HK40113932A - Short tandem repeat sequence sequencing and analysis methods - Google Patents

Description

Short tandem repeat sequence sequencing methods and analysis methods

Technical Field

This invention relates to the field of biology. Specifically, it relates to methods for sequencing and analyzing short tandem repeat sequences.

Background Technology

Short tandem repeats (STRs), also known as microsatellite DNA or simple repeat sequences (SRSs; simple sequences of repeats, SSRs), exhibit significant variations in distribution among different races, populations, and individuals due to differences in their core units (also called "repetition units" or "motif sequences") and the number of repetitions. This constitutes the genetic polymorphism of STRs. On average, there is one STR locus every 15–20 kb in the human genome, with nearly 8,000 STR loci distributed across the 23 pairs of human chromosomes [Ma Wei, Zhang Liang, Li Yan, Xu Fei. Human Short Tandem Repeats (STRs) and Their Research Progress [J]. Journal of Dalian Medical University, 2007, 29(1):78-81.]. Given their polymorphism and wide distribution, STR loci can serve as highly specific and sensitive genetic markers. Combined with PCR technology, STR testing has been widely used in many fields such as genetic mapping, gene localization, forensic identification, and prenatal diagnosis. For example, in the field of forensic identification, approximately a few dozen autosomal STRs and Y-chromosome STRs are currently mainly used.

The main methods for detecting STRs include PCR and sequencing, but sequencing-based STR detection methods still need further research and improvement.

Summary of the Invention

This invention aims to at least partially solve at least one of the aforementioned technical problems or provide a practical STR detection solution. In one aspect of this invention, a method for sequencing short tandem repeat (STR) sequences is proposed. According to an embodiment of the invention, the method includes: acquiring a library, wherein the insert fragment of the library contains a short tandem repeat sequence and a specific sequence linked to the short tandem repeat sequence; performing sequencing-while-synthesizing on the library on a solid surface to obtain sequencing data, the sequencing data comprising multiple reads, wherein the solid surface carries at least one of a first sequencing primer and a second sequencing primer, the first sequencing primer having the following nucleotide sequence: (S0)x, wherein S0 represents the motif sequence of the short tandem repeat sequence or its complementary sequence, and x is not less than 1; the second sequencing primer having the following nucleotide sequence: S1-(S0)y, wherein S1 is the specific sequence or a sequence corresponding to the specific sequence, S0 is the motif sequence of the short tandem repeat sequence or its complementary sequence, and y is greater than or equal to 0.

Conventional sequencing primers typically do not contain the sequence to be tested. For example, commercial sequencing-by-synthesis methods or platforms, such as those from ILLUMINA, BGI Genomics, and ThermoFisher, typically use sequencing primers designed based on the adapters at the ends of the library. These primers are sequences that can match the adapter or a portion of the adapter. Based on this, primer extension and corresponding signal detection identify the base type and sequence of part or all of the insert/test sequence.

Unlike conventional sequencing primers, the inventors, based on the following understanding, prediction, and testing, discovered that the target sequence STR is composed of tandem repeat units of 2-6 bases and that the "misalignment" behavior between short tandem repeat sequences occurs largely randomly. Therefore, they ingeniously designed this first sequencing primer. Specifically, the first sequencing primer is, for example, a part of the sequence to be tested, such as a polynucleotide sequence formed by tandem repeat units (motif sequences) of several target STRs. Given that testing revealed a degree of randomness in the first sequencing primer's hybridization matching to different positions in a library containing the target STR (i.e., misalignment), capturing or hybridizing this library using this first sequencing primer will understandably generate a series of hybridization complexes. Furthermore, extending the first sequencing primer to sequence these hybridization complexes will yield sequencing data containing multiple reads corresponding to these hybridization complexes, or rather, different positions of the target STR. Analysis of this sequencing data can then determine the complete sequence of the target STR, that is, the number of repeats of the STR's motif sequence, thus enabling STR genotyping.

Similarly, based on the above understanding, predictions, and experimental tests, the inventors ingeniously designed a second sequencing primer. This second sequencing primer is, for example, a polynucleotide sequence consisting of a specific sequence or a sequence corresponding to the specific sequence and one or more repeating units (motif sequences) of the target STR tandemly. For example, by using the second sequencing primer 5'-S1-(S0)y-3' with its 5' end fixed on a designated surface to capture or hybridize a library containing the target STR, a hybridization complex with a double strand at its 5' end can be obtained. In other words, the complementary hybridization position between the second sequencing primer and the library is fixed at one end. Thus, the hybridization complex can be further sequenced to determine the sequence of the STR and achieve the detection of the STR.

More specifically, the second sequencing primer is designed to contain a set of sequences of different lengths, with each length containing a specific sequence or a sequence corresponding to the specific sequence, as well as (S0)y of different lengths, i.e., a set of sequences with multiple different values for y. Understandably, by using this set of sequences to capture or hybridize a library containing the target STR, a series of hybridization complexes with double strands at the 5' end can be obtained. Sequencing these hybridization complexes will, understandably, produce sequencing data containing multiple reads corresponding to these hybridization complexes or different positions of the target STR. Furthermore, by analyzing this sequencing data, the complete sequence of the STR can be determined, i.e., the number of repetitions of the motif sequence of the STR can be determined, accurately achieving genotyping of the STR, shortening sequencing time, saving sequencing costs, and showing promising application prospects.

In another aspect of the invention, a method for identifying short tandem repeat sequences is proposed. According to an embodiment of the invention, the method includes: (i) acquiring sequencing data of the short tandem repeat sequence, the sequencing data being obtained according to the short tandem repeat sequence sequencing method described above; (ii) aligning the sequencing data with a reference sequence to classify multiple reads into test-through reads and non-test-through reads, wherein test-through reads contain at least a portion of a specific sequence or a complementary sequence of the specific sequence, and non-test-through reads do not contain a specific sequence or a complementary sequence of the specific sequence; and (iii) identifying the short tandem repeat sequence based on the test-through reads and non-test-through reads.

In another aspect of the invention, a sequencing chip is provided. According to an embodiment of the invention, the sequencing chip includes: a substrate having a surface; a first sequencing primer fixed on the surface, and the first sequencing primer having the following nucleotide sequence: (S0)x, wherein S0 represents the motif sequence of the short tandem repeat sequence or its complementary sequence, and x is not less than 1.

In another aspect of the invention, a sequencing chip is provided. According to an embodiment of the invention, the sequencing chip includes: a substrate having a surface; a second sequencing primer fixed on the surface, and the second sequencing primer having the following nucleotide sequence: S1-(S0)y, wherein S1 is a specific sequence or a sequence corresponding to the specific sequence, S0 is a motif sequence of a short tandem repeat sequence or its complementary sequence, the specific sequence is capable of indicating the position of the short tandem repeat sequence on a reference sequence, and y is greater than or equal to 0.

In another aspect of the invention, a kit is provided. According to an embodiment of the invention, the kit comprises: (1) a first sequencing primer and/or a second sequencing primer as described above in the short tandem repeat sequencing method; or (2) a chip as described above.

In another aspect, the present invention provides a method for identifying an individual. According to an embodiment of the present invention, the method includes: determining the sequence of a short tandem repeat sequence of a sample to be tested, the sample containing nucleic acid, according to the preceding short tandem repeat sequencing method; and determining, based on the sequence, one or more individuals from which the sample to be tested originates.

Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention.

Attached Figure Description

The above and/or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which:

Figure 1 shows a schematic diagram of the STR amplification product structure according to an embodiment of the present invention;

Figure 2 shows a schematic flowchart of a short tandem repeat sequence sequencing method according to an embodiment of the present invention;

Figure 3 shows a schematic diagram of the principle of mismatched hybridization according to an embodiment of the present invention;

Figure 4 shows a schematic diagram of constructing a sequencing library according to an embodiment of the present invention;

Figure 5 shows a schematic diagram of a sequencing library structure according to an embodiment of the present invention;

Figure 6 shows a schematic diagram illustrating the relationship between penetration test ratio and the number of repeat sequences according to an embodiment of the present invention;

Figure 7 shows a schematic diagram of the distribution obtained by simulation using 1MReads under a Gaussian distribution with read length distribution of avg=40 and std=5 according to an embodiment of the present invention.

