Sequencing scaffolds and methods

Abstract

A sequencing scaffold for attaching a target nucleic acid strand (204) comprises at least one nucleic acid scaffold (100, 102, 104, 200a, 200b), the at least one nucleic acid scaffold (100, 102, 104, 200a, 200b) having a plurality of attachment regions (118, 119) at predetermined locations, each attachment region comprising at least one attachment site (122, 124, 124a, 124b), the attachment sites (122, 124, 124a, 124b) being configured to hybridize directly or indirectly to the target nucleic acid strand (204), and the nucleic acid scaffold (100, 102, 104, 200a, 200b) having a three-dimensional lattice structure, and attachment sites (122, 124, 124a, 124b) are arranged in three dimensions on the lattice structure of the nucleic acid backbone (100, 102, 104, 200a, 200b). In further aspects, methods of sequencing are provided.

Description

Technical Field

[0001] The present invention relates to a sequencing scaffold for attaching a target nucleic acid chain and a method for sequencing the target nucleic acid chain. Background Art

[0002] Nucleic acid sequencing has been continuously improving from early methods such as Maxam-Gilbert or Sanger sequencing to massively parallel sequencing such as 454 sequencing, Illumina sequencing, or DNA nanoball sequencing. While earlier sequencing methods allowed for higher read lengths, they were severely limited in terms of throughput. In contrast, massively parallel sequencing methods use miniaturized and parallelized methods to sequence a large number of short reads, which are then assembled into longer sequences. However, there are ongoing efforts to further improve these methods to provide higher throughput methods, thereby achieving faster sequencing results. Summary of the invention

[0003] It is therefore an object to provide devices and methods allowing improved high-throughput sequencing.

[0004] The above objects are achieved by the subject-matter of the independent claims. Advantageous embodiments are defined in the dependent claims and in the following description.

[0005] The sequencing support for attaching the target nucleic acid chain includes at least one nucleic acid backbone, the at least one nucleic acid backbone having a plurality of attachment regions at predetermined positions, each attachment region comprising at least one attachment site, and the nucleic acid backbone having a three-dimensional lattice structure, and the attachment regions are arranged in a three-dimensional manner on the lattice structure of the nucleic acid backbone.

[0006] Therefore, the nucleic acid backbone provides an anchor point for the target nucleic acid (meaning a nucleic acid chain that is sequenced, for example, by a pyrophosphate sequencing method). The lattice structure of the backbone extends in a three-dimensional manner, and the attachment regions are distributed in a three-dimensional manner on the backbone. It should be understood that this is in contrast to the surface of the wall of a microarray, a flow cell or a microfluidic channel that only provides a two-dimensional surface to which the target nucleic acid chain can be attached. Therefore, compared with a two-dimensional attachment surface, the number of attachment regions in a given space can be greatly increased by providing a three-dimensional lattice structure.

[0007] Preferably, the sequencing scaffold comprises at least first, second and third orientation indicator markers. These orientation indicator markers may be, for example, fluorescent dyes and are capable of determining the orientation of the scaffold in space.

[0008] It is particularly preferred that the orientation indicators are spaced apart from each other or are arranged at opposite ends of the skeleton. For example, the orientation indicators, in particular the corresponding fluorescent dyes, can further differ from each other in optical properties such as excitation wavelength, emission wavelength or emission lifetime. Thus, the orientation indicators are attached to the skeleton and can be distinguished from each other. Thus, the orientation indicators are able to identify the corresponding ends of the skeleton and thus its orientation in space.

[0009] Preferably, the attachment areas are arranged on the frame in several, in particular parallel planes, and these planes are arranged at a distance from each other in a direction perpendicular to the planes. This enables regularly spaced attachment areas and a high density of attachment areas to be achieved within a given volume.

[0010] Preferably, the distance between the planes is in the range of 5 nm to 6400 nm, preferably in the range of 10 nm to 3200 nm, more preferably in the range of 50 nm to 800 nm. This enables a particularly high density of attachment areas within a given volume. The spacing between the planes can be selected based on the resolution of a readout device (e.g., a microscope) used to read out the sequencing reaction when sequencing the target nucleic acid.

[0011] Preferably, the maximum spatial extent of the backbone is in the range of 10 nm to 10000 nm, preferably in the range of 0.1 µm to 5 µm. This enables a particularly compact scaffold that can be integrated into flow devices for sequencing, such as microfluidic chips.

[0012] Preferably, the sequencing support is attached to a wall of a flow device or microfluidic chip, wherein the wall is suitable for enabling optical readout, such as by microscopy. For example, the wall may be optically transparent.

