Image sensor structure and associated method

Abstract

The image sensor structure includes an imaging layer having an array of photodetectors disposed therein. A device stack is disposed above the imaging layer. An array of light guides is disposed within the device stack. Each light guide is associated with a photodetector. An array of nanowells is disposed above the device stack. Each nanowell is associated with a first light guide of the array of light guides. A first primer set is disposed across a first well region of each nanowell. A second primer set is disposed across a second well region of each nanowell. The second well region is adjacent to the first well region. The first and second primer sets are operable to bind forward strand clusters of forward polynucleotide strands in the first well region and reverse strand clusters of reverse polynucleotide strands in the second well region.

Description

Technical Field

[0001] (Cross - Reference to Related Applications) This application is the present application of U.S. Provisional Application No. 63 / 123,741, entitled "IMAGE SENSOR STRUCTURES AND RELATED METHODS", filed on December 10, 2020. This application claims the priority of the provisional application, and the content of the provisional application is incorporated herein by reference.

Background Art

[0002] An image sensor structure can be coupled with such a microfluidic device as a flow cell to form a sensor system. The sensor system can be, for example, a biosensor system. Such a sensor system often utilizes a high - density array of nanowells disposed on an upper layer of a passivation stack of one or more layers (referred to herein as the "passivation stack") of the image sensor structure to execute a controlled reaction protocol on polynucleotide chains disposed within the nanowells. The reaction protocol can, for example, determine the order of nucleotides within the chain.

[0003] In an example of such a reaction protocol, a polynucleotide chain (such as a cluster of DNA fragments, a nucleic acid molecular chain, etc.) disposed in the nanowell array of the image sensor structure can be tagged with an identifiable label (such as a fluorescently labeled nucleotide base) sent to the chain via the flow of fluid through the flow cell. Then, one or more excitation lights can be directed onto the labeled chain within the nanowell. Then, the labeled chain can emit photons of radiated light indicating the order of nucleotide bases within the chain, which can be sent through the passivation stack to the optical guide of the image sensor structure associated with (e.g., located directly beneath) each nanowell.

[0004] An optical guide directs synchrotron photons to a photodetector located within the image sensor structure and associated with the optical guide. The photodetector detects the emitted photons. Device circuits within the image sensor structure then process and transmit data signals using these detected photons. The data signals can then be analyzed to reveal the sequence of nucleotide bases in the chain. An example of such a sequencing process is known as synthetic sequencing.

[0005] In an example of preparing a polynucleotide chain for sequencing, a first adapter and a different second adapter are often added to the ends of the polynucleotide chain to form what is known as a DNA library. The adapters are complementary to forward and reverse primers, such as oligonucleotide fragments (oligonucleotides), and are immobilized in the nanowells of a flow cell by their 5' ends. Thus, the DNA library to be sequenced is hybridized (seed) to the forward and reverse primers and amplified on a solid support to form DNA clusters.

[0006] The forward and reverse primers contain chemical cleavage sites so that the forward or reverse strand can be cleaved and removed independently. Sequencer of the forward and reverse strands is performed in order, first by removing the reverse strand, blocking its 3' end, sequencing the forward strand, then, after the cluster is re-amplified, removing the forward strand, blocking its 3' end, and sequencing the reverse strand.

[0007] However, because the synthesis of forward and reverse chains occurs sequentially, the process is very time-consuming. Additionally, the larger the nanowell (for example, for larger clusters or multiple clusters), the more likely polyclonality is to occur (i.e., two or more types of chains are initially seeded in the nanowell and then simultaneously amplified into a polyclonal cluster). Furthermore, closer clusters become more interconnected, increasing the probability of crosstalk (i.e., light emitted from one cluster enters the optical guide of another cluster and is recorded on an unrelated photodetector).

[0008] Therefore, an image sensor structure is needed that enables a faster sequencing process than sequentially synthesizing the forward and reverse chains of the polynucleotide. Furthermore, if the faster sequencing process may involve larger nanowells, the probability of polyclonality needs to be reduced. Additionally, if the faster sequencing process may involve multiple clusters in close proximity to each other, the probability of crosstalk needs to be reduced. [Overview of the Initiative]

[0009] This disclosure provides advantages, benefits, and alternatives to the prior art by providing an image sensor structure that enables simultaneous paired-end sequencing (or reading) of adjacent forward and reversed-chain clusters. Simultaneous paired-end sequencing allows the user to sequence both the forward and reversed complementary chains of a cluster at the same time. Additionally, the image sensors of this disclosure include various structures that enable polyclonality and reduced crosstalk for adjacent forward and reversed-chain clusters.

[0010] An image sensor structure according to one or more aspects of the present disclosure includes an imaging layer. The imaging layer includes an array of photodetectors disposed inside. A device stack is disposed on top of the imaging layer. An array of optical guides is disposed within the device stack. Each optical guide is associated with a photodetector in the array of photodetectors. An array of nanowells is disposed on top of the device stack. Each nanowell in the array of nanowells is associated with a first optical guide in the array of optical guides. A first set of primers is disposed across the entire first well region of each nanowell. A different second set of primers is disposed across the entire second well region of each nanowell. The second well region is adjacent to the first well region at the region interface. The first and second sets of primers are operable to bind forward-chain clusters of forward polynucleotide chains within the first well region and adjacent reverse-chain clusters of reverse polynucleotide chains within the second well region.

[0011] In some examples of image sensor structures, each nanowell in an array of nanowells is associated with a second optical guide in an array of optical guides. The first well region is positioned on the first optical guide. The second well region is positioned on the second optical guide.

[0012] In some examples of image sensor structures, the area of ​​the first well region is smaller than the area of ​​the second well region.

[0013] In some examples of image sensor structures, the first and second well regions have substantially equal widths. The first well region has a length of 90% or less of the length of the second region.

[0014] In some examples of image sensor structures, the first well region includes a first section positioned on the entire first optical guide. The first section has a first section width. A second section extends from the first section to the region interface. The second section has a second section width smaller than the first section width. The second well region includes a third section positioned on the entire second optical guide. The third section has a third section width. A fourth section extends from the third section to the region interface. The fourth section has a fourth section width smaller than the third section width. The second section width of the first well region and the fourth section width of the second well region are substantially equal.

[0015] In some examples of image sensor structures, the first and third sections have a substantially circular shape. The widths of the first and third sections are the diameters of the first and third sections, respectively.

[0016] In some examples of image sensor structures, the widths of the second and fourth sections are 50% or less of the widths of the first and third sections, respectively.

[0017] In some examples of image sensor structures, an opaque layer is placed between the array of optical guides and the first and second well regions of each nanowell. The opaque layer extends beneath the entire region interface between the first and second well regions. The opaque layer covers less than the entire upper surface of the first and second optical guides associated with each nanowell.

[0018] In some examples of image sensor structures, the opaque layer does not cover any portion of the upper surface of the first and second optical guides associated with each nanowell.

[0019] In some examples of image sensor structures, an opaque layer covers more than 10% of the upper surface of the first and second optical guides associated with each nanowell.

[0020] In some examples of image sensor structures, a first optical guide is associated with a first photodetector in an array of photodetectors. A second optical guide is associated with a second photodetector in the array of photodetectors. Each nanowell is associated with first and second optical guides having a width smaller than the pitch between the first and second photodetectors. The first and second optical guides extend at acute angles to each other from the nanowells associated with them to the first and second photodetectors associated with them.

[0021] In some examples of image sensor structures, the first well region is positioned on a first portion of the first optical guide. The second well region is positioned on a second portion of the first optical guide.

[0022] In some examples of image sensor structures, a waveguide layer is positioned between the array of optical guides and the first and second well regions of each nanowell. The first waveguide is located within the waveguide layer and extends below the first well region. The first waveguide is operable to irradiate excitation light onto forward-chain clusters coupled within the first well region. The second waveguide is located within the waveguide layer and extends below the second well region. The second waveguide is operable to irradiate excitation light onto reverse-chain clusters coupled within the second well region.

[0023] In some examples of image sensor structures, a first waveguide can be operated to irradiate excitation light onto forward-chain clusters of forward polynucleotide chains bound within a first well region. Additionally, a second waveguide can be operated to irradiate excitation light onto reverse-chain clusters of reverse polynucleotide chains bound within a second well region.

[0024] In some examples of image sensor structures, the passivation stack is placed on top of the device stack, and the array of nanowells is placed inside the passivation stack.

[0025] In some examples of image sensor structures, the first well region of each nanowell is positioned on a first portion of the associated first optical guide. The second well region of each nanowell is positioned on a second portion of the associated first optical guide. An array of first waveguides is positioned on top of the device stack. Each first waveguide is associated with a nanowell in the array of nanowells. Each first waveguide is operable to irradiate excitation light onto a forward-chain cluster of forward polynucleotide chains bound within the first well region of the nanowell associated with the first waveguide. An array of second waveguides is positioned on top of the device stack. Each second waveguide is associated with a nanowell in the array of nanowells. Each second waveguide is operable to irradiate excitation light onto a reverse-chain cluster of reverse polynucleotide chains bound within the second well region of the nanowell associated with the second waveguide.

[0026] In some examples of image sensor structures, a waveguide layer is positioned between the array of optical guides and the first and second well regions of each nanowell. Each first waveguide in the array of waveguides is located within the waveguide layer and extends beneath the first well region of the nanowell associated with the first waveguide. Each second waveguide in the array of waveguides is located within the waveguide layer and extends beneath the second well region of the nanowell associated with the second waveguide.

[0027] In some examples of the image sensor structure, a passivation stack is disposed over the device stack, and an array of nanowells is disposed within the passivation stack. Each first waveguide of the first waveguide array is disposed within the passivation stack adjacent to a side surface of the associated nanowell of the first waveguide. Each second waveguide of the waveguide array is disposed within the passivation stack adjacent to a side surface opposite the side surface of the associated nanowell of the second waveguide.

[0028] In some examples of the image sensor structure, each first waveguide of the first waveguide array is operable to irradiate an excitation light onto a cluster of polynucleotide chains bound within a first or second well region of a nanowell adjacent to the associated nanowell of the first waveguide. Each second waveguide of the second waveguide array is operable to irradiate an excitation light onto a cluster of polynucleotide chains bound within a first or second well region of a nanowell adjacent to the associated nanowell of the second waveguide.

[0029] In some examples of the image sensor structure, the first well region and the second well region are bounded by the well walls, except for the region interface.

[0030] Another image sensor structure according to one or more aspects of the present disclosure includes an image stack disposed on a device stack. The device stack is disposed on an imaging layer. An array of optical guides is disposed within the device stack. Each optical guide is associated with a photodetector of an array of photodetectors. An array of nanowells is disposed on the device stack. Each nanowell of the array of nanowells is associated with first and second optical guides of the array of optical guides. A first primer set is disposed throughout a first well region of each nanowell. The first well region is disposed over the first optical guide. A different second primer set is disposed throughout a second well region of each nanowell. The second well region is disposed over the second optical guide and is adjacent to the first well region at a region interface. The first and second primer sets are operable to couple a forward strand cluster to the first well region and a reverse strand cluster adjacent to the second well region. The area of the first well region is smaller than the area of the second well region.

[0031] In some examples of the image sensor structure, the first well region includes a first section having a substantially circular shape disposed over the entire first optical guide. The first section has a first section diameter. A second section extends from the first section to the region interface. The second section has a second section width that is smaller than the first section diameter. The second well region includes a third section having a substantially circular shape disposed over the entire second optical guide. The third section has a third section diameter. A fourth section extends from the third section to the region interface. The fourth section has a fourth section width that is smaller than the third section diameter. The second section width of the first well region and the fourth section width of the second well region are substantially equal.