Figure 8 shows a schematic diagram of the read length distribution obtained by simulation using 10MReads under a Gaussian distribution with avg=40 and std=5 according to an embodiment of the present invention.

Figure 9 shows a schematic diagram of the distribution obtained by simulation using 1M Reads under a Gaussian distribution with read length distribution of avg=40 and std=10 according to an embodiment of the present invention;

Figure 10 shows a schematic diagram of the distribution obtained by simulation using 10MReads under a Gaussian distribution with read length distribution of avg=40 and std=10 according to an embodiment of the present invention.

Detailed Implementation

The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number or order of the indicated technical features. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this invention, unless otherwise stated, "a plurality of" means two or more.

In this paper, the term "chip" refers to a solid substrate with a surface capable of fixing, connecting, or supporting the sample to be tested. Examples include glass slides, magnetic beads, or reaction chambers with liquid-containing spaces. Preferably, a chamber with liquid-containing spaces, capable of supporting biochemical reactions or detection, may also be called a flow cell or flow-phase cell. The substrate or solid substrate can be any solid support suitable for fixing nucleic acid sequences, such as nylon membranes, glass slides, plastics, silicon wafers, magnetic beads, etc.

In this article, the term "template strand" refers to the nucleic acid sequence to be tested; it is sometimes also called "hybrid strand," which is the opposite of the fixed strand, such as a single-stranded or double-stranded DNA or a hybrid complex that is complementary to a probe or fixed strand immobilized on a surface and attached to that surface.

In this article, "sequencing" refers to sequence determination, generally meaning the determination of the base order in a nucleic acid sequence, including DNA sequencing and/or RNA sequencing; including sequencing-by-synthesis (SBS). Sequencing-by-synthesis involves repeatedly binding one or more nucleotides (including nucleotide analogs) to a template strand and collecting the corresponding reaction signals. Sequencing can be performed using sequencing platforms, including single-molecule sequencing platforms.

In this paper, the term "motif sequence" can also be referred to as "repetitive sequence," which refers to the repeating unit of a short tandem repeat sequence. Typically, a motif sequence consists of 2 to 6 bases. In this paper, the term "repetition number" of a short tandem repeat sequence is also called "cycle number" or "motif number," which refers to the number of motif sequences within the short tandem repeat sequence.

In this article, “reads” are the base sequences that are read.

In this document, the term "primer" or "sequencing primer" is sometimes also referred to as a fixed strand or probe, and is a nucleotide fragment with a pre-defined or known sequence. Preferably, the primer is single-stranded. In some embodiments, the primer may also be double-stranded. If double-stranded, it may be pretreated to isolate the single strands before preparing the extension product. In some embodiments, the primer is selected from RNA, DNA, PNA, or LNA.

Short tandem repeat sequencing methods

In one aspect of the present invention, a method for sequencing short tandem repeat sequences is proposed. According to an embodiment of the present invention, the method includes: acquiring a library, wherein the insert fragment of the library contains short tandem repeat sequences and specific sequences linked to the short tandem repeat sequences; performing sequencing-while-synthesizing on a solid surface to obtain sequencing data of the short tandem repeat sequences, wherein the chip simultaneously or independently carries a first sequencing primer and a second sequencing primer.

In this paper, the term "specific sequence" or "sequence corresponding to the specific sequence" refers to a sequence that indicates the position of the short tandem repeat sequence on the reference sequence. The specific sequence or the sequence corresponding to the specific sequence can, for example, be a relatively conserved sequence upstream and/or downstream of the target STR on the reference sequence. This positional relationship is generally determined through sequence alignment. The analysis of sequencing data to determine the target STR sequence includes determining this positional relationship through sequence alignment. Specifically, it can be used as an identifier to identify the composition of the linked STR sequences, and the number of STR repeats can be determined based on whether the specific sequence is detected during sequencing. Furthermore, the specific sequence can also be used to identify the position of the linked STR sequences. For example, in the human genome, the same STR sequence may be distributed at different positions on the same gene, or on different genes or different chromosomes, making it impossible to distinguish STR sequences at different positions based on sequencing results. Since each STR sequence is linked to a specific sequence, sequencing analysis of the STR sequence and the specific sequence can accurately distinguish STR sequences at different positions.

In this paper, the reference sequence is a pre-determined sequence containing chromosome number and the location information of the target site (such as the short tandem repeat sequence here) on the chromosome. The reference sequence can be a pre-assembled DNA and/or RNA sequence determined by oneself, or a publicly available DNA and/or RNA sequence determined by others. It can be any reference template within the biological category to which the target individual belongs, such as all or at least a portion of a publicly available genome assembly sequence of the same biological category. If the target individual is human, its reference sequence can be all or a portion of a human genome reference sequence (also called a reference genome or reference chromosome set), such as HG19 provided by the NCBI database. The sequence alignment or comparison here includes the process of locating a read or a portion of a read onto the reference sequence, as well as the process of obtaining the location/matching results of the read or a portion of a read.

This sequencing method for STR detection has no requirements on read length; in principle, any sequencing platform with a read length greater than one motif sequence (generally 2-6 bp) can be used. Furthermore, after surface hybridization, there is no need to amplify the hybridization product; sequencing can be performed directly on the hybridization product.

This invention does not strictly limit the connection method between the specific sequence and the STR sequence; they can be directly connected or separated by a spacer sequence (short sequence). The length of the spacer sequence should not be too long, ensuring that the sequencing data does not consist entirely of spacer sequences or that the length of the specific sequence read is too short to accurately identify the specific sequence. Furthermore, the short tandem repeat sequence can be linked to a specific sequence at one end or at both ends. Taking the Th01 locus sequencing library in Figure 1 as an example, (AATG)n is the target STR sequence (Th01 locus), AATG is the motif sequence, and the sequences at both ends of the motif sequence are specific sequences. Specific primers are used to amplify the target STR region, forming an amplification product with at least one specific sequence at the 5' and 3' ends. The amplified product is then used to construct a library to obtain the sequencing library. Sequencing libraries typically contain insert fragments/test fragments and adapters. During this process, one or more tags (indexes or barcodes) can be introduced at one or both ends of the insert fragment to ensure that multiple sequencing libraries can be mixed and sequenced on the same flow cell or the same lane, thereby improving sequencing throughput.

According to an embodiment of the present invention, the first sequencing primer has the following nucleotide sequence: (S0)x, wherein S0 represents the motif sequence of a short tandem repeat sequence or its complementary sequence, and x is not less than 1.

Referring to Figure 2, because the sequencing library contains multiple short tandem repeat sequences, when sequencing is performed using the library as a template strand, the first sequencing primer (STR)x will undergo misalignment hybridization with the template strand. That is, the first sequencing primer will randomly hybridize with any part of the short tandem repeat sequences in the library, resulting in different hybridization patterns. Due to the limited sequencing length, two scenarios will occur: specific sequences are detected (referred to as "test-through," corresponding to the test-through read) and no specific sequences are detected (referred to as "non-test-through," corresponding to the non-test-through read). Analyzing the test-through and non-test-through reads can reveal sequence information of the short tandem repeat sequences, such as the repeat number.

In this article, "test-through" refers to the detection of a specific sequence, meaning that at least a portion of the specific sequence linked to a short tandem repeat sequence is detected during the sequencing process. The corresponding base sequence obtained is called a "test-through read". Test-through reads are specifically divided into two cases: a set of specific sequences and partial STR sequences, and a case where all reads are specific sequences.

In this article, "untested" refers to the situation where no specific sequence was detected. It means that no specific sequence linked to the short tandem repeat sequence was detected during the sequencing process, and the corresponding base sequence obtained is called "untested read".

Therefore, the method according to the embodiments of the present invention can accurately achieve high-throughput short-read sequence sequencing, saving sequencing time and sequencing costs.