[0013] Preferably, the attachment regions are spaced apart from one another in the range of 1 nm to 5000 nm, preferably in the range of 5 nm to 1000 nm, more preferably in the range of 10 nm to 500 nm. This enables a particularly dense arrangement of the target nucleic acids on the backbone.

[0014] Preferably, for each attachment region, the attachment sites are spaced in the range of 1 nm to 500 nm, preferably in the range of 10 nm to 200 nm. The spacing between the attachment sites can be selected according to the resolution capability of the readout device used to read out the sequencing reaction when sequencing the target nucleic acid. Particularly preferred ranges can correspond to the lateral resolution achievable with different microscopic modalities, such as MINFLUX (<1 nm to 10 nm), single molecule localization microscopy (1 nm to 25 nm), structured illumination and / or STED microscopy (50 nm to 100 nm), high NA (numerical aperture) light microscopy (about 200 nm) and low NA light microscopy (about 500 nm). Importantly, the attachment sites can be far away from each other so that the readout device can resolve them individually.

[0015] Similar ranges regarding the typical axial optical resolution (lower than the typical lateral optical resolution) of optical readout achievable with different microscopic modalities can be applied. In particular, the attachment sites are spaced apart from each other corresponding to the axial resolution achievable with different microscopic modalities. For example, a nucleic acid backbone can be generated such that the attachment sites are arranged on the backbone in several planes, wherein the distances between these planes are in a direction perpendicular to the planes and greater than the typical axial optical resolution of the optical readout. Thus, the optical readout of the sequencing reaction can also be resolved in the axial direction of the optical readout system.

[0016] For example, using microscopic readout modalities such as MINFLUX, single-molecule localization microscopy, structured illumination or STED, a very dense packing of attachment sites can be achieved, where the attachment sites are spaced a few nanometers apart and where the readout can still assign the signal from the sequencing reaction to a specific target nucleic acid attached to a specific attachment site. In this way, the number of reads generated from a single flow cell channel can be greatly increased. Appropriate spacing of attachment sites in a three-dimensional manner can also take into account the length of the nucleic acid target that should be attached and sequenced, where a 300 bp long DNA oligonucleotide is about 100 nm long.

[0017] Preferably, the backbone comprises a scaffold chain and a staple chain, the scaffold chain may be a longer nucleic acid chain (e.g., M13 phage genomic DNA), the staple chain being configured to bind to the scaffold chain at a predetermined position to fold the scaffold chain into a predetermined shape. Thus, the backbone may be a DNA origami backbone. The size of these DNA origami structures may range from a few nanometers to micrometers. To make such DNA origami-based structures, a longer DNA molecule (scaffold chain) is folded at precisely identified positions by a so-called staple chain. DNA origami can be designed to provide a self-assembling backbone of a particular predetermined shape. This enables simple and reproducible synthesis and assembly of the backbone. The staple chain may be position-selectively functionalized. In this case, the position resolution is limited by the size of the nucleotides in the nanometer or sub-nanometer range. This has been used in the prior art to generate fluorescent standards, in which fluorescent dyes are attached to precisely positioned bands on DNA origami. These standards are referred to as "nano rulers" and are used to calibrate imaging systems such as confocal or super-resolution microscopy (e.g., STED), for example, as disclosed in US2014 / 0057805 A1.

[0018] DNA origami provides a scaffold for the target nucleic acid chain. Preferably, the DNA origami backbone comprises at least one scaffold chain and a plurality of staple chains, wherein the staple chains are complementary to at least a portion of the scaffold chains, and the staple chains are configured to form a predetermined conformation of the scaffold chains. In particular, the staple chains are oligonucleotides. This enables the generation of a nucleic acid backbone having a predetermined two-dimensional or three-dimensional shape that can self-assemble. In addition, this enables the use of special staple chains (also referred to as docking chains) as attachment sites to achieve site-specific placement of attachment sites on the backbone. In the sense of this document, a staple chain that partially contains a sequence (which is not bound to the scaffold chain and is used as an attachment site or other form of functionalization) may also be referred to as a docking chain.

[0019] Alternatively, the nucleic acid backbone may comprise or consist of DNA bricks, which typically comprise four domains (1-4). These DNA bricks are oligonucleotides of shorter length with overlapping hybridization stretches to bind to each other and assemble the backbone. In the case of DNA bricks, attachment sites may be formed by extending a particular DNA brick by at least one other domain (i.e., a fifth domain) that serves as an attachment site and has no hybridization pairing domain in the set of DNA bricks used to assemble the DNA brick-based nucleic acid backbone.