[0032] In some examples of image sensor structures, an opaque layer is placed between the array of optical guides and the first and second well regions of each nanowell. The opaque layer extends beneath the entire region interface between the first and second well regions. The opaque layer covers less than a portion of the entire upper surface of the first and second optical guides associated with each nanowell.

[0033] In some examples of image sensor structures, the opaque layer does not cover any portion of the upper surface of the first and second optical guides associated with each nanowell.

[0034] In some examples of image sensor structures, a first optical guide is associated with a first photodetector in an array of photodetectors. A second optical guide is associated with a second photodetector in the array of photodetectors. Each nanowell is associated with first and second optical guides having a width smaller than the pitch between the first and second photodetectors. The first and second optical guides extend at acute angles to each other from the nanowells associated with them to the first and second photodetectors associated with them.

[0035] Another image sensor structure according to one or more aspects of the present disclosure includes an image stack positioned on top of a device stack. The device stack is positioned on top of the imaging layer. An array of optical guides is positioned within the device stack. Each optical guide is associated with a photodetector in an array of photodetectors. An array of nanowells is positioned on top of the device stack. Each nanowell in the array of nanowells is associated with a first optical guide in the array of optical guides. A first set of primers is positioned across the entire first well region of each nanowell. The first well region is positioned above a first portion of the first optical guide. A different second set of primers is positioned across the entire second well region of each nanowell. The second well region is positioned above a second portion of the first optical guide. The second well region is adjacent to the first well region at its region interface. The first and second sets of primers are operable to couple forward-chain clusters in the first well region and reverse-chain clusters adjacent to the second well region.

[0036] In some examples of image sensor structures, a waveguide layer is positioned between the array of optical guides and the first and second well regions of each nanowell. The first waveguide is located within the waveguide layer and extends below the first well region. The first waveguide is operable to irradiate excitation light onto forward-chain clusters coupled within the first well region. The second waveguide is located within the waveguide layer and extends below the second well region. The second waveguide is operable to irradiate excitation light onto reverse-chain clusters coupled within the second well region.

[0037] In some examples of image sensor structures, a first waveguide array is positioned on top of the device stack. Each first waveguide is associated with a nanowell in the array of nanowells. Each first waveguide is operable to irradiate excitation light onto a forward-chain cluster of forward polynucleotides bound within the first well region of the nanowell associated with the first waveguide. A second waveguide array is positioned on top of the device stack. Each second waveguide is associated with a nanowell in the array of nanowells. Each second waveguide is operable to irradiate excitation light onto a reverse-chain cluster of reverse polynucleotides bound within the second well region of the nanowell associated with the second waveguide.

[0038] A method for simultaneous paired-end sequencing according to one or more aspects of the present disclosure includes seeding a first primer set with a polynucleotide chain into a first well region of a nanowell of an image sensor structure. A second primer set in a second well region of the nanowell is inactivated to prevent seeding of other polynucleotide chains in the second well region. The first polynucleotide chain is amplified into multiple forward and reverse chains throughout the first well region. The reverse chain is cleaved from the first well region to form a forward chain cluster in the first well region. A second primer set in the second well region is activated to enable seeding and amplification in the second well region. The forward chain cluster in the first well region is amplified into the second well region to form multiple forward and reverse chains in the second well region. The forward chain is cleaved from the second well region to form a reverse chain cluster in the second well region. The forward chain cluster in the first well region and the reverse chain cluster in the second well region are sequenced substantially simultaneously.

[0039] In some examples of this method, inactivating the second primer set includes masking the second well region, and activating the second primer set includes unmasking the second well region.

[0040] In some examples of the method, a first complementary nucleotide with a first fluorescent tag is bound to a nucleotide in a forward-chain cluster within a first well region. A second complementary nucleotide with a second fluorescent tag is bound to a nucleotide in a reverse-chain cluster within a second well region. Excitation light is emitted substantially simultaneously onto the forward-chain and reverse-chain clusters, causing the first and second tags to fluoresce. The light from the first tag is directed to a first photodetector through a first optical guide, and the light from the second tag is directed to a second photodetector through a second optical guide, determining the sequences of the forward-chain and reverse-chain nucleotides, respectively.

[0041] In some examples of the method, the first well region has a smaller area than the second well region.

[0042] In some examples of the method, the first well region includes first and second sections. The first section is positioned over the entire first optical guide. The first section has a first section width. The second section extends from the first section to the region interface between the first and second well regions. The second section has a second section width smaller than the first section width. The second well region includes third and fourth sections. The third section is positioned over the entire second optical guide. The third section has a third section width. The fourth section extends from the third section to the region interface. The fourth section has a fourth section width smaller than the third section width.

[0043] In some examples of this method, opaque layers are placed between the first and second optical guides, and between the first and second well regions. The opaque layer extends beneath the entire region interface between the first and second well regions. The opaque layer covers less than the entirety of the first and second optical guides.

[0044] In some examples of this method, the nanowells have a width smaller than the pitch between the first and second photodetectors. The first and second optical guides extend at acute angles to each other to the first and second photodetectors associated with them.

[0045] In some examples of this method, the first well region is positioned on a first portion of the first optical guide. The second well region is positioned on a second portion of the first optical guide.

[0046] In some examples of this method, a first complementary nucleotide with a first fluorescent tag is bound to a nucleotide in the forward-chain cluster. A second complementary nucleotide with a second fluorescent tag is bound to a nucleotide in the reverse-chain cluster. Substantially larger excitation light is emitted onto the forward-chain cluster than onto the reverse-chain cluster, causing substantially larger synchrotron radiation to fluoresce from the first tag than onto the second tag. The synchrotron radiation is delivered from the first tag through a first optical guide to a first photodetector to determine the nucleotides in the forward-chain. Substantially larger amounts of excitation light are emitted onto the reverse-chain cluster than onto the forward-chain cluster, causing substantially larger amounts of synchrotron radiation to fluoresce from the second tag than onto the first tag. The synchrotron radiation is delivered from the second tag through a first optical guide to a first photodetector to determine the nucleotides in the reverse-chain.

[0047] In some examples of this method, a waveguide layer is positioned between a first optical guide and first and second well regions. The first waveguide is positioned in the waveguide layer and extends below the first well region but not below the second well region. The second waveguide is positioned in the waveguide layer and extends below the second well region but not below the first well region. Excitation light is radiated onto the forward-chain cluster through the first waveguide. Excitation light is radiated onto the reverse-chain cluster through the second waveguide.

[0048] In some examples of this method, a first waveguide is positioned on a first optical guide. A second waveguide is positioned on the first optical guide. Excitation light is emitted onto the forward-chain cluster through the first waveguide. Excitation light is emitted onto the reverse-chain cluster through the second waveguide.

[0049] In some examples of this method, a first complementary nucleotide with a first fluorescent tag is bound to a nucleotide in the forward-chain cluster. A second complementary nucleotide with a second fluorescent tag is bound to a nucleotide in the reverse-chain cluster. Excitation light is emitted substantially simultaneously on the forward-chain cluster in the first well region and on the reverse-chain cluster in the second well region, causing synchrotron radiation to fluoresce from the first and second tags. The combined synchrotron radiation from the first and second tags is delivered to a first photodetector through a first optical guide. Signal processing techniques are used to determine the forward and reverse-chain nucleotides associated with the combined synchrotron radiation detected by the first photodetector.

[0050] In some examples of this method, sequencing substantially simultaneously involves receiving synchrotron radiation from a first tag in the forward chain to a first photodetector and from a second tag in the reverse chain to a second photodetector, and determining the sequences of the nucleotides in the forward and reverse chains, respectively.

[0051] In some examples of this method, sequencing involves substantially simultaneously receiving synchrotron radiation from a first tag in the forward chain to a first photodetector and from a second tag in the reverse chain to a first photodetector, thereby determining the sequences of the nucleotides in the forward and reverse chains, respectively.

[0052] In some examples of this method, the synchrotron radiation received by the first photodetector from the forward-sequenced first tag is received substantially simultaneously with the synchrotron radiation received by the first photodetector from the reverse-sequenced second tag.

[0053] In some examples of this method, the synchrotron radiation received by the first photodetector from the forward-sequenced first tag is received before the synchrotron radiation from the reverse-sequenced second tag is received by the first photodetector.

[0054] It should be understood that all combinations of the aforementioned concepts and further concepts, which are described in more detail below (on the premise that such concepts are not contradictory), are considered to be part of the subject matter disclosed herein and / or can be combined to achieve specific interests in a particular aspect. Specifically, all combinations of the claimed subject matter appearing at the end of this disclosure are considered to be part of the subject matter disclosed herein. [Brief explanation of the drawing]

[0055] This disclosure will be better understood by reading the following detailed description in conjunction with the attached drawings. [Figure 1] An example of a set of primer molecules immobilized in the well region of an image sensor structure according to the embodiments described herein is shown. [Figure 2] Examples of polynucleotide chains that can be introduced into the well region of an image sensor structure according to the embodiments described herein are shown. [Figure 3] An example of a cluster of polynucleotide chains bound in a well region as a forward chain, according to the embodiments described herein, is shown. [Figure 4] An example of a cluster of polynucleotide chains bound to the well region as a reverse chain, according to the embodiments described herein, is shown. [Figure 5] An example of a forward chain sequenced to determine the order of its nucleotides, according to the embodiments described herein, is shown. [Figure 6] Examples of adjacent forward-chain and reverse-chain clusters arranged on nanowells according to the embodiments described herein are shown. [Figure 7A] An example of a cross-sectional side view of a sensor system having a flow cell coupled to an image sensor structure, according to an embodiment described herein, is shown. [Figure 7B] An example of a top view of the image sensor structure in Figure 7A along the line 7B-7B in Figure 7A, according to the embodiments described herein, is shown. [Figure 8]An example of a top view of a first well region and an adjacent second well region within a nanowell of an image sensor structure according to an embodiment described herein is shown, wherein the first and second well regions have substantially equal areas. [Figure 9] Another example of a top view of a first well region and an adjacent second well region within a nanowell of an image sensor structure according to the embodiments described herein is shown, wherein the first and second well regions have substantially unequal areas. [Figure 10] Another example of a top view of a first well region and an adjacent second well region within a nanowell of an image sensor structure according to the embodiments described herein is shown, wherein the first and second well regions generally have a dogbone shape. [Figure 11] An example of a graph of the contrast ratio (between a first well region having forward-chain clusters and a second well region having reverse-chain clusters) versus the distance from the optical guide center associated with the first well region, according to the embodiments described herein, is shown. [Figure 12] An example of a cross-sectional view of an image sensor structure having an opaque layer positioned between an array of optical guides and the first and second well regions of a nanowell, according to an embodiment described herein, is shown. [Figure 13A] An example of a top perspective view of the opaque layer of the image sensor structure shown in Figure 12, according to an embodiment described herein, is shown. [Figure 13B] Another example of a top perspective view of the opaque layer of the image sensor structure in Figure 12, according to the embodiments described herein, is shown. [Figure 13C] Another example of a top perspective view of the opaque layer of the image sensor structure in Figure 12, according to the embodiments described herein, is shown. [Figure 14A] An example of a graph of contrast ratio versus distance from the optical guide center associated with the first well region, according to the embodiments described herein, is shown, where the opaque layer is not located between the first and second well regions and the optical guide array. [Figure 14B]An example of a graph of contrast ratio versus distance from the optical guide center associated with the first well region, according to the embodiments described herein, is shown, where the opaque layer is positioned between the first and second well regions and the optical guide array. [Figure 15] This figure shows an example of an image sensor structure having first and second well regions arranged on first and second optical guides according to an embodiment described herein, wherein the optical guides form acute angles with respect to each other. [Figure 16] An example of an image sensor structure having nanowells comprising first and second well regions positioned on first and second portions of an optical guide, according to the embodiments described herein, wherein the first and second waveguides are each positioned within a waveguide layer and each extends below the first and second well regions. [Figure 17] An example of an image sensor structure having nanowells having first and second well regions arranged on first and second portions of an optical guide, according to the embodiments described herein, wherein the first and second waveguides are arranged adjacent to and on both sides of the nanowells within a passivation stack. [Figure 18] An example of a cross-sectional view of an image sensor structure having the nanowell and optical guide array shown in Figure 17, according to an embodiment described herein, is shown. [Figure 19] An example of a cross-sectional view of an image sensor structure having first and second well regions positioned on first and second portions of a shared optical guide, according to an embodiment described herein, is shown. [Figure 20A] An example of a scatter plot of bright clusters according to the embodiments described herein is shown. [Figure 20B] An example of a scatter plot of dark clusters according to the embodiments described herein is shown. [Figure 21] An example of a scatter plot showing 16 distributions formed by intensity values ​​from bright and dark clusters, according to the embodiments described herein, is shown. [Figure 22] An example of a flowchart for a simultaneous paired-end sequence determination method according to the embodiments described herein is shown. [Figure 23]An example of another flowchart for a simultaneous paired-end sequencing method according to the embodiments described herein is shown. [Figure 24] An example of another flowchart for a simultaneous paired-end sequencing method according to the embodiments described herein is shown. [Figure 25] An example of another flowchart for a simultaneous paired-end sequencing method according to the embodiments described herein is shown. [Modes for carrying out the invention]

