double strand with the template. In some cases, direct hybridization between the adapter anchor probe and the template can interfere with primer annealing, for example, annealing of the AD1 primer in the adapter anchor probe to the AD1 adapter.

[0088] b. Targeted Probe Proximity Ligation and Amplification Using TSR

[0089] The TSR at the 3' end of the first bridge probe can be extended like a primer to produce a first extension product (see, e.g., Figure 8B ). A second adaptor (AD2) can be attached to the first extension product and the AD2 primer can be used with the AD1 primer to amplify the template (see, e.g., Figure 8C AD2 may be double-stranded and attached to the template and the first extension product, which may be favored by phosphorylation of the 5' end of the template (see, e.g., Figure 8C ). In some cases, AD2 is single-stranded and is attached to the 5' end of the template. In other embodiments, primers that anneal to the complementary sequences of AD1 and TS can be used to amplify the target sequence.

[0090] Figure 8A-8C An example of an extension reaction using TSR (3' end of the bridge probe) is shown. The TSR at the 3' end of the bridge probe can serve as an extension primer to synthesize a first extension product having a sequence complementary to the template (see, e.g., Figure 8B The 5' end of the template may be phosphorylated to facilitate ligation of double-stranded AD2 (see, e.g., Figure 8C ). In some cases, single-stranded AD2 is ligated to the 5' end of the template. The template can be amplified by a primer set that anneals to the AD1 and AD2 sequences (or the complementary sequence of AD2).

[0091] c. Hybridization and proximity ligation of multiple target probe bridge probes

[0092] The method can further include hybridizing a second target-specific region (TRS2) of a second bridge probe to a second target sequence (TS2) of the template nucleic acid molecule, wherein a second adapter landing sequence (ALS2) of the second bridge probe binds to a second bridge binding sequence (BBS2) of the adapter anchor probe (see, e.g., Figure 9-12 The second bridge probe may further include a linker connecting the second target specific region (TSR2) and ALS2 (see, e.g., Figure 9-12 ). The second bridge probe can include a second linker between the second target-specific region (TSR2) and the second adapter landing sequence (ALS2).

[0093] The first target-specific region (TSR1) of the first bridge probe may be in the 3' portion of the first bridge probe and the second target-specific region (TSR2) of the second bridge probe may be in the 3' portion of the second bridge probe, e.g. Fig. 9 The first target-specific region (TSR1) of the first bridge probe can be in the 5' portion of the first bridge probe and the second target-specific region (TSR2) of the second bridge probe can be in the 5' portion of the second bridge probe (see, e.g., Fig.10 ). The first target-specific region (TSR1) of the first bridge probe can be in the 5' portion of the first bridge probe and the second target-specific region (TSR2) of the second bridge probe can be in the 3' portion of the second bridge probe (see, e.g., Fig.12 ). The first target-specific region (TSR1) of the first bridge probe can be in the 3' portion of the first bridge probe and the second target-specific region (TSR2) of the second bridge probe can be in the 5' portion of the second bridge probe (see, e.g., Fig.11 ). The linker of the first bridge probe can further hybridize with the linker of the second bridge probe. The hybridization of the first bridge probe and the second bridge probe can stabilize the four nucleic acid molecule assembly and can promote the connection of the 5' end of the adapter anchor probe to the 3' end of the template (see, for example, Fig.11 ).

[0094] More than two bridge probes can be used in the methods described herein. For example, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 25, 50, 75, 100 or more bridge probes can be used to bridge the template and adapter anchor probe in the methods described herein.

[0095] IV. Addition of Adaptors by Direct Hybridization

[0096] The method disclosed herein can include direct hybridization of the capture probe and the template nucleic acid, and adding or incorporating the adapter of the capture probe into the extension product. The capture probe can include an adapter, and the primer can hybridize with the adapter. Amplification can be performed using primers for sequencing and / or library construction. The capture probe can further include a molecular barcode. Adaptors can also be added without connecting the capture probe to the template nucleic acid.

[0097] Template nucleic acid molecules 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 performed by direct hybridization with a capture probe. The capture 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 a bead. The bead can be a streptavidin bead.

[0098] Figure 19A-Figure 19CVarious methods of adding or incorporating adapters after direct hybridization of the capture probe to the template nucleic acid are shown. A nucleic acid comprising a target sequence (hash tag, TS) can be used as a template. The capture probe can include a target specific region (TSR) complementary to the TS of the template and an adapter (AD). Fig.19A Hybridization or capture of a template nucleic acid to a capture probe is shown, followed by direct attachment of an adaptor to the template. Fig.19C Attachment of a capture probe comprising an adapter to a template is shown after hybridization with the template. A nucleic acid comprising a target sequence (hash tag, TS) can be used as a template. The capture probe can include a target specific region (TSR) complementary to the TS of the template and an adapter (AD).

[0099] a. Adding adapters after exonuclease digestion

[0100] Disclosed herein is a method comprising hybridizing a target-specific region of a capture probe with a target sequence of a template nucleic acid molecule, wherein the capture probe further comprises a first adapter located at the 5' end of the target-specific region; after hybridization with the target-specific region, the template nucleic acid molecule is contacted with a 3' to 5' exonuclease; and the 3' end of the template nucleic acid molecule is extended to produce a first extension product. The method further comprises hybridizing a primer comprising the first adapter with the first extension product; and extending the primer comprising the first adapter to produce a second extension product. In some cases, the 3' end of the capture probe can be extended to produce a second extension product.

[0101] Fig.19B Hybridization or capture of a template nucleic acid molecule to a capture probe is shown, followed by digestion of the single stranded 3' overhang using a 3' to 5' exonuclease. After digestion, the template nucleic acid is extended using the adapter of the capture probe as a template, thereby incorporating the adapter into the extension product.

[0102] Figure 22A-22G Further shown are the steps of hybridization, digestion and extension. Fig.22E The hybridization of the target-specific primer to the second extension product is shown, wherein the target-specific primer is attached to the second adaptor; and the target-specific primer is extended to produce a third extension product. The second extension product and the third extension product can be further amplified to produce Figure 22G amplification product.

[0103] In some cases, the disclosed methods may include attaching a second adaptor to the 5' end of the first extension product, such as Fig.22F The 5' end of the first extension product can be phosphorylated to facilitate attachment of the second adaptor. The second adaptor can be a double-stranded adaptor, wherein one of the two strands of the second adaptor is attached to the second extension product. After attachment of the second adaptor, the first extension product and the second extension product can be amplified to produce Figure 22Gamplification product.

[0104] b. Adding adapters after capture probe extension

[0105] The hybridized or captured template nucleic acid molecule can also be amplified by extending the 3' end of the capture probe to produce a first extension product without the need for a separate extension primer.The capture probe can include an adaptor.

[0106] The method may further include attaching a second adapter to the 3' end of the first extension product, thereby producing a first extension product attached to the second adapter. The 5' end of the template nucleic acid may be phosphorylated to facilitate attachment or connection of the second adapter to the template. The second adapter may be a double-stranded adapter. One of the two chains of the double-stranded second adapter may be attached to the template nucleic acid molecule. The second adapter primer may hybridize with the second adapter attached to the first extension product and extend to produce a second extension product. The first extension product and the second extension product attached to the second adapter may be amplified to produce an amplification product.

[0107] In some cases, the target-specific primer can hybridize with the first extension product attached to the second adaptor. The target-specific primer can be extended to synthesize a complementary strand (second extension product) of the first extension product attached to the second adaptor. The first extension product and the second extension product can be amplified to form more amplification products.

[0108] Through direct or indirect interactions (see Fig.25A , top and middle) extend the hybridized target probe attached to the adapter, and the adapter can be added to the extension product. Thymine can be added to the DNA template by terminal nuclease transferase (see Fig.25A , bottom). The polyadenine (poly A) oligonucleotide can hybridize to the 3'-poly T of the extension template. Fig.25B As shown, the poly A oligonucleotide can be attached to the adaptor such that when the 3' end of the poly A is extended, the first extension product can include the first adaptor. A polycytosine (poly C) can be added to the 3' end of the extension product, which can complementarily bind to a polyguanine (poly G) primer attached to the second adaptor (see Fig.25C The poly-G-adapter can then be used as a template to allow the nucleotide addition process to proceed and as a second adaptor to be added to the first extension product (see Fig.25D ).