[0020] Preferably, the staple strands comprise attachment sites, in which case they may also be referred to as docking strands. The attachment site is a nucleic acid sequence, preferably a nucleic acid sequence of a staple strand. Preferably, the target nucleic acid may be attached to the staple strand of the backbone at a predetermined attachment site. Since the staple strand is positioned at a predetermined position, the position of the attachment site may also be predetermined. Therefore, the attachment site is an oligonucleotide sequence configured to bind to the target nucleic acid strand.

[0021] In a preferred embodiment, the backbone comprises a double-stranded oligonucleotide and the attachment site comprises a single-stranded oligonucleotide. Thus, the target nucleic acid can bind only at the attachment site and random binding to the rest of the backbone is avoided.

[0022] Preferably, each attachment site comprises a unique oligonucleotide sequence. In particular, the attachment sites may be random. This allows a large number of different target nucleic acid strands to hybridize directly to the attachment sites.

[0023] Preferably, each attachment region comprises at least a first set of attachment sites configured to hybridize with a first adapter oligonucleotide and a second set of attachment sites configured to hybridize with a second adapter oligonucleotide. This enables particularly efficient sequencing of the target strand.

[0024] More preferably, the first and second oligonucleotide adaptors are configured to be ligated to the target nucleic acid strand.

[0025] Preferably, the scaffold comprises a second nucleic acid backbone, and the nucleic acid backbones are linked to each other. The link can be a nucleic acid link or a chemical non-nucleic acid link. This enables the assembly of a larger scaffold using several backbones.

[0026] On the other hand, a method for sequencing a target nucleic acid chain is provided, the method comprising the following steps: providing a support comprising at least one nucleic acid backbone; directly or indirectly attaching the target nucleic acid chain to an attachment site of at least one nucleic acid backbone of the support; sequencing the target nucleic acid chain according to a sequencing method, the sequencing method comprising at least a step of amplifying the attached target nucleic acid chain and a step of iteratively optically reading the attachment site.

[0027] In particular, the sequencing method may be a method that requires optical readout of a sequencing reaction of a target nucleic acid strand attached to a corresponding attachment site, for example, a pyrophosphate sequencing method or a dye terminator method, such as Illumina or 454 sequencing.

[0028] Preferably, a first adapter oligonucleotide and a second adapter oligonucleotide are ligated to each target nucleic acid strand prior to the step of attaching the target nucleic acid strands to the attachment sites.

[0029] The method has the same advantages as the sequencing scaffold described above and can be supplemented with the features of the dependent claims directed to the sequencing scaffold. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In the following, specific embodiments will be described with reference to the accompanying drawings, in which: Figure 1 is a schematic diagram of several nucleic acid backbones with orientation indicator markers and attachment regions; Figure 2A is a first schematic diagram of a nucleic acid backbone of a sequencing scaffold with attached target nucleic acid strands; Figure 2B is a first schematic diagram of a nucleic acid backbone of a sequencing scaffold with attached target nucleic acid strands and docking strands; Figure 3 is based on Figure 2A , Figure 2B A schematic diagram of a nucleic acid backbone having sequencing information of a target nucleic acid strand attached thereto; Figure 4 is a schematic diagram of DNA nanoball sequencing on a sequencing scaffold after rolling circle amplification; Figure 5A is a schematic diagram of steps S500 to S504 of a sequencing method for sequencing a scaffold; Figure 5B is a schematic diagram of step S506 of the sequencing method; Figure 5C is a schematic diagram of step S508 of the sequencing method; Figure 5D is a schematic diagram of step S510 of the sequencing method; Figure 5E is a schematic diagram of step S512 of the sequencing method; Fig. 5F is a schematic diagram of step S514 of the sequencing method; Figure 5G is a schematic diagram of step S516 of the sequencing method; Figure 5H is a schematic diagram of step S518 of the sequencing method; Fig.5I is a schematic diagram of step S520 of the sequencing method; and Figure 5J is a schematic diagram of step S522 of the sequencing method. DETAILED DESCRIPTION

[0031] Figure 1Schematic diagram of several three-dimensional nucleic acid backbones 100, 102, 104 of sequencing scaffolds. In general, the nucleic acid backbones 100, 102, 104 are composed of a plurality of nucleic acid strands 120. In particular, the backbones 100, 102, 104 are based on structural DNA nanotechnology (e.g., DNA tile-based structures, DNA origami-based structures, DNA brick-based structures, or other DNA nanotechnology-based structures), which allow for the generation of predetermined, stable three-dimensional shapes.