According to an embodiment of the present invention, the first sequencing primer carried on the solid surface is represented as L-(S0)x, where L represents a linker group used to link the first sequencing primer to the solid surface. (S0)x is connected to the solid surface through the linker group L, i.e., L-(S0)x, thereby achieving the purpose of fixation.

According to embodiments of the present invention, x is 3 to 20 or 3 to 10. For example, x is 3, 4, 4.5, 5, 6, 6.2, 7, 8, 9, 9.7, 10, 11, 12, 13, 13.2, 14, 14.9, 15, 16, 17, 18, 19.

It should be noted that x in the first sequencing primer (S0)x can be an integer, meaning that the primer matches any one or more complete motif sequences on the template strand; or it can be a non-integer, meaning that the primer matches any one or more complete motif sequences connected to the template strand as well as a portion of a motif sequence. Preferably, it is an integer.

This invention does not strictly limit the chip temperature during the sequencing-by-synthesis reaction; it can be flexibly selected according to actual conditions, for example, it can be 37 degrees Celsius. The binding temperature of the first or second sequencing primer to the template strand is preferably higher than the chip's reaction temperature to avoid strand unwinding, which could affect the sequencing reaction.

According to an embodiment of the present invention, the library is not amplified on the chip before performing the synthesis-sequencing reaction on the chip.

According to an embodiment of the present invention, the sequencing-by-synthesis reaction is performed on a single-molecule sequencing platform.

Running a single-molecule sequencing platform (SMS) involves the following steps: placing the hybridization product under conditions suitable for polymerization, including introducing a fluorescently labeled reversible termination base into the detection region via a flow cell; extending the sequencing primers under the catalysis of DNA polymerase to incorporate the reversible termination base into the template strand/hybridization complex; and detecting the corresponding fluorescent signal. This process is repeated multiple times to determine the DNA template sequence.

Referring to Figure 3, in common next-generation sequencing technologies, there is no misalignment hybridization. A cluster (a clonal cluster containing thousands of identical sequences) generates the same signal to detect a single base at the same position in multiple identical molecules within that cluster. In current mainstream high-throughput sequencing platforms, misalignment hybridization is extremely detrimental to sequencing and analysis. Once misalignment hybridization occurs, meaning the primer hybridizes to multiple positions on multiple molecules within a cluster, multiple different DNA positions within the cluster will undergo extension reactions and emit corresponding extension signals during a single extension. This makes it difficult to distinguish the extension signals and identify the corresponding bases, leading to sequencing failure. Therefore, it is preferable to use single-molecule sequencing, i.e., without amplifying the target molecule into clusters on the surface. This eliminates the problem of mixed signals from multiple DNA copies, allowing for accurate sequencing results.

According to an embodiment of the present invention, the second sequencing primer has the following nucleotide sequence: S1-(S0)y, wherein S1 is a specific sequence or a sequence corresponding to the specific sequence, S0 is the motif sequence of a short tandem repeat sequence or its complementary sequence, and y is greater than or equal to 0. S1 in the second sequencing primer can bind to the specific sequence on the template strand to fix the hybridization position, and sequencing begins from the (S0)y end of the primer. Due to the limited sequencing length, two situations may occur: the specific sequence is detected (correspondingly, a test read is obtained) and the specific sequence is not detected (correspondingly, an untested read is obtained). Analyzing the test reads and untested reads can reveal the sequence information of the short tandem repeat sequence, such as the repeat number.

According to an embodiment of the present invention, the solid surface further carries a second sequencing primer, denoted as L-S1-(S0)y, wherein the second sequencing primer is linked to the solid surface using L. The solid surface simultaneously carries both the first and second sequencing primers, and S1-(S0)y is connected to the solid surface via the linker group L.

According to an embodiment of the invention, L does not contain oligonucleotides. According to another embodiment of the invention, L contains oligonucleotides. According to yet another embodiment of the invention, L contains at least one selected from polynucleotides, polyamino acids, polyethylene glycol, and polyethylene glycol-polyamino acid copolymers.

According to an embodiment of the invention, S1 is derived from at least one of the following: (a) an upstream sequence of a short tandem repeat sequence; (b) a downstream sequence of a short tandem repeat sequence; and (c) a complementary sequence of (a) or (b).

The term "derived from" should be interpreted broadly. It can mean that S1 is completely identical to the upstream/downstream sequence or its complementary sequence of STR, or that there is a difference or deletion of one or more bases, as long as it can be guaranteed that S1 and (S0)y can match the same strand (positive or negative strand) of the inserted fragment at the same time.

According to embodiments of the present invention, for a given short tandem repeat sequence, a variety of second sequencing primers are carried on a solid-phase surface, each of which has a different y value. By using a series of second sequencing primers for sequencing, and based on the analysis of test-broken and untested reads, information about the short tandem repeat sequence, such as the repeat number, is obtained.

Understandably, the value of y and the number of y values (corresponding to a series of second sequencing primers of different lengths) are not particularly restricted, as long as there exists a y value that can produce the corresponding test-through read. For example, y can start from 0 and take values continuously up to a number not less than the maximum repeat count of that type of STR locus. For example, according to publicly available databases, the maximum repeat count of a certain STR is m. For instance, currently, the repeat count of the repeat units contained in various STR loci commonly selected for forensic applications is generally less than 60. Therefore, y can take values up to 60, 61, or even higher, to ensure that there are second sequencing primers that can ensure that the sequencing data of that STR(s) contain the corresponding test-through read.

According to an embodiment of the present invention, the value of y is determined based on the following formula: y = a + k × d, where a is a first predetermined constant and a is an integer; d is a plurality of different integers in the range of 0 to 100; k is a second predetermined constant and k is greater than 0.

According to embodiments of the present invention, k is determined based on the read length of sequencing-by-synthesis and/or the length of the motif sequence. Specifically, as the sequencing length of the sequencing-by-synthesis reaction increases, the value of k increases; as the length of the motif increases, the value of k decreases. This reduces the number of possible values for y, meaning fewer types of second sequencing primers need to be designed or synthesized while still ensuring the sequencing data contains the corresponding test-break reads, thus reducing detection costs and possessing high industrial practical value.

According to an embodiment of the present invention, k is determined based on the following formula: where b is the sequencing length of the sequencing-by-synthesis reaction, and t is the length of the motif sequence. The above function is a floor function, and k is an integer not exceeding a certain value. Thus, the maximum k for each STR locus can be obtained based on various STR loci and the sequencing platform used, thereby designing the minimum number of second sequencing primers required for each locus detection. This allows for sequencing and genotyping of a specified STR using the minimum number of second sequencing primers, significantly reducing detection costs and possessing high industrial practical value.

According to an embodiment of the present invention, a is an integer less than k, and/or d is a plurality of consecutive integers in the range of 0 to 10.

According to an embodiment of the present invention, a second sequencing primer is carried on the solid-phase surface, denoted as L-S1-(S0)y, where L represents a linker group used to connect the second sequencing primer to the solid-phase surface. S1-(S0)y is fixed to the solid-phase surface by the linker group L, and S1 can bind to a specific sequence on the template strand to fix the hybridization position. Sequencing begins from the (S0)y end of the primer. Due to the limited sequencing length, two situations may occur: the specific sequence is detected (correspondingly, a test read is obtained) and the specific sequence is not detected (correspondingly, an untested read is obtained). Analyzing the test reads and untested reads can reveal the sequence information of the short tandem repeat sequence, such as the repeat number.

According to an embodiment of the invention, S1 is derived from at least one of the following: (a) an upstream sequence of a short tandem repeat sequence; (b) a downstream sequence of a short tandem repeat sequence; and (c) a complementary sequence of (a) or (b).

According to embodiments of the present invention, for a given short tandem repeat sequence, a variety of second sequencing primers are carried on a solid-phase surface, each of which has a different y value. By using a series of second sequencing primers for sequencing, and based on the analysis of test-broken and untested reads, information about the short tandem repeat sequence, such as the repeat number, is obtained.

According to an embodiment of the present invention, the value of y is determined based on the following formula: y = a + k × d, where a is a first predetermined constant and a is an integer; d is a plurality of integers in the range of 0 to 100, and k is a second predetermined constant.