[0056] Herein, specific embodiments are described in order to provide an overall understanding of the structure, function, manufacturing and use principles of the methods, systems, and devices disclosed herein. One or more embodiments are shown in the accompanying drawings. Those skilled in the art will understand that the methods, systems, and devices specifically described herein and illustrated in the accompanying drawings are non-limiting examples, and the scope of this disclosure is defined solely by the claims. Features illustrated or described in relation to one embodiment may be combined with features of other embodiments. Such modifications and variations are intended to be included within the scope of this disclosure.

[0057] The terms “substantially,” “approximately,” “about,” “relatively,” or other similar terms, which may be used throughout this disclosure including the claims, are used to describe and take into account small variations from a standard or parameter, such as variations in the process. Such small variations include zero-point variations from the standard or parameter. For example, they may refer to ±10%, such as ±5%, ±2%, ±1%, ±0.5%, ±0.2%, ±0.1%, ±0.05%, etc.

[0058] Referring to Figure 1, an example of a set of primer molecules (i.e., a primer set) 102 immobilized (e.g., via a chemical anchor 103) in the well region 104 of an image sensor structure 100 is shown according to the embodiments described herein. The primer set 102 may include at least two types of oligonucleotides (i.e., oligos) 106, 108 that can activate the well region 104 of the image sensor structure 100 to immobilize a polynucleotide chain 110 (see Figure 2) to the image sensor structure 100. Oligos 106 and 108 may be referred to herein as forward oligos and reverse oligos or primers, respectively.

[0059] Referring to Figure 2, an example of a polynucleotide chain 110 that can be introduced into the well region 104 of the image sensor structure 100 via a flow cell 112 (see Figure 7A) according to the embodiments described herein is shown. The polynucleotide chain 110 may include a polynucleotide fragment (such as a DNA or RNA fragment) 114 of an unknown sequence of nucleotide 116.

[0060] The polynucleotide chain 110 may also include a pair of forward-end adapters 118 and reverse-end adapters 120. The pair of adapters 118, 120 may be complementary to oligos 106, 108.

[0061] Referring to Figure 3, an example of a forward chain cluster 121 of polynucleotide chains 110 bound to well region 104 as a forward chain 122 is shown according to the embodiment described herein. When polynucleotide chains 110 are bound as a forward chain 122, the forward oligo 106 can replace the forward end adapter 118 to fix the forward chain 122 to the floor of well region 104. The reverse end adapter 120 remains attached to the free end of the forward chain 122.

[0062] Referring to Figure 4, an example of a reverse chain cluster 123 of a polynucleotide chain 110 bound to a well region 104 as the reverse chain 124 is shown according to the embodiment described herein. When the polynucleotide chain 110 is bound as the reverse chain 124, the reverse oligo 108 replaces the reverse end adapter 120 to fix the chain 110 to the floor of the well region 104. The forward end adapter 118 remains attached to the free end of the reverse chain 124.

[0063] Referring to Figure 5, an example of a forward chain 122 sequenced to determine the order of nucleotides 116 according to the embodiments described herein is shown. Each nucleotide 116 of the forward chain 122 of a specific type of nitrogenous base (e.g., 116-1 to 116-9 and above) associated with it is identified by a circled letter A, G, C, or T, which represents the base type of nucleotide 116: adenine, guanine, cytosine, and thymine, respectively.

[0064] To identify the sequence of bases of nucleotide 116 in the forward chain 122, multiple second nucleotides 126 (e.g., 126-1 to 126-6 and beyond) having bases equipped with a fluorescent tag 128 are introduced via a flow cell 112 (see Figure 7A). The bases (A, G, C, T) of nucleotide 126 compete for addition to the growing second polynucleotide 130, which is the complement of the forward chain 122. When the bases of the second nucleotide 126 (e.g., nucleotide 126-6 with base C and fluorescent tag 128) are each added to the growing second polynucleotide 130 chain, the fluorescent tag 128 is excited by excitation light. The unique signal of the fluorescent tag 128 identifies the base type of the second nucleotide 126. Once the base is identified, the fluorescent tag 128 is removed. Identifying each second nucleotide 126 of the second polynucleotide 130 also identifies the complementary nucleotide 116 associated with it in the forward chain 122. This process of identifying a base is referred to herein as "base calling."

[0065] However, in each cycle of this sequencing process, the degree of uncertainty for each base call increases as the length of the second polynucleotide chain 130 increases. At some point, the degree of uncertainty becomes excessively large and continues. Therefore, in embodiments where each end of the chain is sequenced sequentially (i.e., not in parallel or simultaneously), the forward chain 122 is washed away, and the reverse chain 124 is fixed in the well region 104 in place of the forward chain 122. The sequencing process is then continued with the reverse chain 124 in the same manner.

[0066] Referring to Figure 6, an example of adjacent clusters 121 and 123 of the forward chain 122 and a reverse chain 124, each placed on a nanowell 136, is shown according to the embodiments described herein. In this example, the forward and reverse chains 122 and 124 are placed within a first well region 132 and an adjacent second well region 134 of the nanowell 136.

[0067] The first and second well regions 132 and 134 are separated by a region interface 138. In one example, the region interface 138 is the boundary line between two directly adjacent well regions 132 and 134. In another example, the region interface 138 is small enough to span the region interface 138 from one well region 132 to the other well region 134.

[0068] A first primer set 140 is placed throughout the first well region 132. A second, different primer set 142 is placed throughout the second well region 134. The difference between the first and second primer sets 140, 142 allows the primer sets to bind forward-chain clusters 121 in the first well region 132 and reverse-chain clusters 123 in the second well region 134. Detailed examples of the differences in primer sets 140, 142 and chemicals used to control the generation of forward-chain and reverse-chain clusters 122, 124 in adjacent first and second well regions 132, 134, respectively, are provided in International Patent Application No. PCT / US2019 / 036105, International Publication No. 2020 / 005503, entitled “FLOW CELLS,” by Fisher et al., assigned to Illumina, Inc. and incorporated herein by reference on International Filing Date June 6, 2019.

[0069] In an example of the difference between the first primer set 140 and the second primer set 142, the first primer set 140 may include a first primer that is not cleavable and a second primer that is cleavable. On the other hand, the second primer set may include a first primer that is cleavable and a second primer that is not cleavable.

[0070] In a more specific example, the first primer set 140 may include a first forward oligonucleotide primer 106 having a length of approximately 20 nucleotides with specific sequences of adenosine, guanosine, cytidine, and thymidine, and a first reverse oligonucleotide primer 108 having a length of approximately 20 nucleotides with different sequences of adenosine, guanosine, cytidine, thymidine, and one or more deoxyuridines. The second primer set 142 may include a second forward oligonucleotide primer 106, which includes a second reverse oligonucleotide primer 108 having a length of approximately 20 nucleotides with specific sequences of adenosine, guanosine, cytidine, thymidine, and one or more deoxyuridines, and a second reverse oligonucleotide primer 108 having a length of approximately 20 nucleotides with different sequences of adenosine, guanosine, cytidine, and thymidine. The N-glycosidic bond at the deoxyuridine site is enzymatically cleavable. The sequences of the first forward primer 106 in the first primer set 140 and the second forward primer 106 in the second primer set 142 are identical except for the substitution of thymidine with cleavable deoxyuridine. The sequences of the first reverse primer 108 in the first primer set 140 and the second reverse primer 108 in the second primer set 142 are identical except for the substitution of thymidine with cleavable deoxyuridine. Examples of chemically cleavable nucleic acid bases, modified nucleic acid bases, or linkers include vicinal diols, disulfides, silanes, azobenzenes, photocleavable groups, allyl T (thymine nucleotide analogs having an allyl functional group), allyl ethers, or azide-functionalized ethers.

[0071] During operation, the first primer set 140 in the first well region 132 is activated, allowing polynucleotide chains 110 to be seeded into the first primer set 140 in position 122. That is, the primer set 140 can hybridize with one or more polynucleotide chains 110. The second primer set 142 is inactivated (for example, by masking the second well region 134 or by not having seeding primers, etc.) so that seeding does not occur in the second well region 134.

[0072] Next, the seeded forward chain 122 is amplified throughout the first primer set 140, forming forward chain clusters 121 and reverse chain 124 within the first well region 132. Then, the reverse primer 108 is enzymatically cleaved, leaving only the forward chain 122 hybridized (bound) to the forward primer 106 within the first well region 132.

[0073] For the purposes of this specification, the term “activation” means “means or methods that enable seeding of a primer set with a polynucleotide chain into a well region (or alternatively, enabling 3' extension or amplification).” The term “inactivation” means the opposite, in that it means “means or methods that prevent seeding of a primer set with a polynucleotide chain (or alternatively, preventing 3' extension or amplification).” Inactivating a primer set can be achieved, for example, by masking the primer set so that a polynucleotide cannot be seeded into it. Alternatively, a primer set can be inactivated by not including seeding primers in the primer set. Activating a primer set can be achieved, for example, by unmasking the primer set to expose a primer set that is set on a polynucleotide chain that could potentially be seeded into a well region.

[0074] Furthermore, for the purposes of this specification, the term “masking” means or method of placing a temporary protective layer over a primer set in a well region to physically prevent access to the primer set by the polynucleotide chain. The term “unmasking” means “means or method of removing the protective layer to expose the primer set on the polynucleotide chain for the purpose of seeding.”

[0075] Furthermore, it should be understood that amplification and / or sequencing of polynucleotide chains (e.g., forward or reverse chains) may not necessarily produce exact copies of the chain or exact copies of the reverse complement of the chain. This is because errors can be introduced into the amplification and / or sequencing process due to various factors, which can introduce defects (e.g., incorrect bases) into the polynucleotide sequence of bases. For example, up to one defect per million, ten defects per million, or one defect per million may be introduced into the sequenced or amplified chain. Thus, a cluster of forward or reverse chains 121, 123 may not contain exact copies of each chain within the cluster, but may contain substantially the same overlap as each chain within the cluster.

[0076] Once the amplification of the forward chain 122 is substantially complete within the first well region 132, the second primer set 142 is then activated (by unmasking the second well region 134, etc.) to allow seeding of the second well region 134. For example, any protective layer on the second primer set 142 can be removed (unmasked) to expose both the second forward primer 106 and the second reverse primer 108 within the second primer set 142.