[0032] The framework can have various geometric shapes, for example, a tetrahedral framework 100, a cubic (especially cube) framework 102, or a polyhedral framework 104. These three-dimensional geometric shapes of the frameworks 100, 102, 104 enable the attachment sites to be arranged in a three-dimensional manner on the frameworks 100, 102, 104. Compared with two-dimensional structures (such as sheet-like or rod-like structures), these three-dimensional structures can provide a greater number of attachment sites for a given substrate area. The geometric shape can be arbitrary and can include solid, wireframe-like or hollow structures.

[0033] The nucleic acid backbone 100, 102, 104 is capable of generating a plurality of attachment regions 118, 119 at predetermined locations along the backbone 100, 102, 104. The attachment regions 118, 119 are nucleic acid segments that allow complementary oligonucleotides (e.g., target nucleic acid strands) to hybridize to the backbone 100, 102, 104 at these predetermined locations.

[0034] In particular, the backbone 100, 102, 104 may include an attachment region 118 having an integrated attachment site 122, which is a short sequence fragment of the nucleic acid strand 120 of the backbone 100, 102, 104. Alternatively, the attachment region 118 of the backbone 100, 102, 104 may include a linked attachment site 124, which is arranged on a nucleic acid staple strand (also referred to as a docking strand) of the backbone 100, 102, 104, which is partially complementary to a portion of the nucleic acid strand 120 of the backbone. Thus, the target nucleic acid strand may hybridize directly or indirectly with the nucleic acid strand 120 of the backbone 100, 102, 104.

[0035] Alternatively or additionally, the backbone 100, 102, 104 may include an attachment region 119. The attachment region 119 includes a plurality of attachment sites. Specifically, the attachment region 119 includes a plurality of first attachment sites 124a and a plurality of second attachment sites 124b. These attachment sites 124a, 124b support specific target nucleic acid amplification strategies and chemical methods on the backbone 100, 102, 104. For example, a plurality of attachment regions 119 are spatially distributed on the nucleic acid backbone 100, 102, 104, and the corresponding attachment sites 118a, 118b of each attachment region 119 are densely stacked, that is, their spacing is preferably in the range of 1 nm to 50 nm. This allows a given target nucleic acid chain to be amplified in a given attachment region 119 using the chemical method described in further detail in the description of FIG. 5.

[0036] Preferably, each attachment region 118, 119, in particular the corresponding attachment sites 122, 124, 124a, 124b, can be hybridized with a specific predetermined target nucleic acid (such as a gene or genomic site set associated with disease diagnosis). This allows the capture of relevant sequence fragments of a liquid biopsy incubated with the backbone 100, 102, 104 of the sequencing scaffold. Alternatively, the attachment region can be configured to hybridize the target nucleic acid indirectly with the backbone 100, 102, 104. For example, the target nucleic acid can be fragmented and then ligated to a universal oligonucleotide (also referred to as a linker), which in turn hybridizes with the attachment regions 118, 119, in particular with the corresponding attachment sites 122, 124, 124a, 124b.

[0037] To enable the orientation of the skeleton 100, 102, 104 to be determined, the skeleton 100, 102, 104 may optionally include a first orientation indicator 110, a second orientation indicator 112, and a third orientation indicator 114. The skeleton 100 may further include a fourth orientation indicator 116.

[0038] Orientation indicator markers 110, 112, 114, 116 can be used to determine the orientation, directionality or polarity of skeleton 100, 102, 104. Orientation indicator markers 110, 112, 114, 116 can include at least one dye, especially fluorescent dye, such as fluorescein or fluorescent protein. In addition, the dye of each of orientation indicator markers 110, 112, 114, 116 can have different characteristics. These characteristics can include fluorescence emission characteristics, excitation characteristics or life characteristics. This makes it possible to distinguish orientation indicator markers 110, 112, 114, 116 in the optical readout of skeleton 100, 102, 104, for example, generated by a microscope, a cell counter or an imaging cell counter. Orientation indicator markers 110, 112, 114, 116 are arranged to be spaced apart from each other. Preferably, each orientation indicator marker 110, 112, 114, 116 is arranged at a specific end of skeleton 100, 102, 104. Thus, the orientation indicator markers 110, 112, 114, 116 can distinguish the ends of the backbone 100, 102, 104. Ultimately, this can determine the orientation, directionality or polarity of the backbone 100, 102, 104. The orientation indicator markers 110, 112, 114, 116 can be attached to a specific one of the attachment sites of the backbone 100, 102, 104 by a suitable oligonucleotide.