According to an embodiment of the present invention, k is determined based on the read length of the sequencing-by-synthesis reaction and/or the length of the motif.

According to an embodiment of the present invention, k is determined based on the following formula: where b is the read length of the sequencing-by-synthesis reaction, and t is the length of the motif sequence. Thus, the maximum k for each STR locus can be obtained based on various STR loci and the sequencing platform used, thereby designing the minimum number of second sequencing primers required for each locus detection. This allows for sequencing and genotyping of a specified STR using only the minimum number of second sequencing primers, significantly reducing detection costs and possessing high industrial practical value.

According to an embodiment of the present invention, a is an integer not greater than k, and/or d is a plurality of consecutive integers in the range of 0 to 10.

According to an embodiment of the present invention, the solid surface further carries the first sequencing primer, denoted as L-(S0)x, wherein the first sequencing primer is connected to the solid surface using the L. In addition to the second sequencing primer being carried on the solid surface, the first sequencing primer is also carried, wherein the (S0)x of the first sequencing primer is connected to the solid surface via L.

According to embodiments of the present invention, x is 3 to 20 or 3 to 10. In some embodiments, x is an integer.

According to embodiments of the present invention, L does not contain oligonucleotides, or L contains oligonucleotides, or L contains at least one of polynucleotides, polyamino acids, polyethylene glycol, and polyethylene glycol-polyamino acid copolymers.

According to an embodiment of the present invention, the length of the first sequencing primer is greater than 20 nt, and/or the length of the second sequencing primer is greater than 20 nt.

According to an embodiment of the present invention, the library does not need to be amplified on the solid surface before performing synthesis-sequencing on the solid surface.

According to an embodiment of the present invention, sequencing-by-synthesis is performed on a single-molecule sequencing platform.

According to embodiments of the present invention, the first sequencing primer and the second sequencing primer are independently immobilized on the same or different solid-phase surfaces.

According to embodiments of the present invention, for a given short tandem repeat sequence, the sequencing data includes first sequencing data corresponding to a first sequencing primer and/or second sequencing data corresponding to a second sequencing primer, wherein the number of reads contained in the first sequencing data is not less than the number of reads contained in the second sequencing data. Specifically, for a given short tandem repeat sequence, the sequencing data contains at least 100,000, at least 1 million, or at least 10 million sequencing reads. When the sequencing throughput reaches the above levels, even if the sequencing read length is much lower than the total STR length, this sequencing method can accurately distinguish the repeat number of the short tandem repeat sequence, such as the genotypes of (STR) ₆₂ and (STR) ₆₃ , by sequence frequency.

According to an embodiment of the present invention, the short tandem repeat sequence sequencing method further includes: step 10: aligning the sequencing data with a reference sequence to classify the plurality of reads into test-through reads and non-test-through reads, wherein the test-through reads contain at least a portion of the specific sequence or a complementary sequence of the specific sequence, and the non-test-through reads do not contain the specific sequence or a complementary sequence of the specific sequence; and step 12: determining the number of motif sequences contained in the short tandem repeat sequence based on the test-through reads and non-test-through reads.

As mentioned earlier, the first sequencing primer contains a polynucleotide sequence formed by the tandem repeat units (motif sequences) of several target STRs. Its matching to different positions in the library containing the target STR (i.e., mishybridization) has a certain degree of randomness. Therefore, capturing or hybridizing this library using the first sequencing primer will generate a series of hybridization complexes. Further, extending the first sequencing primer to sequence these hybridization complexes will yield sequencing data containing multiple reads corresponding to these hybridization complexes, or rather, different positions of the target STR. Due to the limited sequencing length, two scenarios may occur: specific sequences are detected (correspondingly, test-through reads are obtained) and no specific sequences are detected (correspondingly, untest-through reads are obtained). By comparing and analyzing this sequencing data with a reference sequence, the complete sequence of the target STR can be determined, that is, the number of repeats of the motif sequence of the STR can be determined, thus enabling genotyping of the STR.

The second sequencing primer contains a polynucleotide sequence formed by the tandem of a specific sequence or its complementary sequence and one or more repeating units (motif sequences) of the target STR. For example, using a second sequencing primer 5'-S1-(S0)y-3' with its 5' end fixed to a designated surface to capture or hybridize a library containing the target STR, a hybridization complex with a double-stranded 5' end can be obtained. That is, the complementary position of the second sequencing primer and the library is fixed. Thus, the hybridization complex can be further sequenced, and the sequencing data will contain multiple reads corresponding to these hybridization complexes or different positions of the target STR. Due to the limited sequencing length, there will be two situations: the specific sequence is detected (correspondingly, a probe read is obtained) and the specific sequence is not detected (correspondingly, an unprobe read is obtained). By comparing and analyzing the sequencing data with the reference sequence, the complete sequence of the target STR can be determined, that is, the number of repeats of the motif sequence of the STR can be determined, thus realizing the genotyping of the STR.

According to an embodiment of the present invention, step 10 includes: step 102: aligning the read segment with a first reference sequence, wherein if the read segment has a segment matching the first reference sequence and the segment length is not less than 10 bp, the read segment is indicated to be the test-through read segment; step 104: aligning the read segment with a second reference sequence, wherein if the read segment has a segment matching the second reference sequence and the segment length is greater than or equal to 7 bp, the read segment is indicated to be the non-test-through read segment; the first reference sequence includes at least a portion of the specific sequence or its complementary sequence, and the second reference sequence is (S0)p, where p is an integer from 2 to 100.

According to an embodiment of the present invention, the error rate of the comparison is set to be less than or equal to 0.1 or 0.2.

According to an embodiment of the present invention, for sequencing data corresponding to a given short tandem repeat sequence generated by the first sequencing primer, step 12 includes: step 122: determining the number of test-through reads and the number of optional untest-through reads; step 124: determining the number of motif sequences contained in the short tandem repeat sequence based on the number of test-through reads.

According to an embodiment of the present invention, step 124 includes: step 1242: determining the proportion of the test-break reads in the sequencing data corresponding to a given short tandem repeat sequence generated by the first sequencing primer based on the number of test-break reads; step 1244: determining the number of motif sequences contained in the short tandem repeat sequence based on the proportion.

According to an embodiment of the present invention, step 1244 includes determining the number of motif sequences contained in the short tandem repeat sequence based on the ratio and according to a predetermined ratio-motif number standard relationship.

Sequencing analysis is performed on multiple sequencing libraries with known motif numbers using the corresponding first sequencing primers to determine the proportion of test-break reads in the generated sequencing data. Based on this proportion and the motif number, a standard relationship between proportion and motif number is determined, such as obtaining a functional relationship between proportion and motif number.

Taking the first sequencing primer (S0) ₄ as an example, when it is fixed on a chip, for a sample with genotype (S0)n, (n-3) hybridization patterns will be generated. In subsequent single-molecule sequencing, if the read length is infinite, n-3 sequences will be generated. The frequency of each sequence is 1/(n-3). When the read length is finite but the total length of the STR sequence exceeds the sequencing read length, assuming the read length is L (bp), the number of S0 bases is x, and the number of repeats is n, then the types of undetected sequences are (rounded down; if this value is less than or equal to 0, it is counted as 0), the types of detected sequences are, then the undetected ratio is, and the detected sequence ratio is, then the proportion of each detected sequence is 1/(n-3).

Assuming a sequencing read length of 40 and an allele distribution of 6-n at the TH01 locus, using (AATG) ₆ as the first sequencing primer, the sequencing results are expected to be as follows:

Table 1. Wearability Ratio Analysis

According to an embodiment of the present invention, for sequencing data corresponding to a given short tandem repeat sequence generated by the second sequencing primer, step 12 includes: step 126: determining the sequence of the short tandem repeat sequence based on the second sequencing primer corresponding to the test-break read and the sequence information of the test-break read.