[0077] Because the first and second well regions 132 and 134 are adjacent, the forward chain 122 at the boundary or region interface 138 can then arch into the second well region 134 and bind to the second primer set 142. The second primer set 142 then amplifies the reverse chain cluster 123 of the reverse chain 124 throughout the second well region 134 via bridge amplification. That is, the cluster reaction can then be performed to form clusters of the forward chain 122 and reverse chain 124 hybridized to the second forward primer 106 and the second reverse primer 108 in the second well region 134. The second forward primer 106 is then enzymatically cleaved, leaving only the reverse chain 124 hybridized (bound) to the second reverse primer 108 in the second well region 134. The results, as shown in Figure 6, are a forward-chain cluster 121 in the first well region 132 and a reverse-chain cluster 123 in the second well region 134.

[0078] Advantageously, by providing adjacent clusters 121 and 123 of the forward 122 and reverse 124, respectively, simultaneous paired-end sequencing (or reading) of adjacent forward clusters 121 and reverse clusters 123 becomes possible. Simultaneous paired-end sequencing allows the user to sequence both types of polynucleotide chains 110, forward 122 and reverse 124, in parallel and simultaneously, rather than sequentially. This can significantly increase the throughput of the sequencing process described herein. Additionally, or alternatively, this can significantly reduce errors in the sequencing process.

[0079] Referring to Figure 7A, an example of a cross-sectional side view of a sensor system 200 having a flow cell 112 coupled to an image sensor structure 100 according to an embodiment described herein is shown. The image sensor structure 100 is operable to perform simultaneous paired-end sequence determination of adjacent forward-chain clusters 121 of forward-chain 122 and reverse-chain clusters 123 of reverse-chain 124, as shown in Figure 6.

[0080] The flow cell 112 of the sensor system 200 includes a flow cell cover 150 fixed to the flow cell side wall 152. The flow cell side wall 152 is joined to the passivation stack 156 of the image sensor structure 100, and a flow channel 158 can be formed between them.

[0081] The passivation stack 156 includes an array of nanowells 136 arranged thereon. Polynucleotide chains 110 (such as DNA segments, oligonucleotides, or other nucleic acid chains) can be arranged within the nanowells 136 as both forward-chain clusters 121 of forward-chain 122 and reverse-chain clusters 123 of reverse-chain 124.

[0082] The flow cell cover 150 includes an inlet port 160 and an outlet port 162 sized to allow a fluid flow 164 to pass through and exit the channel 158. The fluid flow 164 can be used to execute a number of different controlled reaction protocols on forward and reverse chains 122, 124 placed in the nanowell 136. The fluid flow 164 can also deliver nucleotides 126 having a fluorescent tag 128 (see Figure 5) which can be used to tag the polynucleotide chains 122, 124.

[0083] The image sensor structure 100 of the sensor system 200 includes an imaging layer 168 disposed on a base substrate 170. The imaging layer 168 may be a dielectric layer such as SiN and may include an array of photodetectors 172 disposed inside. The photodetectors 172 used herein may be semiconductors such as photodiodes, complementary metal oxide semiconductor (CMOS) materials, or both. The photodetectors 172 detect photons of synchrotron radiation 174 emitted from fluorescent tags 128 coupled to chains 122, 124 in the nanowells 136. The base substrate 170 may be glass, silicon, or other similar materials.

[0084] The device stack 176 is positioned on top of the imaging layer 168. The device stack 176 may include a plurality of dielectric layers (not shown) that interface the photodetector 172 with process data signals using the detected photons.

[0085] Furthermore, an array of optical guides 180 is arranged within the device stack 176. Each optical guide 180 is associated with at least one photodetector 172 of the array of photodetectors. For example, an optical guide 180 can be oriented perpendicular to the imaging layer 168 and placed directly on its associated photodetector 172. Alternatively, an optical guide 180 can form an acute angle with respect to the imaging layer 168 and approach its associated photodetector 172 from a certain angle. The optical guides 180 direct photons of synchrotron radiation 174 from the forward and reverse-chained fluorescent tags 128 122, 124, arranged within the nanowell 136, to their associated photodetectors 172.

[0086] The passivation stack 156 is configured to shield the device stack 176 and the optical guide 180 from the fluid environment of the flow cell 112. The passivation stack 156 may consist of one or more layers. In the example shown in Figure 7A, the passivation stack includes a single passivation layer 182. However, other layers, such as a chemical protective layer (not shown), may also be included in the passivation stack 156. The passivation layer 182 may consist of silicon nitride (SiN). The chemical protective layer (not shown) may consist of tantalum pentoxide (Ta2O5).

[0087] The array of nanowells 136 is placed within a passivation stack 156, and each nanowell 136 in the array of nanowells is associated with at least one optical guide (first optical guide) 180A of the array of optical guides 180. In the example shown in Figure 7A, each nanowell 136 is associated with the first optical guide 180A and the second optical guide 180B.

[0088] As shown in Figure 6, the first primer set 140 is placed across the entire first well region 132 of each nanowell 136. A different second primer set 142 is placed across the entire second well region 134 of each nanowell 136. The second well region 134 is adjacent to the first well region 132 at the region interface 138. The first and second primer sets 140 and 142 are operable to bind forward-chain clusters 121 of forward polynucleotide chains 122 within the first well region 132 and to bind adjacent reverse-chain clusters 123 of reverse polynucleotide chains 124 within the second well region 134.

[0089] The first and second well regions 132 and 134 can be positioned on a portion of a single optical guide 180A. However, in the example shown in Figure 7A, the first well region 132 is positioned on the first optical guide 180A, and the second well region 134 is positioned on the second optical guide 180A.

[0090] During operation, various types of excitation light 186 are emitted onto the forward and reverse chains 122 and 124 within the nanowell 136, causing the fluorescent tag 128 to fluoresce with synchrotron radiation 174. Most of the photons of synchrotron radiation 174 are sent out through the passivation stack 156 and can be incident on associated optical guides 180A and 180B. The optical guides 180 filter out most of the excitation light 186 and direct the synchrotron radiation 174 to associated photodetectors 172 located directly below the optical guides 180.

[0091] The embodiments described herein show excitation light 186 emitted from the front of the image sensor structure 100 to excite the fluorescent tag 128 and the fluorescence emission light 174. However, within the scope of this disclosure, the excitation light 186 can be emitted from the back of the image sensor structure (i.e., back illumination) to excite the fluorescent tag 128 and the fluorescence emission light 174.

[0092] The photodetector 172 detects synchrotron radiation photons. The device circuit 178 within the device stack 176 then converts the detected photons into a data signal, which is electrically transmitted to an external readout device. The data signal can then be analyzed to simultaneously determine the order of both the forward and reverse strands of nucleotides 122 and 124.

[0093] Referring to Figure 7B, an example of a top view of the image sensor structure of Figure 7A, cut along line 7B-7B of Figure 7A, is shown. In the example of Figure 7B, the first and second well regions 132, 134 are substantially equal in area and directly adjacent to each other in well region 138. The well regions are rectangular in shape because the floor 185 of the nanowell 136 is rectangular. Each well region 132, 134 is substantially centered on its associated optical guides 180A and 180B.

[0094] Except for the region interface 138, the first well region 132 can be bounded by the wall 181 of the nanowell 136. Also, except for the interface region 138, the second well region 134 can be bounded by the wall 183 of the nanowell 136. Essentially, throughout the entire image sensor structure 100, multiple pairs of first and second well regions 132, 134 can be defined by the walls 181, 183 of the nanowell 136.

[0095] However, since the nanowell 136 spans two optical guides 180A and 180B, the risk of polyclonality in the two-well region is greater compared to a nanowell that spans only one optical guide. In other words, two or more types of polynucleotide chains 110 (see Figure 2) that enter the nanowell 136 simultaneously are seeded and amplified together, which increases the risk of a significant increase in the signal-to-noise ratio.

[0096] Additionally, the region interface 138 extends across the entire maximum width of the first and second well regions 132 and 134 (represented by arrow 188). This increases the risk of crosstalk from the reverse chain 124 entering the optical guide 180A (particularly the reverse chain at the large boundary / region interface 138), contaminating the signal from the forward chain 122.

[0097] Referring to Figure 8, an example of a top view of a first well region 132 and an adjacent second well region 134 within a nanowell 136 of an image sensor structure 100 according to the embodiments described herein is shown, where the first and second well regions 134, 136 have substantially equal areas. Figure 8 is essentially an enlarged view of the first and second well regions 132, 134 of Figure 7B. The rectangular region of well region 132 extends beyond the circular boundary of the optical guide 180A. Much of the area beyond the circular boundary of the optical guide 180A may not be required for the strong signal from the forward chain 122 amplified in well region 132.

[0098] Referring to Figure 9, another example is a top view of a first well region 132 and an adjacent second well region 134 within a nanowell 136 of an image sensor structure 100 according to an embodiment described herein, wherein the first and second well regions have substantially unequal areas. In Figure 9, the area of ​​the first well region 132 is smaller than the area of ​​the second well region 134. Since the rectangular regions of the first and second well regions 132 and 134 have substantially equal widths 188, the area can be changed by changing the length of the first well region 132 (indicated by arrow 190) and the length of the second well region 134 (indicated by arrow 192). In this case, the length 190 of region 132 is shorter than the length 192 of region 134, so the area of ​​region 132 is smaller than the area of ​​region 134.

[0099] For example, the first and second well regions 132 and 134 may have substantially equal widths 188, and the first well region 132 may have a length 190 which is less than or equal to 90% of the length 192 of the second region 134. In this example, it should be noted that the length 190 is not so small that the first well region does not completely cover the associated optical guide 180A. Fully positioning the forward chain 122 associated with the first well region 132 over the optical guide 180A helps the photodetector 172 generate a strong signal even if the area of ​​the well region 132 is reduced.

[0100] Reducing the area of ​​the first well region 132 lowers the risk of undesirable polyclonality. Furthermore, the second well region is not activated after the forward chain 122 has been fully amplified, and polynucleotides do not flow through the channel 158. Therefore, once the reverse chain 124 begins to amplify within the second well region 134, polyclonality is unlikely to carry over from the first well region to the second well region.

[0101] Refer to Figure 10. Another example is a top view of a first well region 132 and an adjacent second well region 134 within a nanowell 136 of an image sensor structure 100 according to an embodiment described herein, wherein the first and second well regions 132, 134 generally have a dogbone shape. The dogbone shape reduces the area of ​​the well regions 132, 134, which helps reduce polyclonality, but provides physical continuity between the two well regions 132, 134 to allow cluster growth between them. Additionally, the smaller the width 194 of the dogbone in the interface region 138, the smaller the crosstalk that may occur. This is because the walls of the nanowell 136 on either side of the neck of the dogbone may act to help block crosstalk from larger circular regions.

[0102] In the dogbone configuration, the first well region 132 includes a first section 196 and a second section 198. The first section 196 is positioned over the entire first optical guide 180A. This helps to ensure a strong signal in the optical guide 180A.

[0103] The first section 196 has a first section width 201. Note that the first section 196 does not have to be circular. Rather, the first section may be square, rectangular, or any other suitable shape.

[0104] The second section 198 extends from the first section 196 to the region interface 138. The second section 198 has a second section width 194 that is smaller than the first section width 201.

[0105] The second well region 134 includes the third section 202 and the fourth section 204. The third section 202 is positioned above the entire second optical guide 180B. This helps to ensure a strong signal in the optical guide 180B.

[0106] The third section 202 has a third section width 206. Note that the third section 202 does not have to be circular. Rather, the third section may be square, rectangular, or any other suitable shape.