[0109] V. Addition of Adaptors by Indirect Ligation

[0110] Target probe hybridization can be promoted by the synergistic interaction of the template nucleic acid and one or more probes forming the assembly. The multi-complex assembly can stabilize the hybridization interaction between the template and the target probe. The bridge probe can include a target-specific region hybridized with the target region of the template and an adapter landing sequence (ALS) hybridized with the bridge binding sequence (BBS) of the adapter anchor probe. The hybridization between the template and the bridge probe and between the bridge probe and the adapter anchor probe can form a multi-complex assembly. The bridge probe can further include AD1, a unique identifier, MB and / or 5' phosphate. Extension products can be synthesized using bridge probes. The adapter can also be added or incorporated into the extension product without connecting the bridge probe or the adapter anchor probe to the template nucleic acid. More than two bridge probes can be used in the methods disclosed herein. For example, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 25, 50, 75, 100 or more bridge probes can be used to bridge the template and the adapter anchor probe.

[0111] 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 performed by hybridization with bridge probes, via indirect association with adapter anchor probes. The bridge probe and / or adapter anchor 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 a bead. The bead can be a streptavidin bead.

[0112] Figures 20A-20D Different methods of adding or incorporating adapters into the template nucleic acid following hybridization or capture with bridge probes and adapter anchor probes are shown. Fig. 20A Ligation of adaptors to captured template nucleic acids is shown. Fig.20D Proximity ligation of the adapter anchor probe including the adapter to the template after hybridization is shown.

[0113] The method can further include hybridizing the second target-specific region of the second bridge probe to a second target sequence of the template nucleic acid molecule, wherein the second adapter landing sequence of the second bridge probe can bind to the second bridge binding sequence of the adapter anchor probe (see, e.g., Figure 21A-Figure 21E ). The second bridge probe can further include a linker connecting the second target-specific region and the second adaptor landing sequence.

[0114] a. Adding adapters after bridge probe extension

[0115] Disclosed herein is a method comprising hybridizing a first target-specific region of a first bridge probe to a first target sequence of a template nucleic acid molecule, wherein a first adapter landing sequence of the first bridge probe can bind to a first bridge binding sequence of an adapter anchor probe; hybridizing a second target-specific region of a second bridge probe to a second target sequence of a template nucleic acid molecule, wherein a second adapter landing sequence of the second bridge probe can bind to a second bridge binding sequence of an adapter anchor probe; and extending the 3' end of the first bridge probe to generate a first extension product. The first bridge probe may include a first adapter.

[0116] The method may further include attaching a second adaptor to the 3' end of the first extension product. The second adaptor may be a double-stranded adaptor. One of the two strands of the second adaptor may be attached to the template nucleic acid molecule. Prior to attaching the second adaptor to the template, the 5' end of the template nucleic acid molecule may be phosphorylated.

[0117] Alternatively, the target-specific primer can hybridize with the first extension product and can be extended to produce a second extension product. The target-specific primer can be attached to the second adapter. The primer including the first adapter can hybridize with the second extension product and can be extended to produce a third extension product. Amplifying the second extension product and the third extension product can produce further amplification products.

[0118] Fig. 20B Incorporation of an adaptor into the extension product by extending the 3' end of the bridge probe using the target nucleic acid molecule as a template is shown.

[0119] Figure 23A-23F Incorporation of one or more adaptors into the extension products is shown. Figures 23A-23C Hybridization and extension of the first bridge probe, and the first extension product are shown. The target-specific primer can hybridize to the first extension product and extend to synthesize a strand complementary to the first extension product, and the primer including the first adapter can hybridize to the second extension product to produce a sequence complementary to the second extension product (see Fig.23D ). The second extension product and the third extension product can be amplified to produce Fig.23F In another embodiment, the second (double-stranded) adapter can be attached to the 3' end of the first extension product and the 5' end of the template nucleic acid molecule (see Fig.23E ). The 5' end of the template nucleic acid molecule can be phosphorylated prior to attachment of the second adaptor. The bridge probe can further include a molecular barcode that can be incorporated into the extension product to uniquely identify the attached template for subsequent sequencing, detection, identification, measurement and / or analysis of the template nucleic acid molecule.

[0120] The adapter can be added or incorporated into the extension product by extending the hybridization bridge probe attached to the adapter. The adapter can also be added to the extension product by first extending the template nucleic acid molecule with a 3' primer. Polythymine (poly-T) oligonucleotides can be added to the 3' end of the DNA template by terminal nucleoside transferase. Polyadenine (poly-A) oligonucleotides can hybridize with the 3'-poly-T of the extension template. Poly-A oligonucleotides can be attached to the adapter so that when the 3' end of the poly-A is extended, the first extension product can include the first adapter. Polycytosine (poly-C) can be added to the 3' end of the first extension product, which can be complementary to the polyguanine (poly-G) primer attached to the second adapter. The poly-G-adapter can serve as a template, and the first extension product can be further extended to add the second adapter.

[0121] b. Adding adapters after exonuclease digestion

[0122] After hybridization with the bridge probe, the template may have a single-stranded 3' overhang. The 3' overhang may be digested with an enzyme that acts as a 3' to 5' exonuclease. A method is provided herein, comprising hybridizing a first target-specific region of a first bridge probe to a first target sequence of a template nucleic acid molecule, wherein a first adapter landing sequence of the first bridge probe binds to a first bridge binding sequence of an adapter anchor probe, wherein the first bridge probe further comprises a first adapter located at the 5' end of the target-specific region; and after hybridization with the target-specific region, contacting the template nucleic acid molecule with a 3' to 5' exonuclease; extending the 3' end of the template nucleic acid molecule using the first adapter as a template, thereby producing a first extension product (see Fig. 20C ).

[0123] Figure 24A-Figure 24F Further shown is the step of incorporating one or more adaptors into the extension product following digestion with a 3' to 5' exonuclease. Figure 24A-24C A plurality of bridge probes are shown hybridized to a template and an adapter anchor probe, followed by extension of the 3' end of the template using the first adapter of the first bridge probe as a template to produce a first extension product comprising a complementary sequence of the first adapter. The first extension product can be hybridized to a primer comprising the first adapter and the 3' end of the primer can be extended to produce a second extension product (see Figure 24D-Figure 24E ). The target-specific primer can hybridize to the second extension product and the 3' end can be extended to produce a third extension product (see Fig.24D ). Before the target-specific primer hybridizes to the first extension product, the second adaptor can be attached to the target-specific primer. The second extension product and the third extension product can be amplified to produce more amplification products (see Fig.24F ).like Fig.24EAs shown, the second (double-stranded) adaptor can be attached to the 5' end of the first extension product and the 3' end of the second extension product. The first extension product may need to be phosphorylated prior to attachment to the second adaptor.

[0124] In some cases, the 3' end of the template nucleic acid molecule can be extended before, simultaneously with, or after the 3' end of the first bridge probe is extended to produce a first extension product and a second extension product. The template nucleic acid molecule can be contacted with a 3' to 5' exonuclease to remove any 3' overhang prior to 3' extension of the template nucleic acid.

[0125] VI. Workflow of Bisulfite Conversion

[0126] Provided herein are methods for bisulfite treatment of nucleic acids. Bisulfite-treated nucleic acids can be used to study the methylation of nucleic acids. Bisulfite treatment can convert non-methylated cytosine into uracil. Methylation of cytosine (e.g., 5'-methylcytosine) can prevent bisulfite from converting methylated cytosine into uracil.

[0127] After the capture probes are attached to the template, the resulting product can be treated with bisulfite (see, e.g., Fig. 13B ). The formation of a double-stranded sequence (e.g., between the TS of the template and the TSR of the capture probe) can protect cytosine in the hybridization region from conversion to uracil during bisulfite treatment (see, e.g., Fig.14 ). The double-stranded sequence formed by hybridization of the bridge probe to the template and the adapter anchor probe can provide protection from bisulfite conversion to uracil in the hybridization region. In addition, since bisulfite treatment can convert non-methylated cytosines to uracil, protecting cytosines from conversion to uracil in the TS region can allow the use of amplification primers designed to anneal to non-bisulfite converted DNA. For capture before bisulfite conversion, probes can also be designed for unconverted sequences. Probes and primers that anneal to unconverted cytosines can be more directly designed and provide better hybridization.

[0128] a. Bisulfite treatment after hybridization and / or ligation of target probe to template nucleic acid

[0129] Template nucleic acids (eg, DNA) can be used for targeted adaptor proximity ligation and enrichment (TALE) and subsequent sequencing (TALE-SEQ) as described herein (see, e.g., Fig. 13B (1310); (1312)). The template nucleic acid (e.g., DNA) can be, for example, genomic DNA or cfDNA. The template nucleic acid (e.g., DNA) can be attached to the capture probe or adapter anchor probe by, for example, proximity ligation as described herein, e.g., Figure 1A-Figure 1217 (1314). The template nucleic acid (e.g., DNA) attached to the capture probe or adapter anchor probe can be treated with bisulfite (1316), extended (1318), and subsequently amplified (1320), e.g., for methylation sequencing. In some cases, bisulfite treatment can occur prior to TSR separation of the capture probe (see, e.g., Figure 4B ). In some cases, bisulfite treatment can be performed after the capture probe or bridge probe is hybridized to the template nucleic acid molecule. The template nucleic acid molecule hybridized to the capture probe or bridge probe can be treated with bisulfite, extended, and subsequently amplified for methylation sequencing.