As mentioned earlier, the second sequencing primer has the following nucleotide sequence: S1-(S0)y. Assuming S0 is AATG and 4nt, and that the maximum repeat count of the short tandem repeat sequence to be tested is 63, and the sequencing length used for sequencing-by-synthesis is 40bp, then the maximum test interval (k) for the short tandem repeat sequence is 40/4 = 10. Thus, the value of y can be determined using the example formula n = a + k × d to determine a series of second sequencing primers. For example, a = 4, k = 10, and d is rounded continuously from 0 until y is not less than 63. This ensures that, under the current sequencing parameters, there exists a second sequencing primer that can produce a test read. Specifically, the series of second sequencing primers is as follows:

STR upstream sequence + [STR] ₄ ; STR upstream sequence + [STR] ₁₄ ; STR upstream sequence + [STR] ₂₄ ;

STR upstream sequence + [STR] ₃₄ ; STR upstream sequence + [STR] ₄₄ ; STR upstream sequence + [STR] ₅₄ ;

[STR] ₄ + STR downstream sequence; [STR] ₁₄ + STR downstream sequence; [STR] ₂₄ + STR downstream sequence;

[STR] ₃₄ + STR downstream sequence; [STR] ₄₄ + STR downstream sequence; [STR] ₅₄ + STR downstream sequence.

The proportion of test segments (test ratio) is determined based on the number of test segments and the total number of segments. The interval in which the repeat count of the STR's motif sequence falls is then determined based on the test ratio. For example: when the test ratio is 1, the repeat count of the STR is between 4 and 14; when the test ratio is 1/2, the repeat count is between 14 and 24; when the test ratio is 1/3, the repeat count is between 24 and 34; when the test ratio is 1/4, the repeat count is between 34 and 44; when the test ratio is 1/5, the repeat count is between 44 and 54; when the test ratio is 1/6, the repeat count is between 54 and 64; and less than 1/6 indicates a repeat count above 64. Then, the number of repeats can be determined based on the test-through read and the corresponding second sequencing primer. For example, if the number of motif sequences or sequences corresponding to motif sequences contained in the test-through read is m, then the number of repeats of the motif sequences contained in the short tandem repeat sequence is the baseline value + m. Here, the "baseline value" is the y-value of the second sequencing primer that generated the test-through read. For example, the number of repeats of the STR can be determined using the second sequencing primer "STR upstream sequence + [STR] ₄ " and the test-through read it produces; here, the baseline value is 4.

(1) Taking the above primer sequence as an example to determine the STR with a motif repeat number of 10.

The hybridization primers that can be detected are the upstream STR sequence + [STR] ₄ and the downstream STR sequence + [STR] ₄ . AATG is required to detect six motif sequences. Other primer sequences are difficult to hybridize with the library or cannot be sequenced after hybridization.

(2) Taking the above primer sequence as an example, the STR with a motif repeat number of 48 was determined.

The primers capable of hybridization and extension to obtain reads are: STR upstream sequence + [STR] ₄ , STR upstream sequence + [STR] ₁₄ , STR upstream sequence + [STR] ₂₄ , STR upstream sequence + [STR] ₃₄ , STR upstream sequence + [STR] ₄₄ , [STR] ₄ + STR downstream sequence, [STR] ₁₄ + STR downstream sequence, [STR] ₂₄ + STR downstream sequence, [STR] ₃₄ + STR downstream sequence, and [STR] ₄₄ + STR downstream sequence. Among these, the primers capable of detection are STR upstream sequence + [STR] ₄₄ and [STR] ₄₄ + STR downstream sequence, which generate 1/5 of all reads. Based on this, it can be concluded that the STR contains more than 44 repeats, and the corresponding primers need to read 4 more motif sequences to detect the repeats. Therefore, the motif sequence repeat count of this STR is 48.

Methods for determining short tandem repeat sequences

In another aspect of the invention, a method for identifying short tandem repeat sequences is proposed. According to an embodiment of the invention, the method includes: (i) acquiring sequencing data of the short tandem repeat sequences, the sequencing data being obtained according to the preceding short tandem repeat sequence sequencing method; (ii) aligning the sequencing data with a reference sequence to classify multiple sequencing reads into test-through reads and non-test-through reads; and (iii) determining the short tandem repeat sequence analysis results of the sample to be tested based on the test-through reads and non-test-through reads.

As mentioned earlier, the first sequencing primer contains a polynucleotide sequence formed by the tandem repeat units (motif sequences) of several target STRs. Its matching to different positions in the library containing the target STR (i.e., mishybridization) has a certain degree of randomness. Therefore, capturing or hybridizing this library using the first sequencing primer will generate a series of hybridization complexes. Further, extending the first sequencing primer to sequence these hybridization complexes will yield sequencing data containing multiple reads corresponding to these hybridization complexes, or rather, different positions of the target STR. Due to the limited sequencing length, two scenarios may occur: specific sequences are detected (correspondingly, test-through reads are obtained) and no specific sequences are detected (correspondingly, untest-through reads are obtained). By comparing and analyzing this sequencing data with a reference sequence, the complete sequence of the target STR can be determined, that is, the number of repeats of the motif sequence of the STR can be determined, thus enabling genotyping of the STR.

The second sequencing primer contains a polynucleotide sequence formed by the tandem of a specific sequence or its complementary sequence and one or more repeating units (motif sequences) of the target STR. For example, using a second sequencing primer 5'-S1-(S0)y-3' with its 5' end fixed to a designated surface to capture or hybridize a library containing the target STR, a hybridization complex with a double-stranded 5' end can be obtained. That is, the complementary position of the second sequencing primer and the library is fixed. Thus, the hybridization complex can be further sequenced, and the sequencing data will contain multiple reads corresponding to these hybridization complexes or different positions of the target STR. Due to the limited sequencing length, there will be two situations: the specific sequence is detected (correspondingly, a probe read is obtained) and the specific sequence is not detected (correspondingly, an unprobe read is obtained). By comparing and analyzing the sequencing data with the reference sequence, the complete sequence of the target STR can be determined, that is, the number of repeats of the motif sequence of the STR can be determined, thus realizing the genotyping of the STR.

According to an embodiment of the present invention, in step (ii), the test-through reads and non-test-through reads are determined by the following methods: (ii-1) the read is aligned with a first reference sequence, and the read has a segment that matches the first reference sequence and the segment length is not less than 10 bp, which indicates that the read is a test-through read; (ii-2) the read is aligned with a second reference sequence, and the read has a segment that matches the second reference sequence and the segment length is greater than or equal to 7 bp, which indicates that the read is a non-test-through read; the first reference sequence contains at least a portion of a specific sequence or its complementary sequence; the second reference sequence is (S0)p, where p is an integer from 2 to 100.

According to an embodiment of the present invention, the error rate of the comparison is set to be less than or equal to 0.1 or 0.2.

According to an embodiment of the present invention, for sequencing data generated by a given first sequencing primer, step (iii) further includes: (iii-1) determining the number of test-through reads and the number of optional untest-through reads; (iii-2) determining the number of motif sequences in the short tandem repeat sequence based on the number of test-through reads.

According to an embodiment of the present invention, step (iii-2) further includes: (iii-2-1) determining the proportion of sequencing data corresponding to a given short tandem repeat sequence generated by the first sequencing primer based on the number of test-through reads; (iii-2-2) determining the number of motif sequences contained in the short tandem repeat sequence based on the proportion.

According to an embodiment of the present invention, in step (iii-2-2), based on the ratio, the number of motif sequences contained in the short tandem repeat sequence is determined according to a predetermined ratio-motif sequence number standard relationship.

Sequencing analysis is performed on multiple sequencing libraries with known motif sequence numbers using the corresponding first sequencing primers to determine the proportion of test-break reads in the generated sequencing data. Based on this proportion and the motif sequence number, a standard relationship between the proportion and the motif sequence number is determined, for example, a functional relationship between the proportion and the motif sequence number is obtained.

Taking the first sequencing primer (S0) ₄ as an example, when it is fixed on a chip, for a sample with genotype (S0)n, (n-3) hybridization patterns will be generated. In subsequent single-molecule sequencing, if the read length is infinite, n-3 sequences will be generated. The frequency of each sequence is 1/(n-3). When the read length is finite but the total length of the STR sequence exceeds the sequencing read length, assuming the read length is L (bp), the number of S0 bases is x, and the number of repeats is n, then the types of undetected sequences are (rounded down; if this value is less than or equal to 0, it is counted as 0), the types of detected sequences are, then the undetected ratio is, and the detected sequence ratio is, then the proportion of each detected sequence is 1/(n-3).