[0107] The fourth section 204 extends from the first section 202 to the region interface 138. The fourth section 204 has a fourth section width 194 that is smaller than the third section width 206. Note that in this example, the width 194 of the second section 198 of the first well region 132 and the width 194 of the fourth section 204 of the second well region 134 are substantially equal. Also in this example, the lengths of the second section 198 of the first well region 132 and the fourth section 204 of the second well region 134 are substantially equal. In other examples, the lengths of the second section 198 of the first well region 132 and the fourth section 204 of the second well region 134 are not substantially equal.

[0108] In some examples, the first section 196 and the third section 202 have a substantially circular shape. In that case, the widths 201 and 206 of the first and third sections are the diameters of the first and third sections 196 and 202, respectively. In some examples, the widths 194 of the second and fourth sections are less than 50% of the widths 201 and 206 of the first and third sections, respectively.

[0109] Referring to Figure 11, an example of a graph 300 of the contrast ratio between the first well region 132 having forward-chain clusters 121 and the second well region 134 having reverse-chain clusters 123, and the distance from the optical guide center 302 associated with the first well region 132, according to the embodiments described herein. The contrast ratio is defined as follows: a. Contrast ratio = signal received from region 132 by the first optical guide 180A i. The brightest signal A divided by the signal received by the first optical guide 180A from the adjacent region 134 (i.e., the second brightest signal B). If bA = brightest signal and B = second brightest signal, then the ratio of contrast i can be described as contrast ratio = A / B. c. The contrast ratio is closely related to the purity score, which can be defined as purity = A / (A+B). d. Therefore, if the contrast ratio is 5:1, the purity score is equal to 5 / (5+1)=83%.

[0110] Both the contrast ratio and purity score relate to the overall purity of the fluorescence signal at the base of the target area. This is the area that is targeted for measurement.

[0111] Graph 300 shows that as the first well region 132 becomes longer and larger in area (i.e., grows further away from the center of the associated optical guide 180A), the contrast ratio and purity score decrease. Therefore, it appears that the smaller the area of ​​the well region 132, the purer the signal and the less crosstalk there is.

[0112] However, this must be balanced by the fact that as the area of ​​well region 132 becomes smaller, the amplification of the signal from that region decreases, while the signal from the adjacent well region 134 is amplified. Therefore, if a well region is too small, it may be overwhelmed by the brightness of the adjacent well region.

[0113] Referring to Figure 12, an example of a cross-sectional view of an image sensor structure 100 having an opaque layer 400 positioned between an array of optical guides 180 and the first and second well regions 132, 134 of the nanowells 136, according to the embodiments described herein, is shown. Figure 12 is substantially the same as Figure 7, except that an opaque layer 400 positioned between the passivation stack 156 and the optical guides 180 is added. In some examples, the opaque layer may be made of tantalum, chromium, titanium, aluminum, etc. As used herein, the term “opaque” means blocking all or substantially all light or radiant energy of one or more wavelength ranges or all wavelengths from passing through it.

[0114] Referring to Figures 13A, 13B, and 13C, a top perspective view of the opaque layer 400 of the image sensor structure of Figure 12, according to an embodiment described herein, is shown. In each of the three configurations of the opaque layer shown in Figures 13A to 13C, the opaque layer 400 is positioned between the array of optical guides 180 of each nanowell 136 and the first and second well regions 132, 134. Furthermore, each configuration of the opaque layer 400 extends beneath the entire region interface 138 of the first and second well regions 132, 134. In addition, each opaque layer 400 covers less than the entire top surface of the first and second optical guides 180A, 180B associated with each nanowell 136.

[0115] Since the first and second well regions 132 and 134 are adjacent to the region interface 138, most of the crosstalk occurs in that area. Therefore, the opaque layer 400 is placed over the entire interface region 138 to reduce such crosstalk.

[0116] Specifically, in Figure 13A, the opaque layer 400 does not cover any portion of the upper surface of the first and second optical guides 180A and 180B associated with each nanowell 136. Rather, the opaque layer 400 follows the outer periphery of each of the first and second optical guides 180A and 180B.

[0117] Specifically, in Figure 13B, the opaque layer 400 covers a portion of the upper surface of the first and second optical guides 180A and 180B. For example, the opaque layer 400 can cover more than 10%, more than 15%, or more than 25% of the upper surface of the first and second optical guides 180A and 180B associated with each nanowell 136.

[0118] Specifically, in Figure 13C, the opaque layer 400 covers the neck sections of the first and second well regions 132 and 134 in a dogbone shape, but does not cover any portion of the first and second optical guides 180A and 180B. That is, the opaque layer 400 covers the second section 198 of the first well region 132 and the fourth section 204 of the second well region 134, but follows the periphery of the optical guides 180A and 180B.

[0119] Referring to Figure 14A, an example of a graph 500 of contrast ratio versus distance from the optical guide center associated with the first well region 180A is shown according to the embodiment described herein, in which no opaque layer 400 is placed between the first and second well regions 132, 134 and the array of optical guides 180.

[0120] See also Figure 14B, which shows an example of a graph 502 of contrast ratio versus distance from the optical guide center associated with the first well region 180A according to the embodiments described herein, where the opaque layer 400 is positioned between the first and second well regions 132, 134 and the array of optical guides 180.

[0121] Graph 500 is substantially the same as Graph 300, plotting the contrast ratio versus the distance from the center of the optical guide 180A. Graph 502 plots the same parameters except that the image sensor structure 100 includes an opaque layer 400. As can be seen by comparing the two graphs 500 and 502, the contrast ratio is substantially improved by the opaque layer. This improvement is largely due to the fact that the opaque layer 400 is positioned across the entire interface region 138 where most of the crosstalk occurs.

[0122] Referring to Figure 15, an example of a cross-sectional view of an image sensor structure 100 having first and second well regions 132, 134 arranged on first and second optical guides 180A, 180B according to the embodiments described herein is shown, where the optical guides 180A, 180B form an acute angle 510 with respect to each other. In this example, the passivation stack 156 includes two layers, namely a first passivation layer 182 and a chemical protection layer. The first and second optical guides 180A, 180B are arranged within a device stack 176 and are positioned at an angle toward the photodetector 172 associated with them.

[0123] The pitch 512 between the photodetectors 172 can only be reduced to a certain extent, which becomes a limiting factor in how small the nanowell 136 can be when it spans two photodetectors 172. In the example shown in Figure 15, the nanowell 136 can be fabricated smaller than the pitch 512 between the photodetectors 172 because of the angle 510.

[0124] Accordingly, the image sensor structure 100 in Figure 15 includes a first optical guide 180A associated with a first photodetector 172A of the array of photodetectors 172. A second optical guide 180B is associated with a second photodetector 172B of the array of photodetectors 170. Each nanowell 136 associated with the first and second optical guides 180A, 180B has a width 514 smaller than the pitch 512 between the first photodetector 172A and the second photodetector 172B. The first and second optical guides 180A, 180B extend from the nanowell 136 associated with them to the first and second photodetectors 172A, 172B associated with them at an acute angle 510 with respect to each other.

[0125] Referring to Figure 16, an example of a cross-sectional view of an image sensor structure 100 having a nanowell 136, comprising first and second well regions 132, 134 arranged on first and second portions 526, 528 of a first optical guide 180A associated with the nanowell 136, is shown, where first and second waveguides 520, 522 are arranged within a waveguide layer 524 and extend below the first and second well regions 132, 134, respectively.

[0126] The nanowells 136 and their corresponding structures in Figure 16 may represent arrays of nanowells 136 arranged within a passivation stack 156 and on top of a device stack 176 of an image sensor structure 100. The device stack 176 may include an array of optical guides 180 arranged inside. Each nanowell 136 may be associated with a first optical guide 180A of the array of optical guides 180. Each first optical guide 180A may be associated with a photodetector 172 of an array of photodetectors 172.

[0127] The image sensor structure 100 in Figure 16 includes a first well region 132 positioned on a first portion 526 of an associated first optical guide 180A. A second well region 134 is positioned on a second portion 528 of the associated first optical guide 180A. A waveguide layer 524 is positioned between the array of optical guides 180 in each nanowell 136 and the first and second well regions 132, 134. A first waveguide 520 is positioned within the waveguide layer 524 and extends below the first well region 132. The first waveguide 520 is operable to irradiate excitation light onto a forward chain cluster 121 of forward polynucleotide chains 122 bound within the first well region 132. A second waveguide 522 is positioned within the waveguide layer 524 and extends below the second well region 134. The second waveguide 522 is operable to irradiate excitation light onto the reverse chain cluster 123 of the reverse polynucleotide chain 124 bound within the second well region 134.

[0128] The first and second waveguides 522 and 524 can continuously irradiate the first and second well regions 132 and 134. In this way, the first waveguide 520 can excite mainly the front chain 122 coupled within the first well region 132, which can be read by the photodetector 172. Subsequently, the second waveguide 522 can excite mainly the back chain 124 coupled within the second well region 134, which can also be read by the same photodetector 172. In this manner, the nanowell 136 does not need to spread across two photodetectors and can be substantially smaller due to improved polyclonality.

[0129] During operation, as the excitation light 186 passes through the first waveguide 520, the majority of the excitation light is focused on the forward chain 122 of the forward chain cluster 121 in the first well region 132. However, at least a portion of the excitation light 186 from the first waveguide 520 may be incident on the reverse chain cluster 123 in the second well region 134. Therefore, the synchrotron radiation 174 read by the photodetector 172 may be emitted mainly from the forward chain 122 in the first well region 132, but a smaller proportion of the synchrotron radiation 174 read by the photodetector 172 (e.g., less than 25%, less than 15%, or less than 10%) may be emitted from the reverse chain 124 in the second well region 134.

[0130] Furthermore, during operation, as the excitation light 186 passes through the second waveguide 522, the majority of the excitation light is focused on the reverse chain 124 of the reverse chain cluster 123 in the second well region 134. However, at least a portion of the excitation light 186 from the second waveguide 522 may be incident on the forward chain cluster 121 in the first well region 132. Therefore, while the synchrotron radiation 174 read by the photodetector 172 may be mainly emitted from the reverse chain 124 in the second well region 134, a proportion of the synchrotron radiation 174 read by the photodetector 172 (e.g., less than 25%, less than 15%, or less than 10%) may be emitted from the forward chain 122 in the first well region 132.

[0131] Referring to Figure 17, an example of a cross-sectional view of an image sensor structure 100 having a nanowell 136 comprising a first well region 132 and a second well region 134 positioned on first and second portions 526, 528 of an optical guide 180A, respectively. In the illustrated example, the first waveguide 520 and the second waveguide 522 are located within a passivation stack 156. The first and second waveguides 520 and 522 are positioned adjacent to and on opposing sides 530, 532 of the nanowell 136.

[0132] The nanowells 136 and their corresponding structures in Figure 17 may represent an array of nanowells 136 located within a passivation stack 156 and on top of a device stack 176 of an image sensor structure 100. The device stack 176 may include an array of optical guides 180 located inside. Each nanowell 136 may be associated with a first optical guide 180A of the array of optical guides 180. Each first optical guide 180A may be associated with a photodetector 172 of an array of photodetectors 172.

[0133] The combinations of embodiments illustrated in Figures 16 and 17 demonstrate where the first and second waveguides 520 and 522 can be positioned within the image sensor structure 100 on (or above) the device stack 176. For example, in Figure 16, the waveguides 520 and 522 are located within a waveguide layer 524 positioned on top of the device stack 176. Also, as an example, in Figure 17, the waveguides 520 and 522 are located within a passivation stack 156, which is positioned on top of the device stack 176 and forms the walls of the nanowell 136.

[0134] Furthermore, the first and second waveguides 520, 522 may represent arrays of the first and second waveguide guides 520, 522. More specifically, an array of first waveguides 520 may be arranged on a device stack 176, and each first waveguide 520 may be associated with a nanowell 136 of an array of nanowells 136. Each first waveguide 520 may be operable to irradiate excitation light onto a forward-chain cluster 121 (see Figure 6) of forward polynucleotide chains 122 bound within a first well region 132 of the nanowell 136 associated with the first waveguide.