[0039] Figure 2A and Figure 2B Schematic diagram of nucleic acid backbone 200a, 200b of sequencing scaffold. Backbone 200a, 200b has a cubic lattice structure, with attachment sites 202a, 202b in each corner of backbone 200a, 200b. Target nucleic acid strand 204 can be attached to attachment sites 202a, 202b.

[0040] Specifically, Figure 2A An attachment site 202a is shown that can be configured as an integrated attachment site, such as in combination with Figure 1 The target nucleic acid strand 204 is directly attached to the nucleic acid strand 206 of the nucleic acid backbone 200a via the attachment site 202a. Each attachment site 202a also forms a corresponding attachment region.

[0041] Alternatively, the target nucleic acid strand 204 can be indirectly attached to the nucleic acid strand 206 of the nucleic acid backbone 200b. In this case, the docking strand 208 including the attachment site 202b is attached to the nucleic acid strand 206, and the target nucleic acid strand 204 is in turn attached to the docking strand 208 via the attachment site 202b. The attachment site 202b can be configured as a connected attachment site, such as a binding site. Figure 1 Each attachment site 202b also forms a corresponding attachment region.

[0042] For each attachment region, there may be a plurality of docking chains 208, each docking chain of the attachment region being configured to hybridize with the target nucleic acid chain 204. Specifically, in the case of such a plurality of docking chains 208, a plurality of attachment regions may then be arranged and distributed on the nucleic acid backbone 200b. Preferably, the attachment regions are spaced relative to each other in the range of 1 nm to 2000 nm, preferably in the range of 200 nm to 1000 nm. This enables the target nucleic acid chains amplified with clonal groups on the attachment region on the backbone to be particularly densely arranged. The docking chain 208 has an attachment oligonucleotide portion 210, which may be specifically complementary to a specific portion of the nucleic acid chain 206.

[0043] The docking strand 208 can include a primer sequence for initiating sequencing of the target nucleic acid 204 attached to the docking strand 208. The target nucleic acid strand 204 can be hybridized or ligated to the docking strand 208 before the docking strand 208 hybridizes with the target nucleic acid strand 204 hybridized to the attachment site 202 of the backbone 200.

[0044] The attachment of the target nucleic acid strand 204 to any one of the attachment sites 202a, 202b can be mediated by a linker oligonucleotide. The linker oligonucleotide can be connected (ligated) to the target nucleic acid strand 204 before the target nucleic acid strand 204 is attached to the backbone 200a, 200b, and can be mediated by the complementarity (specific attachment) of the attachment site or docking strand 208 with a portion of the target nucleic acid. The latter principle can be used to pull a target nucleic acid collection from a mixture (e.g., from a cell lysate or from a liquid biopsy) onto the sequencing scaffold. In this sense, the sequencing scaffold can be used to purify the target nucleic acid by capturing and subsequently washing before sequencing the target nucleic acid.

[0045] The adapter oligonucleotide may further comprise a universal primer sequence. This allows priming of all target nucleic acid strands 204 in parallel for sequencing by synthesis.

[0046] Figure 3It is a schematic diagram of a nucleic acid backbone 200a, 200b, and the nucleic acid backbone 200a, 200b has the sequencing information of the attached target nucleic acid chain 204. The sequencing information is depicted as a temporal sequence or continuity of optical signals 300, 302, 304, 306 read out separately. The optical signal can be generated by a synthesis method (e.g., such as pyrophosphate sequencing, DNA nanoball sequencing, ILLUMINA sequencing or other forms of sequencing while synthesis) during sequencing. Therefore, the target nucleic acid chain serves as a template during the synthesis of the complementary chain. For each nucleotide incorporated in the complementary chain, the optical signal can be specific to a specific nucleotide. For example, the optical signal 300 of the last incorporated nucleotide can be specific to the incorporation of adenine, and the optical signals 302, 304 can be specific to the incorporation of thymine and the optical signal 306 can be specific to the incorporation of guanine. Therefore, by sequentially reading out the optical signal, the nucleic acid sequence in the complementary chain can be determined, and therefore the nucleic acid sequence of the target nucleic acid chain is determined. Sequential reading can be performed in a cyclic process involving incorporation of dye-labeled nucleotides, readout of the incorporated dye, and removal of the dye, or it can be the result of a continuous read that continuously reads the incorporation of dye-labeled nucleotides at each attachment site or attachment region.