[0130] The target-specific region of the capture probe or bridge 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 non-methylated cytosine after bisulfite treatment.

[0131] Fig.14 1 shows that non-methylated cytosine in TS and TSR sites is protected from conversion to uracil during bisulfite treatment that occurs after TS and TSR hybridization and ligation of the capture probe to the template. In this example, the capture probe is hybridized to the template and the 5' end of the capture probe is adjacently ligated to the 3' end of the template, forming a double-stranded region and a hairpin loop (see, e.g., Fig.14 ). Subsequently, the attached template is treated with bisulfite, during which time non-methylated cytosines in the hybridized TSR-TS region are not converted to uracil, while non-methylated cytosines in the single-stranded region (in the loop) are converted to uracil. Protecting cytosines from conversion to uracil in the TS region can allow the use of probes designed to anneal to non-bisulfite converted DNA. In some cases, one or more cytosine residues in a primer binding site (e.g., an adapter and / or template) are not protected from bisulfite conversion. After bisulfite conversion, the primer binding site in the adapter may include one or more uracils. The primer can be designed to be complementary to an adapter sequence that includes one or more uracils. The primer can be 100% complementary to an adapter sequence that includes one or more uracils, or less than 100% complementary to an adapter sequence that includes one or more uracils.

[0132] After bisulfite treatment, the template may include one or more uracils. A primer annealed to an adapter may be chain extended using a template including one or more uracils. The extended chain may include one or more adenines that are base paired with one or more uracils. The extension product may be denatured from the template. The primer may anneal to and extend the extension product in a region including one or more adenines. The primer may be used to amplify the template using, for example, an adapter primer.

[0133] b. Bisulfite treatment of target probe prior to hybridization and / or ligation to template

[0134] The template nucleic acid molecule can be bisulfite treated prior to hybridization with the capture probe or bridge probe and ligation with the capture probe or adapter anchor probe (see, e.g., Fig.13A (1300)). The DNA can be treated with bisulfite to convert unmethylated cytosine to uracil (1302). The bisulfite treated DNA can be used as input for targeted adaptor proximity ligation and enrichment (TALE) and subsequent sequencing (TALE-SEQ) (1304). The bisulfite treated DNA can be used as input for proximity adaptor ligation (1306), e.g., Figure 1A-Figure 12 17. The TSR of the probe can be designed to anneal to a template in which the non-methylated cytosine present has been converted to uracil. After proximity adaptor ligation, extension (1306) can be performed, followed by target amplification (1308).

[0135] VII. Clean-up / Specificity

[0136] a. Use exonucleases to remove off-target nucleic acid molecules or random ligation products

[0137] In some cases, the 5' end of the capture probe can be ligated to the 3' end of the template without the TSR of the capture probe annealing to the template. The 5' end of the adapter anchor probe can be ligated to the 3' end of the template without the adapter anchor probe associating with the template through the bridge probe. The products of these ligation events may be undesirable. Following the direct hybridization and ligation steps, an exonuclease can be added to the resulting mixture of nucleic acid molecules to digest any nonspecific attachment of the probe to the template.

[0138] In an example, where the TSR of the capture probe has not annealed to the TS of the template, the 5' end of the capture probe can be ligated to the 3' end of the template, thereby generating a linear molecule (e.g., a molecule lacking a stem-loop) (see, e.g., Fig. 15B Any specific attachment resulting from hybridization of the capture probe's TSR to the template's TS can generate a hairpin loop, thereby protecting the template from exonuclease (e.g., 3' to 5' exonuclease) digestion (see, e.g., Fig.15A ). Fig.15A The specific hybridization of the capture TSR to the template TS and the ligation of the 5' end of the capture probe to the 3' end of the template are shown. The hybridization and ligation can form a hairpin loop that protects the template from digestion by exonucleases (e.g., 3' to 5' exonucleases). The 5' end of the template can include a block that prevents cleavage by a 5' to 3' exonuclease. Fig. 15BThe ligation of the 5' end of the capture probe to the 3' end of the template is shown, wherein the TSR of the template is not annealed to the TS of the template, or the template does not include a TS. In the case of using one or more bridge probes, the 5' end of the adapter anchor probe can be ligated to the 3' end of the template, wherein the adapter anchor probe is associated with the template through one or more bridge probes. The ligation does not result in hairpin loop formation, and cannot prevent exonuclease (e.g., 3' to 5' exonuclease) digestion, thereby resulting in degradation of off-target random ligation products. Fig. 15B Exonucleases (e.g., 3' to 5' exonucleases) that cleave free template, free capture probe, and off-target ligation products of capture probe and template are shown. Exonuclease cleavage of off-target ligation products can result in selection of ligation products in which the capture probe specifically hybridizes to the template and the capture probe is attached (e.g., ligated) to the template. After exonuclease treatment, a purification step can be performed to remove the exonuclease from the ligation product before performing subsequent extension and amplification steps.

[0139] b. Solid Phase Extraction

[0140] Provided herein is a method for selecting a template that hybridizes with a bridge probe (or a template that associates with an adapter anchor probe through a bridge probe) before the adapter anchor probe is connected to the template, for example. The method can employ solid phase extraction. The method can be used to increase the possibility that the adapter anchor probe is connected to a template that specifically associates with the adapter anchor probe. Provided herein is a method for combining a capture probe, a bridge probe, or an adapter anchor probe with a solid support. The possibility that the adapter anchor probe is attached (e.g., connected) to the template independently of the bridge probe can introduce suboptimal specificity. In order to reduce such nonspecific connection products and unbound probes, a label (e.g., biotin) and a capture moiety (e.g., streptavidin beads) can be used.

[0141] The capture probe, bridge probe or adapter anchor probe may include a label. The disclosed method may further include capturing the capture probe, bridge probe or adapter anchor probe by a label. The label may be biotin. The label may be a nucleic acid sequence, such as poly A or poly T, or a specific sequence. The length of the nucleic acid sequence may be about 5 to 30 bases. The nucleic acid sequence may include DNA and / or RNA. The label may be located at the 3' end of the capture probe, bridge probe or adapter anchor probe. The label may be a peptide, or a modified nucleic acid that can be recognized by an antibody (e.g., 5-bromouridine), as well as biotin. The label may be conjugated to the capture probe, bridge probe or adapter anchor probe by a reaction such as "click" chemistry. "Click" chemistry can conjugate a reporter molecule (e.g., a fluorescent dye) to a biomolecule (e.g., DNA). Click chemistry can be a reaction between an azide and an alkyne, which can produce a covalent product (e.g., a 1,5-disubstituted 1,2,3-triazole). Copper can be used as a catalyst.

[0142] The marker can be captured on a solid support. The solid support can be magnetic. The solid support can include beads, flow cells, glass, plates, devices including one or more microfluidic channels, or columns. The solid support can be magnetic beads.

[0143] The solid support (e.g., beads) can include (e.g., by coating) one or more capture moieties that can bind to a marker. The capture moiety can be streptavidin, and the streptavidin can bind to biotin. The capture moiety can be an antibody. The antibody can bind to a marker. The capture moiety can be a nucleic acid, such as a nucleic acid comprising DNA and / or RNA. The nucleic acid capture moiety can bind to a sequence on, for example, an adapter anchor probe or a bridge probe. In some cases, an anti-RNA / DNA hybrid antibody bound to a solid surface can be used as a capture moiety.

[0144] The label and the capture moiety can be bound by one or more covalent or non-covalent bonds. After the capture probe, bridge probe or adapter anchor probe are captured on the solid support, the solid support can be washed to remove, for example, unbound templates from the sample. In some cases, no washing step is performed. Washing can be strict or gentle. The captured capture probe, bridge probe or adapter anchor probe (and associated template) can be eluted, for example, when the label is biotin and the capture moiety is streptavidin, by adding free biotin to the sample.

[0145] The ligation of the 5' end of the capture probe to the 3' end of the template can occur while the capture probe is captured on the solid support. The ligation of the 5' end of the capture probe to the 3' end of the template can occur after the capture probe / template complex is eluted from the solid support. The 5' end of the capture probe or adapter anchor probe can be phosphorylated while the capture probe or adapter anchor probe is captured on the solid support.