[0135] More specifically, an array of second waveguides 522 may be arranged on a device stack 176, and each second waveguide 522 may be associated with a nanowell 136 of an array of nanowells 136. The second waveguide 522 may be operable to irradiate excitation light onto a reverse chain cluster 123 (see Figure 6) of reverse polynucleotide chains 124 bound within the second well region 134 of the associated nanowell 136 of the second waveguide.

[0136] During operation, when the excitation light 186 passes through the first waveguide 520 positioned adjacent to the side surface 530 of the nanowell 136, the majority of the excitation light is focused on the forward chain 122 of the forward chain cluster 121 in the first well region 132. However, at least a portion of the excitation light 186 from the first waveguide 520 may be incident on the reverse chain cluster 123 in the second well region 134. Therefore, the synchrotron radiation 174 read by the photodetector 172 may be emitted mainly from the forward chain 122 in the first well region 132, but a smaller proportion of the synchrotron radiation 174 read by the photodetector 172 (e.g., less than 25%, less than 15%, or less than 10%) may be emitted from the reverse chain 124 in the second well region 134.

[0137] Furthermore, during operation, when the excitation light 186 passes through the second waveguide 522 positioned adjacent to the side surface 532 of the nanowell 136, most of the excitation light is focused on the reverse chain 124 of the reverse chain cluster 123 in the second well region 134. However, at least a portion of the excitation light 186 from the second waveguide 522 may be incident on the forward chain cluster 121 in the first well region 132. Therefore, while the synchrotron radiation 174 read by the photodetector 172 may be mainly emitted from the reverse chain 124 in the second well region 134, a proportion of the synchrotron radiation 174 read by the photodetector 172 (e.g., less than 25%, less than 15%, or less than 10%) may be emitted from the forward chain 122 in the first well region 132.

[0138] Referring to Figure 18, an example of a cross-sectional view of an image sensor structure 100 having an array of nanowells 136 (136A-E individually), an array of first optical guides 520 (520A-C individually), and a second optical guide 522 (522A-C individually). Each of the arrays of the first and second optical guides 520 and 522 has the same or similar structure as the first and second optical guides, as shown in Figure 17.

[0139] Each first waveguide 520 in the array of the first waveguides 520 is operable to irradiate excitation light onto clusters 121 or 123 of polynucleotide chains 122 or 124 bound within a first or second well region 132 or 134 of a nanowell 136 adjacent to the nanowell 136 associated with the first waveguide (see Figure 6). Additionally, each second waveguide 522 in the array of the second waveguides 522 is also operable to irradiate excitation light onto clusters 121 or 123 of polynucleotide chains 122 or 124 bound within a first or second well region 132 or 134 of a nanowell 136 adjacent to the nanowell 136 associated with the second waveguide (see Figure 6).

[0140] As an example shown in the illustration, the nanowell 136A in Figure 18 is associated with the first and second waveguides 520A and 522A. However, the second waveguide 522A may also be capable of irradiating excitation light onto a forward-chain cluster 121 of forward polynucleotide chains 122 bound within the first well region 132 of the adjacent nanowell 136B.

[0141] Furthermore, as an example, the nanowell 136B in Figure 18 is associated with the first and second waveguides 520B and 522A. However, the first waveguide 520B may also be capable of irradiating excitation light onto a forward-chain cluster 121 of polynucleotide chains 122 bound within the first well region 132 of the adjacent nanowell 136C.

[0142] For the purposes of this specification, the first and second waveguides 520, 522 may function as a single array of waveguides. Each waveguide 520, 522 may be operable to irradiate well regions 132, 134 within a pair of adjacent nanowells 136. Additionally, it is not important which waveguide irradiates which well regions in a pair of adjacent nanowells. For example, waveguide 520 may irradiate two first well regions 132, two second well regions 134, or both the first and second well regions 132, 134.

[0143] Referring to Figure 19, an example of a cross-sectional view of an image sensor structure 100 having first and second well regions 132, 134 positioned on first and second portions 526, 528 of an optical guide 180A, according to the embodiments described herein, is shown. In this case, the forward chain 122 in the first well region 132 and the reverse chain 124 in the second well region 134 can be excited simultaneously. Thus, the photodetector 172 is shared by both the first and second well regions 132, 134 and simultaneously receives both a bright signal A (or bright cluster signal) and a second bright signal B (or dark cluster signal) from those well regions. Assuming that the intensity of each signal is approximately the same, the purity score of the combined signal is approximately 50%, and the contrast ratio is approximately 1:1.

[0144] However, even with these low purity scores and contrast ratios, there are signal processing techniques that can be used to determine two types of bases that fluoresce from a combined signal. Such techniques are described in detail in U.S. Patent Application Publication 2019 / 0212295(A1), “SYSTEMS AND DEVICES FOR HIGH-THROUGHPUT SEQUENCING WITH SEMICONDUTOR-BASED DETECTION” by Dehlinger et al., assigned to Illumina, Inc., filed on January 7, 2019, and incorporated herein by reference in their entirety. One such technique is shown in Figures 20A, 20B, and 21.

[0145] Figures 20A and 20B are scatter plots 600A and 600B showing the base calls of bright and dark clusters using the respective pixel signals detected by the shared photodetector 172 according to one embodiment. The X-axis of scatter plots 600A and 600B represents the AT pixel signal detected during the second irradiation phase of the sampling event, which causes illumination from a given cluster representing nucleotide bases A and T. The Y-axis of scatter plots 600A and 600B represents the CT pixel signal detected during the first irradiation phase of the sample event, which causes illumination from a given cluster representing nucleotide bases C and T.

[0146] Scatter plot 600A shows four distributions 602, 604, 606, and 608 into which a signal processor (not shown) classifies pixel signals from bright clusters. In the illustrated embodiment, distribution 602 represents nucleotide base C in the bright cluster, distribution 604 represents nucleotide base T in the bright cluster, distribution 606 represents nucleotide base G in the bright cluster, and distribution 608 represents nucleotide base A in the bright cluster.

[0147] Scatter plot 600B shows 16 sub-distributions (or distributions) 602A-D, 604A-D, 606A-D, 608A-D, each having four sub-distributions for each of the four distributions 602, 604, 606, and 608 of scatter plot 600A in which the signal processor classifies pixel signals from dark clusters. In the illustrated embodiment, the sub-distribution annotated with the letter "A" represents nucleotide base C in the dark cluster, the sub-distribution annotated with the letter "B" represents nucleotide base T in the dark cluster, the sub-distribution annotated with the letter "C" represents nucleotide base G in the dark cluster, and the sub-distribution annotated with the letter "D" represents nucleotide base A in the dark cluster. In other embodiments, different encodings of the bases may be used. When the signal processor classifies pixel signals from dark clusters into one of the 16 sub-distributions, the classification of the corresponding bright clusters is determined by the distribution containing the sub-distributions of the dark clusters. For example, if a dark cluster is classified as sub-distribution 608B (nucleotide base T), the corresponding bright cluster distribution is 608 (nucleotide base A). As a result, the signal processor base-calls the bright cluster as A and the dark cluster as T.

[0148] Figure 21 is a scatter plot 602 showing 16 distributions (or bins) generated by intensity values ​​from cluster pairs of bright and dark clusters according to one embodiment. In this embodiment, the 16 bins are generated over multiple base call cycles. The signal processor combines the pixel signals from the bright clusters and maps them to one of the 16 bins. When the combined pixel signals are mapped to bin 612 for the base call cycle, the signal processor base calls the bright cluster as C and the dark cluster as C. When the combined pixel signals are mapped to bin 614 for the base call cycle, the signal processor 138 base calls the bright cluster as C and the dark cluster as T. When the combined pixel signals are mapped to bin 616 for the base call cycle, the signal processor 138 base calls the bright cluster as C and the dark cluster as G. When the combined pixel signals are mapped to bin 618 for the base call cycle, the signal processor 138 base calls the bright cluster as C and the dark cluster as A.

[0149] Referring to Figure 22, an example flowchart of the simultaneous paired-end array determination method 650 according to the embodiments described herein is shown. This method can utilize one or more of the examples of image sensor structures 100 described herein.

[0150] Method 650 (Figure 22), as well as the following methods 700 (Figure 23), 750 (Figure 24), and 800 (Figure 25), illustrate various steps for carrying out the present method. However, the order in which the steps of each method (650, 700, 750, 800) are performed may not correspond to the order in which the steps are illustrated in Figures 22 to 25. For example, the following sowing step 652 may be performed after the inactivation step 654.

[0151] The method in 652 involves seeding a first primer set 140 into a first well region 132 of a nanowell 136 of an image sensor structure 100 having a polynucleotide chain 110. The polynucleotide chain 110 (see Figure 2) includes adapters 118 and 120 that bind to complementary primers 106 and 108 (see Figure 1) in the first well region 132 (see Figure 7A).

[0152] In step 654, the second primer set 142 in the second well region 134 of the nanowell 136 is inactivated to prevent seeding of other polynucleotide chains in the second well region. This step may be performed before the seeding step in step 652. One example of inactivating the second well region 134 is to mask the second well region with a protective layer to prevent other polynucleotides from accessing the second well region.

[0153] At 656, the first polynucleotide chain is amplified into multiple forward and reverse strands 106, 108 throughout the entire first well region 132. At 658, the reverse strand 108 is cleaved from the first well region 132 to form a forward cluster 121 within the first well region 132.

[0154] In 660, the second primer set 142 is activated within the second well region 134 to enable seeding and amplification within the second well region 134. One example of activating the second well region 134 is to unmask any protective layer previously placed on the second well region 134.

[0155] In step 662, amplification occurs from the forward-chain cluster 121 in the first well region 132 to the second well region 134, forming multiple forward and reverse chains 106 and 108 in the second well region 134. In step 664, the forward chain 122 is cut from the second well region 134, forming a reverse-chain cluster 123 in the second well region 134.

[0156] During seeding (in 660) and the amplification (662) process in the second well region 134, the loose polynomial chains 110 do not flow into the channel 158 of the flow cell 112. Therefore, significant additional polyclonality resulting from the polynomial chains 110 flowing into the second well region 134 through the channel 158 of the flow cell 112 may occur when seeding (660) and amplification (662) are performed within the second well region 134.

[0157] Once forward and reversed clusters 121 and 123 are formed in the first and second well regions 132 and 134, simultaneous sequencing can be performed in step 666. In other words, in step 666, the forward cluster 121 in the first well region 132 and the reversed cluster 123 in the second well region 134 are sequenced substantially simultaneously.

[0158] Referring to Figure 23, an example flowchart of Method 700 for simultaneous paired-end sequencing, as described herein, is shown. Method 700 is a subset of Method 650, in that it is an extension of the simultaneous sequencing step 666.

[0159] In 702, method 700 includes binding a first complementary nucleotide 126 (see Figure 5) having a first fluorescent tag 128 to nucleotide 116 of forward-chain cluster 121 in a first well region 132. In 704, a second complementary nucleotide 126 having a second fluorescent tag 128 is bound to nucleotide 116 of reverse-chain cluster 123 in a second well region 134.

[0160] In step 706, the method emits excitation light 186 substantially simultaneously onto the forward-chain cluster 121 and the reverse-chain cluster 123 (see Figure 7A) to cause synchrotron radiation 174 to fluoresce from the first and second tags 128. In step 708, synchrotron radiation 174 is emitted substantially simultaneously from the first and second tags 128. Synchrotron radiation 174 from the first tag 128 propagates through the first optical guide 180A (see Figure 7A) to the first photodetector 172. Synchrotron radiation 174 from the second tag 128 propagates through the second optical guide 180B to the second photodetector 174, determining the sequences of the nucleotides of the forward-chain and reverse-chain 122 and 124, respectively.