[0047] Preferably, docking strand 208 can be one of first joint set, and this first joint set all has identical attachment oligonucleotide part 210, or this first joint set has attachment oligonucleotide part that is combined with specific, predetermined first attachment site 202 of backbone 200.Second joint set can provide different attachment oligonucleotide parts that are combined with specific, predetermined second attachment site.Target strand 204 can come from first sample and be attached to first attachment site 202 via this first joint set, and target strand from second sample can be attached to this second attachment site via this second joint set.Then optical signal of each first attachment site and second attachment site can be read out, to determine the sequence of corresponding target strand.Because target strand of different samples is specifically attached to first attachment site or second attachment site, therefore their corresponding sequence can be assigned to corresponding sample.Therefore, different samples can be sequenced simultaneously, and the ability of each sequenced target strand being assigned to specific one sample in this sample is retained simultaneously.This further improves sequencing throughput.

[0048] The optical signal can be detected by, for example, optically reading out the attachment site (particularly the sequencing reaction at the attachment site) via an optical readout device, which can be a microscope, such as a point scanning confocal or a camera / wide field imaging system, such as a spinning disk microscope, a light sheet fluorescence microscope, a light field microscope, or a stereo microscope. In addition, the optical readout can be a non-image-based readout using at least one point detector or a line detector. The optical readout can be performed when the support is in a flow-through device, such as a microfluidic chip.

[0049] Prior to sequencing the target nucleic acid strand 204, the target strand 204 may be amplified while attached to the backbone 200a, 200b, for example by Figure 4 The rolling circle amplification (RCA) used in the DNA nanoball sequencing schematically depicted in FIG is amplified. As shown in the schematic diagram, the RCA reaction amplifies the target nucleic acid chain 204 attached to the backbone 200a, 200b in a circle. This generates a concatemer of multiple copies 400a, 400b, 400c, 400d of the original target nucleic acid chain 204. Now the multiple copies 400a, 400b, 400c, 400d can be subjected to a sequencing-by-synthesis reaction 402 in parallel, which leads to an amplification of the optical signal 404 because each copy 400a, 400b, 400c, 400d generates the same signal 404 at the same time. Long concatemer chains composed of multiple copies 400a, 400b, 400c, 400d tend to roll into so-called DNA nanoballs 403, which can be read as single spots by a microscope.

[0050] The target nucleic acid strand 204 attached to the backbone 200a, 200b can be further amplified according to the chemical methods and principles described in "Bentley, D., Balasubramanian, S., Swerdlow, H. et al., Accurate whole human genome sequencing using reversible terminator chemistry. Nature 456, 53–59 (2008). https: / / doi.org / 10.1038 / nature07517". In this case, multiple attachment sites of the first and second types are required because this method uses a "pair of oligonucleotides in a forked adaptor configuration" to generate double-stranded blunt-ended nucleic acid fragments having different (i.e., first and second) adapter sequences at either end. Here, we refer to this method as ILLUMINA sequencing. The present invention supports such a sequencing method as also schematically shown in FIG. 5 by providing a sequencing scaffold having at least a first attachment site and a second attachment site (complementary to the first adapter sequence and the second adapter sequence), wherein a plurality of first attachment sites and second attachment sites are grouped into attachment regions on the nucleic acid backbone 200. Each region then has a number of first attachment sites and second attachment sites. For example, 10 to 25, 25 to 50, 50 to 100, 100 to 1000 attachment sites to allow for different levels of amplification. Spots of approximately 30 nucleic acid targets will generate a signal (approximately 30 fluorescent dye molecules per round) that can be well distinguished using, for example, widefield, confocal, light sheet, super-resolution or light field microscopy as a readout and moderate excitation (laser) power, combined with typical pixel dwell / exposure times that generate sufficient signal-to-noise and signal-to-background ratios for robust base recognition.

[0051] FIG. 5A to FIG. 5J The steps of the sequencing method are schematically shown.