[0146] The ligation of the 5' end of the adapter anchor probe to the 3' end of the template can occur while the adapter anchor probe (and associated bridge probe and template) is captured on a solid support. The ligation of the 5' end of the adapter anchor probe to the 3' end of the template can occur after the adapter anchor probe (and associated bridge probe and template) is eluted from the solid support. The 5' end of the capture probe or adapter anchor probe can be phosphorylated after elution from the solid support.

[0147] An extension step (e.g., extension of an AD1 primer that anneals to an AD1 adapter in a capture probe) can be performed while the capture probe or adapter anchor probe is captured on the solid support or after the capture probe (and attached template) or adapter anchor probe (and attached template) has been eluted from the solid support.

[0148] Fig.16AShown is the use of streptavidin beads for cleanup after hybridization of the template, bridge probe, and adapter anchor probe, where the 3' end of the adapter anchor probe is biotinylated. The hybridization complex and free adapter and anchor adapter can all bind to the beads. Unbound template and bridge probe can be washed away. Proximity ligation can still occur for the hybridization complex. Ligation can be performed while the complex is on the beads. Ligation can be performed after the complex is eluted from the beads. However, for the free adapter anchor probe, the possibility of ligation to any template DNA can be greatly reduced. In Fig. 16B In the method, the 5' end or 3' end of the first and / or second bridge probe can be biotinylated. Streptavidin beads can be used to remove unhybridized adapter anchor adapters and templates, which can prevent random ligation of adapter anchor probes and templates.

[0149] VIII. Template Nucleic Acid Molecule

[0150] 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, non-coding small RNA, polyribosomal RNA, intronic RNA, pre-mRNA, viral RNA or cell-free RNA.

[0151] The template nucleic acid may be naturally occurring or synthetic. The template nucleic acid may have a modified heterocyclic base. The modification may be a methylated purine or pyrimidine, an acylated purine or pyrimidine, an alkylated ribose or other heterocycle. The template nucleic acid may have a modified sugar moiety. The modified sugar moiety may include a peptide nucleic acid. The template nucleic acid may include a peptide nucleic acid. The template nucleic acid may include a threose nucleic acid. The template nucleic acid may include a locked nucleic acid. The template nucleic acid may include a hexitol nucleic acid. The template nucleic acid may be a flexible nucleic acid. The template nucleic acid may include a glycerol nucleic acid.

[0152] Template nucleic acid molecules can be captured and enriched from low input (e.g., 1 ng nucleic acid material) samples (e.g., cell-free DNA (cfDNA) and circulating tumor DNA (ctDNA)). Low input samples can have 1 ng, 2 ng, 3 ng, 4 ng, 5 ng, 6 ng, 7 ng, 8 ng, 9 ng, 10 ng or more nucleic acid material. Low input samples can have less than 10 ng, 9 ng, 8 ng, 7 ng, 6 ng, 5 ng, 4 ng, 3 ng, 2 ng, 1 ng or less nucleic acid material. Low input samples can have 200 pg to 10 ng nucleic acid material. Low input samples can have less than 10 ng nucleic acid material. Low input samples can be less than 10 ng, 5 ng, 1 ng, 100 pg, 50 pg, 25 pg or less nucleic acid material. In some cases, the input sample can have 1 ng, 10 ng, 20 ng, 30 ng, 40 ng, 50 ng or more nucleic acid molecules. The input sample can have less than 50ng, 40ng, 30ng, 20ng, 10ng, 1ng or less nucleic acid material. Capture and enrichment can be performed by target probe hybridization. The target probe can be a capture probe, a bridge 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 a bead. The bead can be a streptavidin bead.

[0153] The template nucleic acid can be damaged. Damaged nucleic acids can include base changes or deletions, and / or backbone modifications. The template nucleic acid can be damaged by oxidation, radiation, or random mutations. The template nucleic acid can be damaged by bisulfite treatment.

[0154] For damaged DNA, the present disclosure can eliminate the double-stranded DNA repair step, providing higher conversion rates and improved sensitivity due to less DNA loss caused by fewer steps in the process.

[0155] Fig.17A The use of damaged dsDNA (with a gap) or ssDNA as a template for library construction is shown. The damage may be due to bisulfite treatment. For damaged dsDNA, the dsDNA can be denatured so that at least one undamaged strand can be used as a template. The template can then be hybridized and attached to a capture probe and amplified using various primers.

[0156] Template can be derived from cell-free DNA (cfDNA) or circulating tumor DNA (ctDNA). The source of cfDNA can be a fetus or a tumor. The template can be derived from a liquid biopsy, a solid biopsy or a fixed tissue of a subject. The template can be cDNA and can be produced by reverse transcription. The template nucleic acid can be derived from a fluid sample, including but not limited to plasma, serum, sputum, saliva, urine or sweat. The fluid sample can be treated with bisulfite to study the methylation pattern of the template nucleic acid and / or determine the tissue source 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 treated with bisulfite to analyze the methylation pattern of the organ from which the template nucleic acid is derived. The subject can suffer from methylation-related diseases, such as autoimmune diseases, cardiovascular diseases, atherosclerosis, neurological disorders and cancer.

[0157] The template nucleic acid can be derived 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.

[0158] The template nucleic acid can be derived from humans, rats, mice, other animals or specific plants, bacteria, algae, viruses, etc. The template nucleic acid can be derived from primates. Primates can be chimpanzees or gorillas. Other animals can be rhesus monkeys. The template can also be a mixture of genomes of different species (including host-pathogens, bacterial groups, etc.). The template can be a cDNA produced by RNA expressed from the genomes of two or more species.

[0159] The template nucleic acid may include a target sequence. The target sequence is an exon. The target sequence may be an intron. The target sequence may include 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 include a chromosome, a chromosome arm, or a gene. The gene may be a gene associated with a condition (e.g., cancer).

[0160] The template nucleic acid molecule can be dephosphorylated prior to hybridization, for example to reduce the rate of self-ligation.

[0161] IX. Capture Probes

[0162] Capture probe can be used to capture template nucleic acid molecules with target sequence. Capture probe can also be connected to template nucleic acid molecules by proximity. Since the space between the connection ends is relatively wide, the connection rate between free probe and template may be very low. However, compared with the use of free capture probe, hybridization capture probe can increase the possibility of connection between capture probe and template. Capture probe can include DNA. Capture probe can include RNA. Capture probe can include uracil or methylated cytosine. In some cases, capture probe does not include uracil.

[0163] The capture probe may include about 400 nucleotides. The capture probe may include about 300 nucleotides. The capture probe may include about 200 nucleotides. The capture probe may include about 180 nucleotides. The capture probe may include about 150 nucleotides. The capture probe may include about 120 nucleotides. The capture probe may include about 100 nucleotides. The capture probe may include about 90 nucleotides. The capture probe may include about 80 nucleotides. The capture probe may include about 70 nucleotides. The capture probe may include about 50 nucleotides. The capture probe may include about 40 nucleotides. The capture probe may include about 30 nucleotides. The capture probe may include about 20 nucleotides. The capture probe may include about 10 nucleotides. The capture probe may be about 10 to about 400 nucleotides, about 10 to about 200 nucleotides, about 10 to about 120 nucleotides, about 20 to about 120 nucleotides, about 20 to about 60 nucleotides, or about 30 to about 50 nucleotides.

[0164] The temperature at which the capture probe is attached to the template nucleic acid can be about 70°C, about 65°C, about 60°C, about 55°C, about 50°C, about 45°C, or about 45°C to about 55°C.

[0165] The capture probe may further include a label. The label may fluoresce. The fluorescent label may be an organic fluorescent dye, a metal chelate, a carbon nanotube, a quantum dot, a gold particle or a fluorescent mineral. The label may be radioactive. The label may be biotin. The capture probe may be combined with a labeled nucleic acid binding molecule. The nucleic acid binding molecule may be an antibody, an antibiotic, a histone, an antibody or a nuclease.

[0166] A variety of capture probes can be used in the sample. A variety of capture probes can be designed to have similar melting temperatures. The melting temperature of a set of capture probes can be within about 15°C, within about 10°C, within about 5°C, or within about 2°C. The melting temperature of one or more capture probes can be about 85°C, about 80°C, about 75°C, about 70°C, about 65°C, about 60°C, about 55°C, or about 50°C. The melting temperature of the capture probe can be about 50°C to about 85°C, about 55°C to about 80°C, about 45°C to about 60°C, or about 52°C to about 58°C.