[0161] To reduce polyclonality while performing method 650 or 700, the first well region 132 may have a smaller area than the second well region 134. For example, the area of ​​the first well region 132 may be 90% or less, 80% or less, or 70% or less, or the area of ​​the second well region 134.

[0162] To reduce both polyclonality and crosstalk between the first optical guide 180A and the second optical guide 180B, the first and second well regions 132, 134 may have a combined shape that generally resembles a dogbone (see Figure 10). More specifically, the first well region 132 may include a first section 196 and a second section 198. The first section 196 of the well region 132 may be positioned on top of the entire first optical guide 180A, and the first section 196 has a first section width 201. The first section 196 may have an area slightly larger than the area of ​​the optical guide 180A in order to reduce polyclonality. For example, the area of ​​the first section 196 may be 5%, 10%, or 15% or less of the upper surface area of ​​the optical guide 180A.

[0163] The second section 198 of the well region 134 may extend from the first section 196 to the region interface 138 between the first well region 132 and the second well region 134. The second section 198 may have a second section width 194 that is smaller than the first section width 201.

[0164] Additionally, the second well region 134 may include a third section 202 and a fourth section 204. The third section 202 of the second well region 134 may be located over the entire second optical guide 180B and may have a third section width 206. The fourth section 204 may extend from the third section 202 to the region interface 138. The fourth section 204 may have a fourth section width 194 that is smaller than the third section width 206. Also, the width 194 of the fourth section 204 may be substantially equal to the width 194 of the second section 198.

[0165] By reducing the width 194 of the second and fourth sections 198, 204, the interface region 138 is reduced. By reducing the interface region 138, the possibility of any polyclonality in the first well region 132 being amplified in the second well region 134 is substantially reduced. In addition, by reducing the interface region 138, the possibility of crosstalk occurring between the first well region 132 and the second well region 134 is also substantially reduced.

[0166] Another method for reducing crosstalk while performing method 650 or 700 is to place an opaque layer 400 between the first and second optical guides 180A, 180B and the first and second well regions 132, 134. The opaque layer 400 (see Figure 12) may extend beneath the entire region interface 138 of the first and second well regions 132, 134. The opaque layer 400 may cover less than the entire first and second optical guides (see Figures 13A–13C).

[0167] Referring to Figure 24, an example flowchart of Method 750 for simultaneous paired-end sequencing, as described herein, is shown. Method 750 is a subset of Method 650, in that it is an extension of the simultaneous sequencing step 666.

[0168] In 752, method 750 includes binding a first complementary nucleotide 126 (see Figure 5) having a first fluorescent tag 128 to nucleotide 116 of forward-chain cluster 121 in a first well region 132. In 754, a second complementary nucleotide 126 having a second fluorescent tag 128 is bound to nucleotide 116 of reverse-chain cluster 123 in a second well region 134.

[0169] At 756, substantially more excitation light 186 is emitted onto the forward-chain cluster 121 than onto the reverse-chain cluster 123, causing substantially more synchrotron radiation 174 to fluoresce from the first tag 128 than from the second tag 128. At 758, the synchrotron radiation 174 from the first tag 128 is delivered to the first photodetector 172 through the first optical guide 180A to determine the nucleotides of the forward-chain 122. At 760, substantially more excitation light 186 is emitted onto the reverse-chain cluster 123 than onto the forward-chain cluster 121, causing substantially more synchrotron radiation 174 to fluoresce from the second tag 128 than from the first tag 128. At 762, the synchrotron radiation 174 from the second tag 128 is delivered to the first photodetector 172 through the first optical guide 180A to determine the nucleotides of the reverse-chain 124.

[0170] Method 750 can be carried out, for example, using an image sensor structure 100 similar to that shown in Figure 16, where the first well region 132 is located on the first portion 526 of the first optical guide 180A, and the second well region 134 is located on the second portion 528 of the first optical guide 180A. A waveguide layer 524 is located between the first optical guide 180A and the first and second well regions 132 and 134. The first waveguide 520 is located within the waveguide layer 524 and extends below the first well region 132 but not below the second well region 134. The second waveguide 522 is located within the waveguide layer 524 and extends below the second well region 134 but not below the first well region 132.

[0171] Alternatively, method 750 may be carried out using, for example, an image sensor structure 100 similar to that shown in Figure 17, wherein each nanowell 136 in the array of nanowells 136 includes a first well region 132 positioned on a first portion 526 of a first optical guide 180A and a second well region 134 positioned on a second portion 528 of the first optical guide 180A. A passivation stack 156 is positioned on the device stack 176 of the image sensor structure 100, and the array of nanowells 136 is positioned within the passivation stack 156. Each first waveguide 520 in the array of first waveguides 520 is positioned within the passivation stack 156 adjacent to the side 530 of the associated nanowell 136 of the first waveguide. Each second waveguide 522 in the array of second waveguides 522 is located in a passivation stack 156 adjacent to the opposing side 532 of the associated nanowell 136 of the second waveguide.

[0172] Using the above structure, excitation light 186 can be emitted mainly onto the forward-chain cluster 121 through the first waveguide 520. Then, synchrotron radiation 174 mainly from the forward-chain cluster 121 can be sent to the photodetector 172 through the first optical guide 180A for analysis. Subsequently, excitation light 186 can be emitted mainly onto the reverse-chain cluster 123 through the second waveguide 522. Then, synchrotron radiation 174 mainly from the reverse-chain cluster 123 can be sent to the photodetector 172 through the first optical guide 180A for analysis.

[0173] Referring to Figure 25, an example flowchart of Method 800 for simultaneous paired-end sequencing, as described herein, is shown. Method 800 is a subset of Method 650, in that it is an extension of the simultaneous sequencing step 666.

[0174] In 802, method 800 includes binding a first complementary nucleotide 126 (see Figure 5) having a first fluorescent tag 128 to nucleotide 116 of forward-chain cluster 121 in a first well region 132. In 804, a second complementary nucleotide 126 having a second fluorescent tag 128 is bound to nucleotide 116 of reverse-chain cluster 123 in a second well region 134.

[0175] In 806, excitation light 186 is emitted substantially simultaneously over the forward-chain cluster 121 in the first well region 132 and over the reverse-chain cluster 123 in the second well region 134, causing synchrotron radiation 174 to fluoresce from the first and second tags 128. In 808, the combined synchrotron radiation 174 from the first and second well regions 132, 134 is delivered from the first and second tags 128 through the first optical guide 18A to the first photodetector 172. Signal processing techniques can then be used to determine the nucleotides of the forward and reverse chains 122, 124 associated with the combined synchrotron radiation 174 detected by the first photodetector 172. Examples of such signal processing techniques are discussed herein with reference to Figures 20A, 20B, and 21. Method 800 can be carried out, for example, using an image sensor structure 100 similar to that shown in Figure 19.

[0176] It will be understood that all combinations of the aforementioned concepts and further concepts discussed in more detail herein are intended to be part of the subject matter of the invention disclosed herein (provided that such concepts are not mutually contradictory). Specifically, all combinations of claimed subject matter appearing at the end of this disclosure are intended to be part of the subject matter of the invention disclosed herein.

[0177] While the present invention has been described with reference to specific embodiments, it should be understood that many modifications may be made within the spirit and scope of the described inventive concept. Therefore, this disclosure is not limited to the described embodiments and is intended to have the entire scope defined by the following claims.

Claims

1. An image sensor structure, An imaging layer comprising an array of photodetectors arranged internally, A device stack disposed on the imaging layer, An array of optical guides arranged within the device stack, wherein each optical guide is positioned on a photodetector of the array of photodetectors, An array of nanowells arranged on the device stack, wherein each nanowell in the array of nanowells is positioned on a first optical guide of the array of optical guides, and each first optical guide is positioned on a first photodetector of the array of photodetectors, A first primer set is arranged throughout the entire first well region of each nanowell, A different second primer set is arranged throughout the entire second well region of each nanowell, wherein the second well region comprises a second primer set adjacent to the first well region at the region interface, The first and second primer sets are operable to bind forward-chain clusters of forward polynucleotide chains into the first well region and adjacent reverse-chain clusters of reverse polynucleotide chains into the second well region, thereby forming an image sensor structure.

2. Each nanowell of the array of nanowells is arranged on a second optical guide of the array of optical guides, and each second optical guide is arranged on a second photodetector of the array of photodetectors, The first well region positioned on the first optical guide, The image sensor structure according to claim 1, comprising: the second well region disposed on the second optical guide.

3. The image sensor structure according to claim 2, wherein the area of ​​the first well region is smaller than the area of ​​the second well region.

4. The first and second well regions having equal widths, The image sensor structure according to claim 2, comprising: a first well region having a length of 90% or less of the length of the second region.

5. The first well region, A first section positioned on the entire first optical guide, having a first section width, and The first well region comprises a second section extending from the first section to the region interface, the second section having a second section width smaller than the width of the first section, The second well region, A third section positioned on the entire second optical guide, the third section having a third section width, and The second well region comprises a fourth section, which extends from the third section to the region interface and has a fourth section width smaller than the width of the third section, The image sensor structure according to claim 2, wherein the width of the second section of the first well region is equal to the width of the fourth section of the second well region.

6. comprising the first section and the third section having a circular shape, The image sensor structure according to claim 5, wherein the widths of the first and third sections are the diameters of the first and third sections, respectively.

7. The image sensor structure according to claim 5, wherein the widths of the second and fourth sections are 50% or less of the widths of the first and third sections, respectively.

8. An opaque layer is disposed between the array of optical guides and the first and second well regions of each nanowell, The opaque layer extending beneath the entire region interface of the first and second well regions, The image sensor structure according to claim 2, further comprising: an opaque layer covering less than the entire upper surface of the first and second optical guides located below each nanowell.

9. The image sensor structure according to claim 8, wherein the opaque layer does not cover any portion of the upper surface of the first and second optical guides located below each nanowell.

10. The image sensor structure according to claim 8, wherein the opaque layer covers more than 10% of the upper surface of the first and second optical guides located below each nanowell.

11. The first optical guide is positioned at the first photodetector of the array of photodetectors, The second optical guide is positioned at the second photodetector of the array of photodetectors, Each nanowell is positioned on the first and second optical guides having a width smaller than the pitch between the first and second photodetectors, The image sensor structure according to claim 2, comprising the first and second optical guides, the first and second optical guides extending from the nanowell to the first and second photodetectors at acute angles to each other.

12. The first well region of each nanowell positioned on the first portion of the first optical guide, The second well region of each nanowell positioned on the second portion of the first optical guide, An array of first waveguides arranged on the device stack, wherein each first waveguide is positioned below the nanowells of the array of nanowells, and each first waveguide is operable to irradiate excitation light onto forward-chain clusters of forward polynucleotide chains bound within the first well region of the nanowells, The image sensor structure according to claim 1, comprising: an array of second waveguides disposed on the device stack, wherein each second waveguide is positioned below the nanowells of the array of nanowells, and each second waveguide is operable to irradiate excitation light onto reverse-chain clusters of reverse polynucleotide chains bound within the second well region of the nanowells.

13. A waveguide layer disposed between the array of optical guides and the first and second well regions of each nanowell, Each first waveguide of the waveguide array, which is arranged within the waveguide layer and extends below the first well region of the nanowell, The image sensor structure according to claim 12, comprising: each second waveguide of the waveguide array, which is disposed within the waveguide layer and extends below the second well region of the nanowell.