[0052] exist Figure 5AIn step S500 of the method shown in , target (double-stranded) nucleic acid chains 500 are introduced. These chains 500 can be obtained from genomic DNA derived from biological samples (e.g., cell lysates, tissue biopsies, liquid biopsies, water samples) via several fragmentation methods. In step S502, a first type of adapter oligonucleotide 502a and a second type of adapter oligonucleotide 502b are ligated to the target nucleic acid chains 500. A "pair of oligonucleotides in a forked adaptor configuration" as described by Bentley et al. in 2008 can be used in this step to ensure that either end of the target nucleic acid chain 500 is connected to a different adapter. The first adapter 502a is represented by a dotted line attached to the target nucleic acid 500, and the second adapter 502b is represented by a dashed line. The first adapter 502a (dotted line) is at least partially complementary to the first attachment site 124a (dotted line), and the second adapter 502b (dashed line) is at least partially complementary to the second attachment site 124b (dashed line). As a result, in step S504 , the (double-stranded) nucleic acid-linker conjugate is melted to generate a single-stranded target nucleic acid-linker conjugate 504 , which in turn hybridizes with the complementary first attachment site 124a (dotted line) of the attachment region 119 .

[0053] exist Figure 5B In step S506 shown in FIG. 1 , the complementary strand 504 b is synthesized, wherein the synthesis is initiated by the universal primer sequence present at the attachment sites 124 a, 124 b. This effectively connects the target nucleic acid strand to the attachment site 124 a of the attachment region 119.

[0054] exist Figure 5C In step S508 shown in FIG. 5 , one of the double strands is melted, thereby allowing the unbound target nucleic acid fragment-adapter conjugate 504 to be released.

[0055] By including FIG. 5D to FIG. 5J In the cyclic process of steps S510-S522 schematically shown in the figure, the physically linked target nucleic acid-linker-conjugate 504b is currently amplified on the attachment area, which generates densely packed spots of multiple copies of the target nucleic acid-linker-conjugate. As described above, the densely packed spots can be sequenced in parallel using the sequencing by synthesis method.

[0056] Figure 5D Step S510 is shown, in which the second linker 502b hybridizes to the adjacent attachment site 124b. Figure 5E Step S512 is shown: DNA synthesis of complementary chain 504a (nth cycle). Fig. 5FStep S514 is shown: melting of complementary strands 504a and 504b (nth cycle). Figure 5G Step S516 is shown: reannealing / rehybridization of complementary strands 504a, 504b (nth cycle). Figure 5H Step S518 is shown: additional DNA synthesis of additional complementary strands 504c, 504d (n+1th cycle). Fig.5I Step S520 is shown: melting of the other complementary strands 504c and 504d (the n+1th cycle). Figure 5J Step S522 is shown: sequencing is performed after the n+mth cycle (the amplification of steps S510 to S518 can continue until all attachment sites 124a, 124b of each attachment region 119 are occupied, or stopped before a sufficient amplification level is reached).

[0057] Sequencing may then be performed by synthesis or by other means, such as, for example, by hybridization or by ligation.

[0058] While amplification may be required in most cases, in some cases simpler non-amplification chemistries are preferred in order to be able to not only sequence target nucleic acid fragments but also to count them, similar to how, for example, in diagnostic applications, levels of water contamination by pathogens can be determined in this way, or in the case of cancer, copy number variation can be assessed.

[0059] Amplification-free sequencing is also possible using high-brightness dyes and particularly sensitive microscopy detection platforms with suitable high-NA, high-transmission optics and sensitive detectors such as back-thinned, back-illuminated CMOS sensors.

[0060] In all figures, elements having the same or similar function are indicated by the same reference numerals. As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items and may be abbreviated as " / ". Individual features of the embodiments and all combinations of individual features of the embodiments with each other and with individual features or groups of features of the preceding description and / or claims are considered to be disclosed.

[0061] Although some aspects are described in the context of an apparatus, it is apparent that these aspects also represent a description of a corresponding method, wherein a block or device corresponds to a method step or a feature of a method step. Similarly, aspects described in the context of a method step also represent a description of a corresponding block or item or feature of a corresponding apparatus.

[0062] Reference numerals list 100, 102, 104, 200a, 200b Nucleic acid backbone 110, 112, 114, 116 Orientation indicator marks 118, 119 Attachment area 122, 202a Integration attachment site Attachment site for 124, 124a, 124b, 202b 204 Target nucleic acid chain 206 Nucleic acid chain of backbone 208 Docking Chain 210 Attachment oligonucleotide part 300, 302, 304, 306, 404 optical signals 400a, 400b, 400c, 400d target nucleic acid strands for rolling circle amplification 402 Sequencing by Synthesis 403 DNA Nanosphere 500 Double-stranded target nucleic acid fragment-adapter conjugate 502a, 502b connectors 504, 504a, 504b, 504c, 504d Single-stranded target nucleic acid-linker conjugate