Assuming a sequencing read length of 40 and an allele distribution of 6-n at the TH01 locus, using (AATG) ₆ as the first sequencing primer, the sequencing results are expected to be as follows:

Table 1. Wearability Ratio Analysis

According to an embodiment of the present invention, for sequencing data corresponding to a given short tandem repeat sequence generated by the second sequencing primer, step (iii) further includes: (iii-a) determining the number of motif sequences contained in the short tandem repeat sequence based on the second sequencing primer corresponding to the test-through read and the sequence information of the test-through read.

As mentioned earlier, the second sequencing primer has the following nucleotide sequence: S1-(S0)y. Assuming S0 is AATG and 4nt, and the maximum number of repetitions of the short tandem repeat sequence to be tested is 63, and the sequencing length used for sequencing-by-synthesis is 40bp, then the maximum test interval (k) for the short tandem repeat sequence is 40/4 = 10. Thus, the value of y can be determined using the example formula n = a + k × d to determine a series of second sequencing primers. For example, a = 4, k = 10, and d is continuously rounded from 0 until y is not less than 63. This ensures that, under the current sequencing parameters, there exists a second sequencing primer that can produce a test read. Specifically, the following series of second sequencing primers are obtained: STR upstream sequence + [STR] ₄ ; STR upstream sequence + [STR] ₁₄ ; STR upstream sequence + [STR] ₂₄ ;

STR upstream sequence + [STR] ₃₄ ; STR upstream sequence + [STR] ₄₄ ; STR upstream sequence + [STR] ₅₄ ;

[STR] ₄ + STR downstream sequence; [STR] ₁₄ + STR downstream sequence; [STR] ₂₄ + STR downstream sequence;

[STR] ₃₄ + STR downstream sequence; [STR] ₄₄ + STR downstream sequence; [STR] ₅₄ + STR downstream sequence.

The proportion of test segments (test ratio) is determined based on the number of test segments and the total number of segments. The interval in which the repeat count of the STR's motif sequence falls is then determined based on the test ratio. For example: when the test ratio is 1, the repeat count of the STR is between 4 and 14; when the test ratio is 1/2, the repeat count is between 14 and 24; when the test ratio is 1/3, the repeat count is between 24 and 34; when the test ratio is 1/4, the repeat count is between 34 and 44; when the test ratio is 1/5, the repeat count is between 44 and 54; when the test ratio is 1/6, the repeat count is between 54 and 64; and less than 1/6 indicates a repeat count above 64. Then, the number of repeats can be determined based on the test-through read and the corresponding second sequencing primer. For example, if the number of motif sequences or sequences corresponding to motif sequences contained in the test-through read is m, then the number of repeats of the motif sequences contained in the short tandem repeat sequence is the baseline value + m. Here, the "baseline value" is the y-value of the second sequencing primer that generated the test-through read. For example, the number of repeats of the STR can be determined using the second sequencing primer "STR upstream sequence + [STR] ₄ " and the test-through read it produces; here, the baseline value is 4.

(1) Taking the above primer sequence as an example to determine the STR with a motif repeat number of 10.

The hybridization primers that can be detected are the upstream STR sequence + [STR] ₄ and the downstream STR sequence + [STR] ₄ . AATG is required to detect six motif sequences. Other primer sequences are difficult to hybridize with the library or cannot be sequenced after hybridization.

(2) Taking the above primer sequence as an example, the STR with a motif repeat number of 48 was determined.

The primers capable of hybridization and extension to obtain reads are: STR upstream sequence + [STR] ₄ , STR upstream sequence + [STR] ₁₄ , STR upstream sequence + [STR] ₂₄ , STR upstream sequence + [STR] ₃₄ , STR upstream sequence + [STR] ₄₄ , [STR] ₄ + STR downstream sequence, [STR] ₁₄ + STR downstream sequence, [STR] ₂₄ + STR downstream sequence, [STR] ₃₄ + STR downstream sequence, and [STR] ₄₄ + STR downstream sequence. Among these, the primers capable of detection are STR upstream sequence + [STR] ₄₄ and [STR] ₄₄ + STR downstream sequence, which generate 1/5 of all reads. Based on this, it can be concluded that the STR contains more than 44 repeats, and the corresponding primers need to read 4 more motif sequences to detect the repeats. Therefore, the motif sequence repeat count of this STR is 48.

It should be noted that the features and advantages described above for sequencing short tandem repeat sequences also apply to this method for identifying short tandem repeat sequences, and will not be repeated here.

chip

In another aspect of the invention, a chip is provided. According to an embodiment of the invention, the chip includes: a substrate having a surface; a first sequencing primer fixed on the surface, and the first sequencing primer having the following nucleotide sequence: (S0)x, wherein S0 represents a motif sequence of a short tandem repeat sequence or its complementary sequence, and x is not less than 1.

This invention designs a first sequencing primer that is part of the sequence to be tested, for example, a polynucleotide sequence formed by the tandem repeat units (motif sequences) of several target STRs. Given that testing has shown that the first sequencing primer's hybridization matching to different positions in a library containing the target STR (i.e., mishybridization) has a certain degree of randomness, capturing or hybridizing this library using the first sequencing primer will understandably generate a series of hybridization complexes. Furthermore, extending the first sequencing primer to sequence these hybridization complexes will yield sequencing data containing multiple reads corresponding to these hybridization complexes, or rather, to different positions of the target STR. Analysis of this sequencing data can then determine the complete sequence of the target STR, i.e., the number of repeats of the motif sequence, thus enabling STR genotyping. This method also offers advantages such as saving sequencing time and sequencing costs.

According to an embodiment of the present invention, the first sequencing primer has the following structure: L-(S0)x, wherein L represents a linker group used to immobilize the first sequencing primer on a chip.

According to an embodiment of the present invention, x is 3 to 20 or 3 to 10.

According to an embodiment of the present invention, x is an integer.

According to an embodiment of the present invention, the chip further includes a second sequencing primer, which is fixed on the surface and has the following nucleotide sequence: S1-(S0)y, wherein S1 is a specific sequence or a sequence corresponding to the specific sequence, S0 is a motif sequence of a short tandem repeat sequence or its complementary sequence, the specific sequence is capable of indicating the position of the short tandem repeat sequence on the reference sequence, and y is greater than or equal to 0.

The inventors designed a second sequencing primer that is, for example, a polynucleotide sequence consisting of a specific sequence or a sequence corresponding to the specific sequence and one or more repeating units (motif sequences) of the target STR tandemly. For example, by using a second sequencing primer 5'-S1-(S0)y-3' with its 5' end fixed on a designated surface to capture or hybridize a library containing the target STR, a hybridization complex with a double strand at its 5' end can be obtained. That is, the complementary hybridization position between the second sequencing primer and the library is fixed at one end. Thus, the hybridization complex can be further sequenced to determine the sequence of the STR and achieve the detection of the STR.

More specifically, the second sequencing primer is designed to contain a set of sequences of different lengths, and each sequence of length contains a specific sequence or a sequence corresponding to the specific sequence, as well as (S0)y of different lengths, i.e., a set of sequences with multiple different values of y. Understandably, by using this set of sequences to capture or hybridize a library containing the target STR, a series of hybridization complexes with double strands at the 5' end can be obtained. Sequencing these hybridization complexes will, understandably, produce sequencing data containing multiple reads corresponding to these hybridization complexes or different positions of the target STR. Furthermore, by analyzing this sequencing data, the complete sequence of the STR can be determined, i.e., the number of repetitions of the motif sequence of the STR can be determined, thus achieving genotyping of the STR.

According to an embodiment of the present invention, the second sequencing primer connected to the surface has the following structure: L-S1-(S0)y, wherein the second sequencing primer is fixed to the surface by L.