14. A passivation stack disposed on the device stack, wherein the array of nanowells is disposed within the passivation stack, Each first waveguide of the array of first waveguides is arranged in the passivation stack adjacent to the side surface of the nanowell, The image sensor structure according to claim 12, comprising: each second waveguide of the waveguide array disposed in the passivation stack adjacent to the side opposite to the nanowell.

15. Each first waveguide in the array of the first waveguides is operable to irradiate excitation light onto clusters of polynucleotide chains bound within the first or second well region of a nanowell adjacent to the nanowell, The image sensor structure according to claim 14, wherein each second waveguide of the array of second waveguides is operable to irradiate excitation light onto clusters of polynucleotide chains coupled within the first or second well region of a nanowell adjacent to the nanowell.

16. The image sensor structure according to claim 1, wherein the first well region and the second well region are bounded by the walls of the wells, except for the region interface.

17. The image sensor structure according to claim 1, further comprising a passivation stack disposed on the device stack, wherein the array of nanowells is disposed within the passivation stack.

18. The image sensor structure according to any one of claims 5 to 16, wherein the area of ​​the first well region is smaller than the area of ​​the second well region.

19. The first and second well regions having equal widths, The image sensor structure according to any one of claims 5 to 16, comprising: a first well region having a length of 90% or less of the length of the second region.

20. The first well region, A first section positioned on the entire first optical guide, having a first section width, and The first well region comprises a second section extending from the first section to the region interface, the second section having a second section width smaller than the width of the first section, The second well region, A third section positioned on the entire second optical guide, the third section having a third section width, and The second well region comprises a fourth section, which extends from the third section to the region interface and has a fourth section width smaller than the width of the third section, The image sensor structure according to any one of claims 8 to 10, wherein the width of the second section of the first well region is equal to the width of the fourth section of the second well region.

21. An opaque layer is disposed between the array of optical guides and the first and second well regions of each nanowell, The opaque layer extending beneath the entire region interface of the first and second well regions, The image sensor structure according to claim 15 or 16, further comprising: an opaque layer covering less than the entire upper surface of the first and second optical guides located below each nanowell.

22. The image sensor structure according to claim 21, wherein the opaque layer does not cover any portion of the upper surface of the first and second optical guides located below each nanowell.

23. The image sensor structure according to claim 21, wherein the opaque layer covers more than 10% of the upper surface of the first and second optical guides located below each nanowell.

24. The image sensor structure according to any one of claims 12 to 14, further comprising a passivation stack disposed on the device stack, wherein the array of nanowells is disposed within the passivation stack.

25. The image sensor structure according to any one of claims 12 to 14, wherein the first well region and the second well region are bounded by the walls of the wells, except for the region interface.

26. An image sensor structure, An imaging layer comprising an array of photodetectors arranged internally, A device stack disposed on the imaging layer, An array of optical guides arranged within the device stack, wherein each optical guide is positioned on a photodetector of the array of photodetectors, An array of nanowells arranged on the device stack, wherein each nanowell in the array of nanowells is positioned on first and second optical guides of the array of optical guides, A first primer set is arranged throughout the entire first well region of each nanowell, wherein the first well region is positioned on the first optical guide, and the first primer set is located thereon. A different second primer set is arranged throughout the entire second well region of each nanowell, wherein the second well region is positioned on the second optical guide and adjacent to the first well region at the region interface, comprising: The first and second primer sets are operable to bind forward-chain clusters of forward polynucleotides to the first well region and to bind adjacent reverse-chain clusters of reverse polynucleotides to the second well region. An image sensor structure in which the area of ​​the first well region is smaller than the area of ​​the second well region.

27. The first well region, A first section having a circular shape positioned on the entire first optical guide, having the diameter of the first section, and The first well region comprises a second section extending from the first section to the region interface, the second section having a second section width smaller than the diameter of the first section, The second well region, A third section having a circular shape positioned on the entire second optical guide, the third section having a third section diameter, and The second well region comprises a fourth section, which extends from the third section to the region interface and has a fourth section width smaller than the diameter of the third section, The image sensor structure according to claim 26, wherein the width of the second section of the first well region is equal to the width of the fourth section of the second well region.

28. An opaque layer is disposed between the array of optical guides and the first and second well regions of each nanowell, The opaque layer extending beneath the entire region interface of the first and second well regions, The image sensor structure according to claim 26 or 27, further comprising: an opaque layer covering less than the entire upper surface of the first and second optical guides located below each nanowell.

29. The image sensor structure according to claim 28, wherein the opaque layer does not cover any portion of the upper surface of the first and second optical guides located below each nanowell.

30. The first optical guide is positioned at the first photodetector of the array of photodetectors, The second optical guide is positioned at the second photodetector of the array of photodetectors, Each nanowell is positioned on the first and second optical guides having a width smaller than the pitch between the first and second photodetectors, The image sensor structure according to claim 26, comprising the first and second optical guides, the first and second optical guides extending at acute angles to each other from the nanowell to the first and second photodetectors.

31. An image sensor structure, An imaging layer comprising an array of photodetectors arranged internally, A device stack disposed on the imaging layer, An array of optical guides arranged within the device stack, wherein each optical guide is positioned on a photodetector of the array of photodetectors, An array of nanowells arranged on the device stack, wherein each nanowell in the array of nanowells is positioned on a first optical guide of the array of optical guides, A first primer set is arranged throughout the entire first well region of each nanowell, wherein the first well region is positioned on a first portion of the first optical guide, A different second primer set is arranged throughout the entire second well region of each nanowell, wherein the second well region is positioned on a second portion of the first optical guide, and the second well region comprises a different second primer set adjacent to the first well region at the region interface, The first and second primer sets are operable to bind forward-chain clusters of forward polynucleotides to the first well region and to bind adjacent reverse-chain clusters of reverse polynucleotides to the second well region, thereby forming an image sensor structure.

32. A first waveguide array disposed on the device stack, wherein each first waveguide is positioned below the nanowells of the nanowell array, and each first waveguide is operable to irradiate excitation light onto forward-chain clusters of forward polynucleotide chains bound within the first well region of the nanowells, The image sensor structure according to claim 31, comprising: an array of second waveguides disposed on the device stack, wherein each second waveguide is positioned below the nanowells of the array of nanowells, and each second waveguide is operable to irradiate excitation light onto reverse-chain clusters of reverse polynucleotide chains coupled within the second well region of the nanowells.

33. It is a method, The first primer set is seeded in the first well region of the nanowell of the image sensor structure using a polynucleotide chain, To inactivate the second primer set in the second well region of the nanowell, thereby preventing the seeding of other polynucleotide chains in the second well region, Throughout the entire first well region, the first polynucleotide chain is amplified into multiple forward and reverse strands, The reverse chain is cut from the first well region to form a forward chain cluster within the first well region, To activate the second primer set in the second well region, enabling seeding and amplification in the second well region, The forward-chain clusters in the first well region are amplified to form a plurality of forward and reverse chains within the second well region, The forward chain is cut from the second well region to form a reverse chain cluster in the second well region, A method comprising simultaneously sequencing the forward-chain cluster in the first well region and the reverse-chain cluster in the second well region.

34. Inactivating the second primer set includes masking the second well region. The method according to claim 33, wherein activating the second primer set comprises unmasking the second well region.

35. Simultaneous sequence determination is The first complementary nucleotide having a first fluorescent tag is bound to the nucleotide of the forward-chain cluster within the first well region, A second complementary nucleotide having a second fluorescent tag is bound to the nucleotide of the reverse-chain cluster within the second well region, By simultaneously emitting excitation light onto the forward-chain cluster and the reverse-chain cluster, the first and second tags emit fluorescence from synchrotron radiation. The method according to claim 33, comprising simultaneously receiving the synchrotron radiation from the first tag through a first optical guide to a first photodetector, and the synchrotron radiation from the second tag through a second optical guide to a second photodetector, in order to determine the sequences of the forward and reverse strands of nucleotides, respectively.

36. The method according to claim 35, wherein the first well region has an area smaller than the area of ​​the second well region.

37. The first well region is, A first section positioned on the entire first optical guide, having a first section width, and A second section extending from the first section to the region interface between the first well region and the second well region, the second section having a second section width smaller than the width of the first section, The aforementioned second well region is, A third section positioned on the entire second optical guide, the third section having a third section width, and The method according to claim 35, further comprising a fourth section extending from the third section to the region interface, the fourth section having a width smaller than the width of the third section.

38. The method according to claim 35, wherein an opaque layer is disposed between the first and second optical guides and the first and second well regions, the opaque layer extends below the entire region interface between the first and second well regions, and the opaque layer covers less than the entirety of the first and second optical guides.

39. The nanowell has a width smaller than the pitch between the first photodetector and the second photodetector. The method according to claim 35, wherein the first and second optical guides extend to the first and second photodetectors at acute angles to each other.

40. The first well region is positioned on the first portion of the first optical guide, The method according to claim 33, wherein the second well region is positioned on the second portion of the first optical guide.

41. Simultaneously determining the arrangement means The first complementary nucleotide having a first fluorescent tag is attached to the nucleotide of the forward-chain cluster, The method involves attaching a second complementary nucleotide having a second fluorescent tag to the nucleotide of the reverse-chain cluster, By emitting a larger amount of excitation light onto the forward-chain cluster than the reverse-chain cluster, a larger amount of synchrotron radiation than the second tag is emitted from the first tag, The synchrotron radiation is received from the first tag through the first optical guide to the first photodetector, and the forward-chain nucleotides are determined. By emitting a larger amount of excitation light onto the reverse-chain cluster than the forward-chain cluster, a larger amount of synchrotron radiation from the second tag than from the first tag is emitted, The method according to claim 33, comprising receiving the synchrotron radiation from the second tag through the first optical guide to the first photodetector to determine the reverse strand nucleotides.

42. A first waveguide is placed on the first optical guide, A second waveguide is placed on the first optical guide, The excitation light is radiated onto the forward-chain cluster through the first waveguide. The method according to claim 41, wherein the excitation light is radiated onto the reverse-chain cluster through the second waveguide.

43. Simultaneously determining the arrangement means The first complementary nucleotide having a first fluorescent tag is attached to the nucleotide of the forward-chain cluster, The method involves attaching a second complementary nucleotide having a second fluorescent tag to the nucleotide of the reverse-chain cluster, By simultaneously emitting excitation light onto the forward-chain cluster in the first well region and the reverse-chain cluster in the second well region, the first and second tags emit fluorescence from synchrotron radiation. The combined synchrotron radiation from the first and second tags through the first optical guide to the first photodetector is received, The method according to claim 33, comprising determining the forward and reverse nucleotides based on the composite synchrotron radiation received by the first photodetector using signal processing technology.

44. Simultaneously determining the arrangement means The method according to claim 33, comprising simultaneously receiving synchrotron radiation from a first tag on the forward strand to a first photodetector and synchrotron radiation from a second tag on the reverse strand to a second photodetector, thereby determining the sequences of the nucleotides on the forward and reverse strands, respectively.

45. Simultaneously determining the sequence means The method according to claim 33, comprising receiving synchrotron radiation from a first tag on the forward strand to a first photodetector and synchrotron radiation from a second tag on the reverse strand to the first photodetector, and determining the sequences of the nucleotides on the forward strand and the reverse strand, respectively.

46. The method according to claim 45, wherein the reception of synchrotron radiation from the forward-sequenced first tag to the first photodetector is received simultaneously with the reception of synchrotron radiation from the reverse-sequenced second tag to the first photodetector.

47. The method according to claim 45, wherein the reception of synchrotron radiation from the forward-sequenced first tag to the first photodetector occurs before the reception of synchrotron radiation from the reverse-sequenced second tag to the first photodetector.

48. Inactivating the second primer set includes masking the second well region. The method according to any one of claims 35 to 47, wherein activating the second primer set comprises unmasking the second well region.

Images