[0167] The capture probe may include a molecular barcode (MB). The capture probe may include an adapter sequence (e.g., for binding an adapter primer). The capture probe may include a target specific region (TSR). The capture probe may include an index for distinguishing samples. The capture probe may include a restriction endonuclease cleavage site, e.g., between the TSR and the adapter sequence. The molecular barcode or index may be 5' of the adapter sequence and 5' of the TSR.

[0168] a. Molecular barcoding

[0169] Molecular barcodes can provide unique identification tools and allow the observation of specific binding between probes and templates. Molecular barcodes can include about 25 nucleotides, about 20 nucleotides, about 15 nucleotides, about 10 nucleotides, about 8 nucleotides, or about 4 nucleotides. Molecular barcodes can include DNA. Molecular barcodes can include RNA. Molecular barcodes can include uracil. In some cases, molecular barcodes do not include uracil. Capture probes can include indexes. Indexes can be used to identify samples. The length of the index can be 2 to 16 nucleotides, or about 6 or about 8 nucleotides.

[0170] b. Adapters and adapter primers

[0171] The adapter in the capture probe can provide a position for the primer binding of amplification. The adapter primer can be complementary to the site on the adapter and can be used to amplify the target sequence in the template. The adapter primer can have a length of about 50 nucleotides. The adapter primer can have a length of about 45 nucleotides. The adapter primer can have a length of about 40 nucleotides. The adapter primer can have a length of about 35 nucleotides. The adapter primer can have a length of about 30 nucleotides. The adapter primer can have a length of about 25 nucleotides. The adapter primer can have a length of about 20 nucleotides. The adapter primer can have a length of about 15 nucleotides. The adapter primer can have a length of about 18 to about 30 nucleotides.

[0172] The adapter may include DNA or RNA. The adapter may be a naturally occurring or synthetic nucleic acid. The adapter may have a modified heterocyclic base. The modification may be a methylated purine or pyrimidine, an acylated purine or pyrimidine, an alkylated ribose or other heterocycle. The adapter may have a modified sugar moiety. The modified sugar moiety may include a peptide nucleic acid. The adapter may include a peptide nucleic acid. The adapter may include a threose nucleic acid. The adapter may include a locked nucleic acid. The adapter may include a hexitol nucleic acid. The adapter may include a flexible nucleic acid. The adapter may include a glycerol nucleic acid.

[0173] c. Target Specific Region (TSR)

[0174] The TSR of a capture probe or bridge probe can be designed to hybridize with a target sequence of a template. TSRs can be designed for a target sequence converted by bisulfite or a target sequence protected by bisulfite. In some cases, 100% of the target-specific region of the probe can be complementary to the target sequence of the template. About 75% of the target-specific region of the probe can be complementary to the target sequence of the template. About 50% of the target-specific region of the probe can be complementary to the target sequence of the template. The target-specific region can include about 35 nucleotides, about 30 nucleotides, about 25 nucleotides, about 20 nucleotides, about 15 nucleotides, about 10 nucleotides, or about 5 nucleotides. The target-specific region anneals to the target sequence described herein.

[0175] X. Adapter / Adapter Primer / Amplification

[0176] The attached, added or incorporated adapters can provide sites for hybridization of primers for amplification. The first adapter (AD1) can be attached to the template via a capture probe or an adapter anchor probe. Primers for AD1 can be used to synthesize a strand complementary to the template. The second adapter (AD2) can be attached to the 5' end of the template and / or the 3' end of the complementary strand to further amplify the template. A library can be constructed using AD1 primers and AD2 primers. Selective amplification can be performed using AD1 primers and primers for TSR or its flanking regions.

[0177] The adapter can be a single-stranded nucleic acid. The adapter can be a double-stranded nucleic acid. The adapter can be a partial duplex, where the long strand is longer than the short strand, or the two strands are equal in length.

[0178] XI. Bridge Probe

[0179] The bridge probe can be used to hybridize the template nucleic acid molecule with the target sequence and the adapter anchor probe. The bridge probe can further allow indirect association of the adapter anchor probe and the template, thereby promoting their attachment. Due to the randomness of the interaction, the connection rate of the free adapter anchor probe and the template may be extremely low. However, compared with the use of free adapter anchor probes, hybridization bridge probes can increase the possibility of connection between the adapter anchor probe and the template. The bridge probe may include DNA. The bridge probe may include RNA. The bridge probe may include uracil and methylated cytosine. The bridge probe may not include uracil.

[0180] 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 the 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.

[0181] The bridge probe may include one or more molecular barcodes. The bridge probe may include one or more binding moieties. The binding moiety may be biotin. The binding moiety may be attached to a support. The support may be a bead. The bead may be a streptavidin bead.

[0182] The bridge probe may include about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 120 nucleotides, about 100 nucleotides, about 90 nucleotides, about 80, about 70 nucleotides, about 50 nucleotides, about 40 nucleotides, about 30 nucleotides, about 20 nucleotides, or about 10 nucleotides.

[0183] A variety of bridge probes can be used to anneal with multiple target sequences in a sample. Bridge probes can be designed to have similar melting temperatures. The melting temperature of a set of bridge probes can be within about 15°C, within about 10°C, within about 5°C, or within about 2°C. The melting temperature of one or more bridge 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 bridge 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.

[0184] The use of adapter anchor probes and one or more bridge probes surrounding a specific bridge probe can help stabilize the hybridization of a specific bridge probe to its target sequence through a synergistic effect. The hybridization temperature for forming multiple bridge probe assemblies can be higher than the melting temperature of a single bridge probe. Higher temperatures can lead to better capture specificity by reducing nonspecific hybridization that may 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 a single bridge probe. The hybridization temperature can be about 5°C to about 20°C higher than the melting temperature of a bridge probe, or about 5°C to about 20°C higher than the average melting temperature of multiple bridge probes.

[0185] The hybridization temperature of the plurality of bridge 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 the plurality of bridge 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.

[0186] The bridge probe may further include a label. The label may fluoresce. The fluorescent label may be an organic fluorescent dye, a metal chelate, a carbon nanotube, a quantum dot, a gold particle or a fluorescent mineral. The label may be radioactive. The label may be biotin. The bridge probe may be combined with a labeled nucleic acid binding molecule. The nucleic acid binding molecule may be an antibody, an antibiotic, a histone, an antibody or a nuclease.

[0187] The bridge probe may include a connector. The connector may include about 30 nucleotides, about 25 nucleotides, about 20 nucleotides, about 15 nucleotides, about 10 nucleotides, or about 5 nucleotides. The connector may include about 5 to about 20 nucleotides.

[0188] The linker may include a non-nucleic acid polymer (eg, a carbon chain). The linker non-nucleotide polymer may include about 30 units, about 25 units, about 20 units, about 15 units, about 10 units, or about 5 units.

[0189] The bridge probe may be blocked at the 3' and / or 5' end. The bridge probe may lack a 5' phosphate. The bridge probe may lack a 3' OH. The bridge probe may include a 3' ddC, a 3' inverted dT, a 3' C3 spacer, a 3' amino group, or a 3' phosphorylation.

[0190] XII. Adapter Anchor Probes

[0191] The adapter anchor probe may include 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 adapter anchor probe may be about 20 to about 70 nucleotides.

[0192] The melting temperature of the adapter anchor probe and the bridge probe can be about 65°C, about 60°C, about 55°C, about 50°C, about 45°C, or about 45°C to about 70°C.

[0193] The adapter anchor probe may include a label. The label may fluoresce. The fluorescent label may be an organic fluorescent dye, a metal chelate, a carbon nanotube, a quantum dot, a gold particle, or a fluorescent mineral. The label may be radioactive. The label may be biotin. The adapter anchor probe may be bound 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.

[0194] The adapter anchor probe may include a molecular barcode (MB). The adapter anchor probe may include an adapter sequence (e.g., for binding an adapter primer). The capture probe may include a bridge binding sequence (BBS). The adapter anchor probe may include 1 to 100 BBSs. The adapter anchor probe may include an index for distinguishing samples. The molecular barcode or index may be 5' of the adapter sequence and 5' of the BBS.

[0195] XIII. Enzymes

[0196] Examples of DNA polymerases that can be used in the methods and kits described herein include Klenow polymerase, Bst DNA polymerase, Bca polymerase, phi 29 DNA polymerase, Vent polymerase, Deep Vent polymerase, Taq polymerase, T4 polymerase, T7 polymerase, or E. coli DNA polymerase 1.

[0197] 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.