According to an embodiment of the invention, S1 is derived from at least one of the following: (a) an upstream sequence of the short tandem repeat sequence; (b) a downstream sequence of the short tandem repeat sequence; and (c) a complementary sequence of (a) or (b).

According to an embodiment of the present invention, for a given short tandem repeat sequence, the chip carries a variety of second sequencing primers, each of which has a different y value.

According to an embodiment of the present invention, the value of y is determined based on the following formula: y = a + k × d, where a is a first predetermined constant and a is an integer, d is a plurality of integers in the range of 0 to 100, and k is a second predetermined constant.

According to an embodiment of the present invention, in a scenario where sequencing-by-synthesis is performed using the chip to determine the short tandem repeat sequence, k is determined based on the read length of the sequencing-by-synthesis and/or the length of the motif sequence.

According to an embodiment of the present invention, k is determined based on the following formula: where b is the read length of the sequencing-by-synthesis reaction, and t is the length of the motif sequence.

According to an embodiment of the present invention, a is an integer not greater than k, and/or d is a plurality of consecutive integers in the range of 0 to 10.

According to embodiments of the present invention, L is free of oligonucleotides, or L contains oligonucleotides, or L contains at least one of polynucleotides, polyamino acids, polyethylene glycol, and polyethylene glycol-polyamino acid copolymers.

According to an embodiment of the present invention, the chip is used in a single-molecule sequencing platform.

Furthermore, the present invention proposes another chip. According to an embodiment of the present invention, the chip includes: a substrate having a surface; a second sequencing primer fixed on the surface, and the second sequencing primer having the following nucleotide sequence: S1-(S0)n, wherein S1 is a specific sequence or a sequence corresponding to the specific sequence, S0 is a motif sequence of a short tandem repeat sequence or its complementary sequence, the specific sequence is capable of indicating the position of the short tandem repeat sequence on a reference sequence, and y is greater than or equal to 0.

The inventors designed a second sequencing primer that is, for example, a polynucleotide sequence consisting of a specific sequence or a sequence corresponding to the specific sequence and one or more repeating units (motif sequences) of the target STR tandemly. For example, by using a second sequencing primer 5'-S1-(S0)y-3' with its 5' end fixed on a designated surface to capture or hybridize a library containing the target STR, a hybridization complex with a double strand at its 5' end can be obtained. That is, the complementary hybridization position between the second sequencing primer and the library is fixed at one end. Thus, the hybridization complex can be further sequenced to determine the sequence of the STR and achieve the detection of the STR.

More specifically, the second sequencing primer is designed to contain a set of sequences of different lengths, and each sequence of length contains a specific sequence or a sequence corresponding to the specific sequence, as well as (S0)y of different lengths, i.e., a set of sequences with multiple different values of y. Understandably, by using this set of sequences to capture or hybridize a library containing the target STR, a series of hybridization complexes with double strands at the 5' end can be obtained. Sequencing these hybridization complexes will, understandably, produce sequencing data containing multiple reads corresponding to these hybridization complexes or different positions of the target STR. Furthermore, by analyzing this sequencing data, the complete sequence of the STR can be determined, i.e., the number of repetitions of the motif sequence of the STR can be determined, thus achieving genotyping of the STR.

According to an embodiment of the invention, S1 is derived from at least one of the following: (a) an upstream sequence of a short tandem repeat sequence; (b) a downstream sequence of a short tandem repeat sequence; and (c) a complementary sequence of (a) or (b).

According to an embodiment of the present invention, for a given short tandem repeat sequence, a chip carries a variety of second sequencing primers, each of which has a different y value.

According to an embodiment of the present invention, the value of y is determined based on the following formula: y = a + k × d, where a is a first predetermined constant and a is an integer, d is a plurality of integers in the range of 0 to 100, and k is a second predetermined constant.

According to an embodiment of the present invention, in a scenario where sequencing-by-synthesis is performed using a chip to determine short tandem repeat sequences, k is determined based on the read length of the sequencing-by-synthesis reaction and/or the length of the motif sequence.

According to an embodiment of the present invention, k is determined based on the following formula: where b is the read length of the sequencing-by-synthesis reaction, and t is the length of the motif sequence.

According to an embodiment of the present invention, a is an integer not greater than k, and/or d is a plurality of consecutive integers in the range of 0 to 10.

According to an embodiment of the present invention, the chip further includes a first sequencing primer, which is attached to the surface and has the following nucleotide sequence: (S0)x, wherein S0 represents the motif sequence of a short tandem repeat sequence or its complementary sequence, and x is not less than 1.

According to an embodiment of the present invention, the first sequencing primer connected to the surface has the following structure: L-(S0)x, wherein the first sequencing primer is fixed on the chip by means of the L.

According to an embodiment of the present invention, x is 3 to 20 or 3 to 10.

According to an embodiment of the present invention, x is an integer.

According to embodiments of the present invention, L does not contain oligonucleotides, or L contains oligonucleotides, or L contains at least one of polynucleotides, polyamino acids, polyethylene glycol, and polyethylene glycol-polyamino acid copolymers.

It should be noted that the features and advantages described above for sequencing or identifying short tandem repeat sequences also apply to this chip, and will not be repeated here.

Reagent test kit

In another aspect of the invention, a kit is provided. According to an embodiment of the invention, the kit includes: (1) a first sequencing primer and/or a second sequencing primer as described in the preceding short tandem repeat sequencing method; or (2) a chip as described above. Thus, this kit can be used to accurately perform high-throughput short-read STR sequence sequencing, saving sequencing time and costs.

It should be noted that the features and advantages described above for the short tandem repeat sequence sequencing method and chip also apply to this kit, and will not be repeated here.

Methods for identifying individuals

In another aspect, the present invention proposes a method for identifying individuals. According to an embodiment of the invention, the method includes: determining the sequences of multiple given short tandem repeat sequences in a sample to be tested, the sample containing nucleic acids, using the aforementioned short tandem repeat sequencing method; and determining, based on the sequences, the origin of one or more individuals from which the sample to be tested originates. Thus, this method can accurately identify individuals, for example, to determine genetic relationships.

The STR testing method or kit provided in any of the above embodiments can be used for various STR testing-based applications, such as forensic individual identification and paternity testing, and can also assist in the diagnosis or screening of genetic variations, such as assisting in prenatal screening.

It should be noted that the features and advantages described above for short tandem repeat sequencing methods also apply to this method, and will not be repeated here.

The present invention will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products. Unless otherwise stated, the specific sequences of single-stranded or double-stranded nucleic acid molecules exemplified herein, including libraries, insert fragments, nucleic acid fragments, target sequences, sites, motif sequences, polynucleotide sequences, adapters, sequencing primers, fixed strands, probes, or hybridization strands, are all written from left to right in a 5' to 3' direction.

Example 1

In this embodiment, the number of STR repeats is determined according to the following method:

1. Building a document library

Refer to Figure 4 for the library creation process to construct the library. The library structure is shown in Figure 5.

2. Fixed chain design

The fixed strands (sequencing primers) were designed as “L-[S0] ₅ ” and “L-[S0] ₆ ”, where L stands for TTTTTTTTTT and S0 stands for AATG. The specific sequences are shown in Table 1.

3. Hybridization chain (synthetic target) design

The hybridization chain design is shown in the table below.

Table 1. Design of fixed-chain and hybrid chains

4. Operating Procedures

The immobilized strand was fixed onto the chip using a single-molecule immobilization procedure, and then the constructed library (hybridized strand) was hybridized onto the chip. After hybridization, sequencing was performed from the 3' end of the immobilized strand according to the single-molecule sequencing procedure. Single-molecule fluorescence sequencing instrument: GenoCare 1600, 80 rounds of two-color sequencing, refer to Table 2.

Table 2 Sequencing conditions

5. Analysis

1) The Fast data from the machine is first compared with the first reference sequence using a local alignment method.

The sequence “AGGGAAATAAGGGAGGAACAGGCCAATGGGAATC” is compared. If the error rate of the alignment result is less than or equal to the preset threshold (0.1 or 0.2) and the length of the matched sequence is greater than or equal to 10 bp, it is considered to be a sequence that has been tested in the Repeat region.