Claims

1. A sequencing scaffold for attaching a target nucleic acid strand (204), comprising: at least one nucleic acid backbone (100, 102, 104, 200a, 200b), the at least one nucleic acid backbone (100, 102, 104, 200a, 200b) having a plurality of attachment regions (118, 119) at predetermined positions, each attachment region comprising at least one attachment site (122, 124, 124a, 124b), wherein the attachment sites (122, 124, 124a, 124b) are configured to hybridize with the target nucleic acid strand (204), and The nucleic acid backbone (100, 102, 104, 200a, 200b) has a three-dimensional lattice structure, and the attachment regions (118, 119) are three-dimensionally arranged on the lattice structure of the nucleic acid backbone (100, 102, 104, 200a, 200b).

2. The sequencing scaffold according to claim 1, comprising at least first, second and third orientation indicating marks (110, 112, 114, 116).

3. The sequencing scaffold according to claim 2, wherein the orientation indicator marks (110, 112, 114, 116) are spaced apart from each other or arranged on opposite ends of the backbone (100, 102, 104, 200a, 200b).

4. The sequencing scaffold according to any one of the preceding claims, wherein the attachment regions (118, 119) are arranged on the backbone (100, 102, 104, 200a, 200b) in several planes, and the planes are arranged at a distance from each other in a direction perpendicular to the planes.

5. The sequencing scaffold according to claim 4, wherein the distance between the planes is in the range of 5 nm to 6400 nm, preferably in the range of 10 nm to 3200 nm, more preferably in the range of 50 nm to 800 nm.

6. The sequencing scaffold according to any of the preceding claims, wherein the maximum spatial extent of the backbone (100, 102, 104, 200a, 200b) is in the range of 10 nm to 10000 nm, preferably in the range of 0.1 µm to 5 µm.

7. The sequencing scaffold according to any of the preceding claims, wherein the attachment regions (118, 119) are spaced relative to each other in the range of 1 nm to 5000 nm, preferably in the range of 5 nm to 1000 nm, more preferably in the range of 10 nm to 500 nm.

8. The sequencing scaffold according to any one of the preceding claims, wherein the backbone (100, 102, 104, 200a, 200b) comprises a scaffold chain and a staple chain, wherein the staple chain is configured to be bonded to the scaffold chain at a predetermined position to fold the scaffold chain into a predetermined shape.

9. The sequencing scaffold of claim 8, wherein at least some of the staple strands comprise the attachment sites (122, 124, 124a, 124b).

10. The sequencing scaffold according to any one of the preceding claims, wherein the backbone (100, 102, 104, 200a, 200b) comprises a double-stranded nucleic acid chain and the attachment sites (122, 124, 124a, 124b) comprise single-stranded oligonucleotides.

11. The sequencing scaffold according to any one of the preceding claims, wherein each attachment site (122, 124, 124a, 124b) comprises a unique oligonucleotide sequence.

12. A sequencing scaffold according to any of the preceding claims, each attachment region (118, 119) comprising at least a first set of attachment sites (124a) and a second set of attachment sites (124b), wherein the first set of attachment sites (124a) is configured to hybridize with a first adapter oligonucleotide (502a) and the second set of attachment sites (124b) is configured to hybridize with a second adapter oligonucleotide (502b).

13. The sequencing scaffold of claim 12, wherein the first and second oligonucleotide adaptors (502a, 502b) are configured to be attached to the target nucleic acid strand (204).

14. The sequencing scaffold according to any one of the preceding claims, comprising a second nucleic acid backbone (100, 102, 104, 200a, 200b), and wherein the nucleic acid backbones (100, 102, 104, 200a, 200b) are connected to each other.

15. A method for sequencing a target nucleic acid strand (204), comprising the following steps: Providing a scaffold comprising at least one nucleic acid backbone (100, 102, 104, 200a, 200b) according to any one of claims 1 to 16; attaching the target nucleic acid strand (204) to an attachment site (122, 124, 124a, 124b) of the at least one nucleic acid backbone (100, 102, 104, 200a, 200b) of the scaffold; The target nucleic acid chain (204) is sequenced according to a sequencing method, which comprises at least the steps of amplifying the attached target nucleic acid chain (204) and iteratively optically reading the attachment sites (122, 124, 124a, 124b).

16. The method of claim 15, wherein the first adapter oligonucleotide (502a) and the second adapter oligonucleotide (502b) are attached to each target nucleic acid strand (204) prior to the step of attaching the target nucleic acid strand (204) to the attachment site (122, 124, 124a, 124b).

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