[0198] Examples of exonucleases that can be used in the methods and kits described herein include exonucleases associated with DNA polymerase I (polA), DNA polymerase II (polB), and DNA polymerase III (dnaQ / mutD), DNA polymerase I large (Klenow) fragment, T4 DNA polymerase, exonuclease I, exonuclease III, exonuclease IV, exonuclease VII, exonuclease IX, exonuclease X, RecBCD exonuclease, RecJ exonuclease, RecE exonuclease, SbcCD endonuclease / exonuclease, ribonuclease D, or TabcD exonuclease. The exonuclease can be a 3' to 5' exonuclease or a 5' to 3' exonuclease.

[0199] The restriction endonucleases that can be used in the methods and kits described herein can be type I, type II, type III, or type IV. The restriction endonucleases can include AciI, HindIII, SspI, MluCI, BspMI, EcoP15, EcoRI, HsdR, HsdM, HhaI, NotI, FokI, or BaeI. The restriction endonucleases can be 4-cut, 5-cut, or 6-cut.

[0200] XIV. Downstream Analysis of Amplification Products

[0201] Amplification products generated using the methods described herein can be further analyzed using a variety of methods, including Southern blot, 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 microarray, 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.

[0202] Second generation sequencing can include 454 sequencing (ROCHE) (using pyrophosphate sequencing), 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), droplet single molecule sequencing using pyrophosphorolysis (BASE4), single molecule electronic detection sequencing (e.g., measuring tunneling current through nanoelectrodes when nucleic acids (DNA / RNA) pass through nanogaps and calculating current differences) (QUANTUM SEQUENCING from QUANTUMBIOSYSTEMS), GenapSys Gene Electornic Nano-IntegratedUltra-Sensitive (GENIUS) technology (GENAPYS), GENEREADER from QIAGEN, sequencing using sequential hybridization and connection of partially random oligonucleotides to centrally determined bases (or base pairs) identified by specific fluorophores (SOLiD sequencing). Sequencing can be double-end sequencing.

[0203] 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.

[0204] The nucleic acid library produced using the methods described herein can be produced from more than one sample. Each library can have different indexes associated with the sample. For example, a capture probe or an adapter anchor probe can include an index that can be used to identify nucleic acid as being from the same sample (e.g., a first group of capture probes or adapter anchor probes including the same first index can be used to produce a first library from a first sample from a first subject, and a second group of capture probes or adapter anchor probes including the same second index can be used to produce a second library from a second sample from a second subject, and the first and second libraries can be merged, sequenced, and the index can be used to distinguish which sample the sequenced nucleic acid comes from). The amplification product produced using the methods described herein can be used to produce libraries from at least 2, 5, 10, 25, 50, 100, 1000 or 10,000 samples, each library having different indexes, and libraries can be merged and sequenced, for example, using second generation sequencing technology.

[0205] Sequencing can generate at least 100, 1000, 5000, 10,000, 100,000, 1,000,000, or 10,000,000 sequence reads. Sequencing can generate about 100 sequence reads to about 1000 sequence reads, about 1000 sequence reads to about 10,000 sequence reads, about 10,000 sequence reads to about 100,000 sequence reads, about 100,000 sequence reads to about 1,000,000 sequence reads, or about 1,000,000 sequence reads to about 10,000,000 sequence reads.

[0206] The sequencing depth may be about 1×, 5×, 10×, 50×, 100×, 1000×, or 10,000×. The sequencing depth may be about 1× to about 10×, about 10× to about 100×, about 100× to about 1000×, or about 1000× to about 10000×.

[0207] XV. Application

[0208] a. Nucleic acid characteristic detection

[0209] One or more nucleic acid features of the amplified nucleic acid products produced using the methods and kits described herein can be analyzed. One or more nucleic acid features can be one or more methylation events. Methylation can be methylation of cytosine in CpG dinucleotides. Methylated bases can be 5-methylcytosine. Cytosine in a non-CpG environment can be methylated. Methylated or non-methylated cytosine can be in a CpG island. A CpG island can be a region with a high frequency of CpG sites in a genome. A CpG island can be at least 200bp, or about 300 to about 3000bp. A CpG island can have a CpG dinucleotide content of at least 60%. A CpG island can be in the promoter region of a gene. 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, for example, methylation patterns of DNA from solid tissues or from biological fluids (e.g., plasma, serum, urine or saliva including, for example, cell-free DNA).

[0210] One or more nucleic acid signatures can be de novo mutations, nonsense mutations, missense mutations, silent mutations, frameshift mutations, insertions, displacements, point mutations, single nucleotide polymorphisms (SNPs), single nucleotide variations (SNVs), new single nucleotide variations, deletions, rearrangements, amplifications, chromosome translocations, interstitial deletions, chromosome inversions, loss of heterozygosity, loss of function, gain of function, dominant negatives or lethal mutations. The amplified nucleic acid product can be analyzed to detect germline mutations or somatic mutations. One or more nucleic acid signatures can be associated with a disease, such as cancer, autoimmune diseases, nervous system diseases, infections (such as viral infections) or metabolic diseases.

[0211] b. Diagnosis / Detection / Monitoring

[0212] The disclosed methods and kits can also be used to diagnose or detect a disease. The disease can be a psychological disorder. The disease can be aging. The disease can be a disease. The disease (e.g., a disease) can be cancer, a neurological disease (e.g., Alzheimer's disease, autism spectrum disorder, Rett syndrome, schizophrenia), immunodeficiency, skin disease, autoimmune disease (e.g., Behcet's disease, systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), multiple sclerosis), infection (e.g., viral infection) or metabolic disease (e.g., hyperglycemia, hyperlipidemia, type 2 diabetes). 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, squamous cell lung cancer, thyroid cancer, or leukemia (see, e.g., Jin and Liu (2018) DNA methylation in human disease. Genes & Diseases, 5: 1-8). The disorder may be Beckwith–Wiedemann syndrome, Prader–Willi syndrome, or Angelman syndrome.

[0213] The methylation pattern of cell-free DNA produced using the methods and kits provided herein can be used as a marker for cancer (see, for example, 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 DNA origin (see, for example, 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 cell 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 Application Publication No. WO2015116837; U.S. Patent Application Publication No. 20170121767). The methods and kits described herein can be used to detect methylation levels in cancer subjects and non-cancer subjects, such as methylation levels of cell-free DNA (see, for example, Vidal et al. A DNA methylation map of human cancer at single base-pair resolution. Oncogenomics 36, 5648-5657; International PCT Application Publication No. WO2014043763). The methods and kits described herein can be used to determine methylation levels or determine the partial contribution of different tissues to a cell-free DNA mixture (see, for example, International PCT Application Publication No. WO2016008451). The methods and kits described herein can be used to determine, for example, the source tissue of cell-free DNA in plasma, such as based on comparing the pattern and abundance of methylation haplotypes (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 classify different cancer types according to their tissue of origin (see, e.g., U.S. Patent Application Publication No. 20170175205A1).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).

[0214] The disclosed method 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., Behcet's disease of the eye), an infection (e.g., a viral infection), or a metabolic disease. The cancer may be in remission. Since the disclosed method can use cfDNA and ctDNA to detect low-level abnormalities, the present disclosure can provide a relatively non-invasive method for monitoring disease. The disclosed method can be used to monitor treatment or therapy. Treatment or therapy can be used for a condition, such as a disease, such as cancer, or any condition disclosed herein.

[0215] Example

[0216] Example 1

[0217] Ligation of adapter anchor probes

[0218] Fig.18A Schematic diagrams of two reaction schemes are provided. In the reaction scheme (1) in the left figure, the bridge probe, template and adapter anchor probe are incubated together. The bridge probe hybridizes with the target sequence on the template, and the bridge probe hybridizes with the adapter landing sequence on the adapter anchor probe (1; upper figure). Hybridization promotes the adjacent connection of the phosphorylated 5' end of the adapter anchor probe and the 3' end of the template (1; upper figure). Then, PCR is used to amplify the connection product (1; lower figure). In the second reaction (2) in the right figure, the template and adapter anchor probe are incubated together without the bridge probe (2; upper figure). Similar reaction steps are performed according to reaction scheme (1).

[0219] In this embodiment, in the reaction scheme (1), the adapter anchor probe (SEQ ID: 2) having a target-specific binding sequence is connected to a template DNA (SEQ ID: 1) having a known target sequence by hybridization of a bridge probe (SEQ ID: 3). The bridge probe includes a target-specific region, which is designed to be complementary to the target sequence of the template. The italic letters in SEQ ID: 1 and SEQ ID: 2 (Table 1) represent the target sequence and target-specific region of the template and the bridge probe, respectively. The bridge probe further includes an adapter landing sequence complementary to the bridge binding sequence of the bridge probe, and an oligo T linker connecting the target-specific region and the adapter landing sequence. The bridge binding sequence and the adapter landing sequence are underlined in SEQ ID: 2 and SEQ ID: 3 in Table 1. In addition to the bridge binding sequence, the adapter anchor probe is also designed to contain an adapter and a molecular barcode with a unique sequence. The bold sequence in SEQ ID: 2 represents the AD1 primer binding site. Primers annealed to AD1 and TSR are used to amplify the target sequence of the template. "N" represents any one of the four DNA nucleotides. The different 3' ends of the template are detected to find the nucleotides that are preferentially ligated and sequenced. The four nucleotides at the 5' end of the adapter anchor probe represent the molecular barcode. Various combinations of molecular barcodes are synthesized and mixed with the template.

[0220] Table 1: Sequence Listing

[0221]

[0222]

[0223] The following are the hybridization and ligation conditions for reaction scheme (1). 1 fmole template, 10 fmole bridge probe, and 200 fmole adapter anchor probe were mixed in a volume of 12 μl, denatured at 95°C for 30 seconds, and then placed on ice. 2 μl of 10×CircLigase buffer, 4 μl of 5M betaine, and 1 μl of 50 mM MnCl2 were added to the denatured DNA mixture to reach a final concentration of 2.5 mM MnCl2, 1 M betaine, 0.033 M Tris-acetic acid (pH 7.5), 0.066 M potassium acetate, and 0.5 mM DTT in a final volume of 20 μl. Hybridization was performed by incubating the mixture at 50°C for 30 min on an MJ thermal cycler. Ligation was completed by adding 100 units of Circligase II to the mixture and incubating at 50°C for 20 minutes on an MJ thermal cycler. Take 1 μl of the ligation product as a template and add 0.2 μM of PCR primers for PCR reaction. Perform 20 cycles of PCR with denaturation conditions of 95°C for 15 seconds, annealing conditions of 58°C for 15 seconds, and extension conditions of 68°C for 30 seconds. The conditions of reaction scheme (2) are the same, except that the bridge probe is not included in the mixture of template and adapter anchor probe. 10 μL of PCR product is run on a 2% agarose gel at 80 V for 20 min, and the DNA bands are observed on a UV light box with 1X GelRed stained gel.

[0224] like Fig.18B As shown, two bands appeared in the 100 bp and 50 bp regions of lane 1, which showed the PCR reaction product of reaction scheme (1). 100 bp is consistent with the size of the connected template and adapter anchor probe, indicating that the connection was successful. The lower band is the expected size of the primer used in the amplification step. No band of about 100 bp was detected in lane 2 of reaction scheme (2), indicating that the bridge probe in reaction scheme (1) promoted the connection of the template and adapter anchor probe.

[0225] Example 2

[0226] Addition of adapters by exonuclease digestion and extension

[0227] Fig.26A schematic diagram of capture probe hybridization and 3' to 5' exonuclease digestion is provided. For hybrid capture, the capture probe is hybridized to the target sequence on the template (E-T1, SEQ ID NO.6 and E-T2, SEQ ID NO.7). For the reaction, the synthetic template E-T1 (input mixture 1, 3, 5, 7 of Table 3) or E-T2 (input mixture 2, 4, 6, 8 of Table 3) is mixed with the capture probe (SEQ ID NO.8) in a ratio of 1:1 at a concentration of (1 μM) in Duplex buffer. The DNA mixture is denatured at 92°C for 2 min and then cooled to room temperature on a thermal cycler.

[0228] Table 2 lists the sequences of the template nucleic acid, capture probe, PCR primers used, and extension products obtained from the experiment. The adapter sequences are in bold, while the target sequences are in italics. Compared to E-T1, the E-T2 template includes more nucleotide sequences downstream of the target specific region (TSR), so when hybridized with the capture probe, E-T2 has a single-stranded 3' overhang.

[0229] Table 2: Sequence Listing

[0230]

[0231] After hybridization, the single-stranded 3' overhang on the template was digested using a 3' to 5' exonuclease. For exonuclease digestion and extension, the DNA mixture was reacted with DNA polymerase I Large (Klenow) fragment or T4 polymerase. Table 3 lists the template and enzyme combinations used for each mixture.

[0232] Table 3: Input mixture and experimental results

[0233]

[0234] Each digestion and extension mixture used approximately 12,000 copies of E-T1 / E-T2 and capture probe annealing products. For the Klenow reaction mixture, 1×NEB buffer 2, 50 μM dNTPs, and 0.1 U / μl Klenow were added and incubated at 25°C for 15 min. For the T4 polymerase mixture, 1×NEB buffer 2.1, 10 μM dNTPs, and 0.06 U / μl T4 polymerase were added and incubated at 12°C for 15 min.

[0235] PCR products were evaluated by qPCR using PCR primers (EGFR forward primer, SEQ ID NO.9 and capture probe reverse primer, SEQ ID NO.10). PCR products that were not reacted with Klenow or T4 polymerase (input mixtures 3, 4, 7 and 8) and whose templates therefore still retained 3' overhangs were not detected to include adapters (see Table 3). In contrast, adapter addition was detected in input mixtures 1, 2, 5 and 6. Products of input mixtures 1, 2, 5 and 6 were also detected to have a sequence of SEQ ID NO.10. Regardless of the nuclease / polymerase used, the cycle threshold (Ct) value of the E-T1 template was lower than that of E-T2, indicating that the E-T1 template is more susceptible to extension because E-T1 does not include a 3' overhang.

[0236] Example 3

[0237] Adding adapters by exonuclease digestion and extension using T4 polymerase

[0238] Fig. 27 A schematic diagram of capture probe hybridization and 3' to 5' exonuclease digestion is provided. The capture probe hybridizes with the target sequence on the template. It is known that fragmented genomic DNA (gDNA) including SEQ ID NO.12 is used as a template. For hybridization capture, 20ng of fragmented (peak size 160bp) template gDNA and 10pmol of capture probe are used. DNA input and hybridization probe are denatured at 95°C for 30 seconds in hybridization buffer, and then the mixture is gradually cooled to 60°C. Hybridization is incubated at 60°C for 1 hour on a thermal cycler. The final hybridization buffer contains 100ng / μl DNA, 1μg / μl bovine serum albumin (BSA), 1μg / μl Ficoll, 1μg / μl polyvinyl pyrrolidone (PVP), 0.075M sodium citrate, 0.75M NaCl, 5×SSC and 1×Denhardt solution.

[0239] For clearing, the hybrid assembly was incubated with streptavidin beads (Thermo Fisher Dynabeads M270 Streptavidin) at room temperature for 10 min. Clearing was performed by 3 washes (wash 1: 5X SSPE, 1% SDS; wash 2: 2X SSPE, 0.1%; wash 3: 0.1X SSPE, 0.01% tritium).

[0240] Then, a 3' to 5' exonuclease (T4 polymerase) was added to the capture assembly to digest the single-stranded 3' overhang on the template and extend the template. The capture assembly was resuspended in 25 μl of T4 polymerase mixture (input mixture 1, 2, 3) and incubated at 12°C for 15 min. The reaction was terminated by heating at 95°C for 1 min. Input mixture 4 was not treated with T4 polymerase.

[0241] qPCR was performed to evaluate the addition of the adapter. Enriched DNA was evaluated by qPCR using the EGFR targeting sequence (EGFR forward primer, SEQ ID NO. 13 and reverse primer, SEQ ID NO. 14). Adaptor addition was evaluated by EGFR and adapter sequence PCR (EGFR forward primer, SEQ ID NO. 13 and probe reverse primer, SEQ ID NO. 15, see Tables 4 and 5). Fig. 27 A schematic diagram of qPCR evaluation is shown. Adaptor addition was detected in all samples treated with T4 polymerase (input mixtures 1, 2, and 3), but not in samples without T4 polymerase reaction (see Table 5). The cycle threshold (Ct) values ​​of the mixtures subjected to exonuclease digestion (input mixtures 1, 2, and 3) were lower than the cycle threshold (Ct) of the undigested template (input mixture 4). The Ct of EGFR PCR was similar in all samples, indicating that EGFR was captured in all reactions, and the Ct of the enriched DNA was compared with the same portion of gDNA that was not captured and enriched, and 65% of EGFR was found to be recovered.

[0242] Table 4: Sequence Listing

[0243]

[0244] Table 5: Input mixture and experimental results

[0245]

[0246] Although preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that these embodiments are provided as examples only. Without departing from the present invention, those skilled in the art will now appreciate that many variations, changes and substitutions may be employed in practicing the present invention. The following claims are intended to define the scope of the present invention, and are intended to encompass methods and structures within the scope of these claims and their equivalents.

Claims

1. A method for target enrichment and adaptor ligation for non-diagnostic and therapeutic purposes, comprising: hybridizing a first target-specific region of a first bridge probe to a first target sequence of a template nucleic acid molecule, wherein a first adaptor landing sequence of the first bridge probe binds to a first bridge binding sequence of an adaptor anchor probe, wherein the first target-specific region of the first bridge probe is in a 3′ portion of the first bridge probe; as well as Attaching the 3' end of the template nucleic acid molecule to the 5' end of the adapter anchor probe using a ligase, thereby generating a template nucleic acid molecule attached to the adapter anchor probe, wherein the adapter anchor probe includes, in order from the 3' end to the 5' end, a first bridge binding sequence, a first adapter, and a molecular barcode; hybridizing a first adaptor primer to a first adaptor of the adaptor anchor probe; as well as The first adaptor primer is extended to generate a first extension product. 2 . The method of claim 1 , further comprising treating the template nucleic acid molecule attached to the adapter anchor probe with bisulfite.

3. The method of claim 1 or 2, wherein the first bridge probe comprises a first linker between the first target-specific region and the first adapter landing sequence. 4 . The method of claim 3 , further comprising attaching a second adaptor to the 3′ end of the first extension product, thereby generating a first extension product attached to the second adaptor.

5. The method of claim 4, further comprising phosphorylating the 5' end of the template nucleic acid molecule prior to attaching the second adaptor.

6. The method of claim 4, wherein the second adaptor is a double-stranded adaptor, wherein one of the two strands of the second adaptor is attached to the template nucleic acid molecule.

7. The method of claim 4, further comprising hybridizing a second adaptor primer to the second adaptor in the first extension product attached to the second adaptor.

8. The method of claim 7, further comprising extending the second adaptor primer to generate a second extension product.

9. The method of claim 8, further comprising amplifying the first extension product and the second extension product attached to the second adaptor, thereby generating an amplification product. 10 . The method of claim 8 , further comprising amplifying the first extension product and the second extension product attached to the second adaptor using the first adaptor primer and the second adaptor primer to produce an amplification product.

11. The method of claim 1, further comprising hybridizing a target-specific primer to the first extension product.

12. The method of claim 11, further comprising extending the target-specific primer to generate a second extension product.

13. The method of claim 12, further comprising amplifying the first extension product and the second extension product to generate amplification products.

14. The method of claim 12, further comprising amplifying the first extension product and the second extension product using the first adapter primer and the target-specific primer to generate amplification products.

15. The method of claim 1 or 2, further comprising isolating the first bridge binding sequence.

16. The method of claim 1 or 2, further comprising extending the 3' end of the first bridge probe to generate a first extension product.

17. The method of claim 1 or 2, wherein the adapter anchor probe comprises a first adapter.

18. The method of claim 17, further comprising attaching a second adaptor to the 5' end of the template nucleic acid molecule.

19. The method of claim 18, further comprising phosphorylating the 5' end of the template nucleic acid molecule prior to attaching the second adaptor.

20. The method of claim 18, wherein the adaptor is a double-stranded adaptor.

21. The method of claim 18, further comprising hybridizing a first adaptor primer to the first adaptor of the adaptor anchor probe.

22. The method of claim 21, further comprising extending the first adapter primer to generate a second extension product.

23. The method of claim 22, further comprising hybridizing a second adaptor primer to the second extension product.

24. The method of claim 23, further comprising extending the second adaptor primer to generate a third extension product.

25. The method of claim 24, further comprising amplifying the second extension product and the third extension product to generate an amplification product.

26. The method of claim 24, further comprising amplifying the second extension product and the third extension product using the first adapter primer and the second adapter primer to produce amplification products.

27. The method of claim 22, further comprising hybridizing a target-specific primer to the second extension product.

28. The method of claim 27, further comprising extending the target-specific primer to produce a third extension product.

29. The method of claim 28, further comprising amplifying the second extension product and the third extension product to generate an amplification product.

30. The method of claim 28, further comprising amplifying the second extension product and the third extension product using the first adapter primer and the target-specific primer to produce amplification products.

31. The method of claim 1 or 2, further comprising hybridizing a second target sequence region of a second bridge probe to a second target sequence of the template nucleic acid molecule, wherein a second adapter landing sequence of the second bridge probe binds to a second bridge binding sequence of the adapter anchor probe.

32. The method of claim 31, wherein the second target-specific region of the second bridge probe is in the 3' portion of the second bridge probe.

33. The method of claim 31, wherein the second target-specific region of the second bridge probe is in the 5' portion of the second bridge probe.

34. The method of claim 31, wherein the second bridge probe comprises a second linker between the second target-specific region and the second adapter landing sequence.

35. The method of claim 31, wherein i) the first target-specific region of the first bridge probe is in the 3' portion of the first bridge probe and the second target-specific region of the second bridge probe is in the 3' portion of the second bridge probe; ii) the first target-specific region of the first bridge probe is in the 5' portion of the first bridge probe and the second target-specific region of the second bridge probe is in the 5' portion of the second bridge probe; iii) wherein the first target-specific region of the first bridge probe is in the 3' portion of the first bridge probe and the second target-specific region of the second bridge probe is in the 5' portion of the second bridge probe; or iv) the first target-specific region of the first bridge probe is in the 5' portion of the first bridge probe and the second target-specific region of the second bridge probe is in the 3' portion of the second bridge probe.

36. A method according to claim 31, wherein the first target-specific region of the first bridge probe is in the 3' portion of the first bridge probe, and the second target-specific region of the second bridge probe is in the 5' portion of the second bridge probe; and wherein the first connector of the first bridge probe and the second connector of the second bridge probe hybridize to each other.

37. The method of claim 2, wherein the target sequence region of the bridge probe is designed based on the target sequence of the template nucleic acid molecule, and wherein the target sequence of the template nucleic acid retains unmethylated cytosine after the bisulfite treatment.

38. The method of claim 1, further comprising treating the nucleic acid molecule with bisulfite, thereby producing the template nucleic acid molecule of claim 1, wherein the template nucleic acid molecule of claim 1 is a product of the bisulfite treatment.

39. The method of claim 38, wherein the target sequence region of the bridge probe is designed based on the target sequence of the template nucleic acid molecule after the bisulfite treatment, wherein non-methylated cytosine in the template nucleic acid is converted to uracil during the bisulfite treatment.

40. The method of claim 1, further comprising dephosphorylating the 5' end of the nucleic acid molecule, thereby producing the template nucleic acid molecule of claim 1.

41. The method of claim 1, wherein the adapter anchor probe further comprises a label.

42. The method of claim 41, further comprising capturing the bridge probe via the label.

43. The method of claim 41, wherein the label is biotin.

44. The method of claim 42, wherein the label is captured on a solid support.

45. The method of claim 44, wherein the solid support is a bead.

46. ​​The method of claim 45, wherein the bead comprises a capture moiety that targets the marker.

47. The method of claim 46, wherein the capture moiety is streptavidin.

48. The method of claim 1, further comprising contacting the nucleic acid molecule with a 3' to 5' exonuclease after attaching the 3' end of the template nucleic acid molecule to the 5' end of the adaptor anchor probe of claim 1, wherein the nucleic acid molecule comprises the adaptor anchor probe and the template nucleic acid molecule.

49. The method of claim 13, 25 or 29, further comprising sequencing the amplification product.

50. The method of claim 49, wherein the sequencing comprises next generation sequencing.

51. The method of claim 1, wherein the template nucleic acid molecule comprises single-stranded DNA.

52. The method of claim 1, wherein the template nucleic acid molecule comprises RNA.

53. The method of claim 1, wherein the template nucleic acid molecule comprises damaged DNA.

54. The method of claim 1, wherein the template nucleic acid molecule is from a formalin-fixed paraffin-embedded (FFPE) sample.

55. The method of claim 1, wherein the template nucleic acid molecule comprises cell-free nucleic acid from a biological sample.

56. The method of claim 55, wherein the cell-free nucleic acid comprises cell-free DNA.

57. The method of claim 56, wherein the cell-free DNA comprises circulating tumor DNA.

58. The method of claim 1, wherein the template nucleic acid molecule has not been subjected to an in vitro DNA repair step prior to claim 1.

59. A kit comprising: a bridge probe comprising a target-specific region that hybridizes to a target sequence of a template nucleic acid molecule, wherein the target-specific region of the bridge probe is in a 3' portion of the bridge probe; An adapter anchor probe comprises a bridge binding sequence hybridizing to the adapter landing sequence of the bridge probe and a 5' end attached to the 3' end of the template nucleic acid molecule, wherein the adapter anchor probe comprises a first bridge binding sequence, a first adapter and a molecular barcode in sequence from the 3' end to the 5' end.

Images