2) Align the remaining sequence with the second reference sequence “AATGAATG”. If the error rate of the alignment result is less than 0.2 and the length of the matched sequence is greater than or equal to 7bp, it is considered that the repeat region has been detected but not penetrated.

3) Analyze the sequence from step two. First, locate the repeat regions in the sequence, i.e., remove the sequence segment "AGGGAAATAAGGGAGGAACAGGCCAATGGGAATC". Then, the program determines the number of repeats. Count the number of reads with repeat values of 0, 1, 2, 3... respectively.

4) Divide the number of reads in step 1 by the sum of the number of reads in steps 1 and 2 to obtain the "test-through ratio": that is, the ratio of sequencing results that are upstream (downstream) specific sequences of STR.

The results are shown in Figure 6. Using (AATG) ₆ primers as the fixed strand, sequencing libraries containing STR repeat sequence lengths of 36, 40, 44, and 48 (i.e., 9, 10, 11, and 12 repeats in the hybridization strand) showed a linear negative correlation between the test-through ratio and the number of repeats. This result is consistent with theoretical predictions. This indicates that the STR repeat number can be determined using this method. Additionally, it's worth mentioning that for a specific STR locus to be tested, the appropriate primer length, hybridization, and/or sequencing reaction conditions can be determined based on the base composition of its motif sequence and experience with polymerase extension reactions to successfully obtain sequencing data. For example, if the AT ratio of the primers designed based on the specific motif sequence is high, and the distribution of sequencing data differs significantly from the prediction, adjustments can be made according to conventional PCR experience, such as designing longer primers containing more motif sequences and/or adjusting the polymerization reaction temperature.

Example 2

In this embodiment, the required throughput is investigated to effectively distinguish STR sequences with 63 repeats and 62 repeats (referred to as "63repeat" and "62repeat" respectively) under Gaussian distributions of read lengths avg=40, std=5 and avg=40, std=10. The default capture sequence repeat=10.

Figures 7 and 8 show the simulated read distributions using 1M and 10M reads, respectively, under a Gaussian distribution with avg=40 and std=5 (this distribution is the reciprocal of the proportion of 0 repeats remaining in the simulated sequencing).

Figures 9 and 10 show the simulated read distributions using 1M and 10M reads, respectively, under a Gaussian distribution with avg=40 and std=10 (this distribution is the reciprocal of the proportion of 0 repeats remaining in the simulated sequencing).

It is evident that when the throughput reaches 10Mreads, even if the sequencing read length is much lower than the total STR length, this sequencing method can accurately distinguish the genotypes of (STR) ₆₂ and (STR) ₆₃ by sequence frequency.

In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims (13)

1. A method for sequencing short tandem repeat sequences, characterized in that it comprises: Obtain a library, wherein the inserted fragments of the library contain short tandem repeat sequences and specific sequences linked to the short tandem repeat sequences; The library is sequenced while synthesizing on a solid surface to obtain sequencing data, which includes multiple reads. The solid surface carries at least one of a first sequencing primer and a second sequencing primer. The first sequencing primer has the following nucleotide sequence: (S0)x, where S0 represents the motif sequence of the short tandem repeat sequence or its complementary sequence, and x is not less than 1. The second sequencing primer has the following nucleotide sequence: S1-(S0)y, where S1 is the specific sequence or the sequence corresponding to the specific sequence, S0 is the motif sequence of the short tandem repeat sequence or its complementary sequence, and y is greater than or equal to 0. 2. The method according to claim 1, wherein the solid-phase surface carries the first sequencing primer, as shown in: L-(S0)x, where, L represents a linker group, used to link the first sequencing primer to the solid surface; Optionally, x is 3–20 or 3–10; Optionally, x is an integer; Optionally, the solid surface further carries the second sequencing primer, as shown below: L-S1-(S0)y, where, The second sequencing primer is attached to the solid surface using the L; Optionally, the L does not contain oligonucleotides; Optionally, the L comprises an oligonucleotide; Optionally, the L comprises at least one of polynucleotides, polyamino acids, polyethylene glycol, and polyethylene glycol-polyamino acid copolymers; Optionally, S1 is derived from at least one of the following: (a) The upstream sequence of the short tandem repeat sequence; (b) the downstream sequence of the short tandem repeat sequence; and (c)(a) or (b) complementary sequences; Optionally, for a given short tandem repeat sequence, the solid surface carries a variety of second sequencing primers, each of which has a different y value; Optionally, the value of y is determined based on the following formula: y = a + k × d, where, a is a first predetermined constant, and a is an integer. d can be multiple distinct integers in the range of 0 to 100. k is a second predetermined constant, and k is greater than 0; Optionally, k is determined based on the read length of the sequencing-by-synthesis process and/or the length of the motif sequence; Optionally, k is determined based on the following formula: in, b is the read length of the sequencing-by-synthesis process. t is the length of the motif sequence; Optionally, a is an integer less than k, and/or d is a plurality of consecutive integers in the range of 0 to 10. 3. The method according to claim 1, wherein the solid-phase surface carries the second sequencing primer, as shown in: L-S1-(S0)y, where, L represents a linker group, used to link the second sequencing primer to the solid surface; Optionally, S1 is derived from at least one of the following: (a) The upstream sequence of the short tandem repeat sequence; (b) the downstream sequence of the short tandem repeat sequence; and (c)(a) or (b) complementary sequences; Optionally, for a given short tandem repeat sequence, the solid surface carries a variety of second sequencing primers, each of which has a different y value; Optionally, the value of y is determined based on the following formula: y = a + k × d, where, a is a first predetermined constant, and a is an integer; d can be multiple integers in the range of 0 to 100. k is a second predetermined constant; Optionally, k is determined based on the read length of the sequencing-by-synthesis reaction and/or the length of the motif; Optionally, k is determined based on the following formula: in, b represents the read length of the sequencing-by-synthesis reaction. t is the length of the motif sequence; Optionally, a is an integer not greater than k, and/or d is a plurality of consecutive integers in the range of 0 to 10; Optionally, the solid surface further carries the first sequencing primer, as shown below: L-(S0)x, where, The first sequencing primer is ligated to the solid surface using the L; Optionally, x is 3–20 or 3–10; Optionally, x is an integer; Optionally, the L does not contain oligonucleotides; Optionally, the L comprises an oligonucleotide; Optionally, the L comprises at least one of polynucleotides, polyamino acids, polyethylene glycol, and polyethylene glycol-polyamino acid copolymers. 4. The method according to any one of claims 1-3, wherein the length of the first sequencing primer is greater than 20 nt, and/or the length of the second sequencing primer is greater than 20 nt; Optionally, the library does not need to be amplified on the solid surface before performing sequencing-by-synthesis on the solid surface; Optionally, the sequencing-by-synthesis is performed on a single-molecule sequencing platform; Optionally, the first sequencing primer and the second sequencing primer are independently immobilized on the same or different solid-phase surfaces; Optionally, for a given short tandem repeat sequence, the sequencing data includes first sequencing data corresponding to the first sequencing primer and/or second sequencing data corresponding to the second sequencing primer, wherein the number of reads contained in the first sequencing data is not less than the number of reads contained in the second sequencing data. Optionally, for a given short tandem repeat sequence, the sequencing data contains at least 100,000, at least 1 million, or at least 10 million reads. 5. The method according to any one of claims 1-4, characterized in that the method further comprises: Step 10: Align the sequencing data with a reference sequence to classify the multiple reads into test-through reads and non-test-through reads. The test-through reads contain at least a portion of the specific sequence or its complementary sequence, while the non-test-through reads do not contain the specific sequence or its complementary sequence. Step 12: Based on the measured and unmeasured reads, determine the number of motif sequences contained in the short tandem repeat sequence; Optionally, step 10 includes: Step 102: Compare the read segment with the first reference sequence. If the read segment has a segment that matches the first reference sequence and the segment length is not less than 10bp, then the read segment is indicated to be the test segment. Step 104: Compare the read segment with the second reference sequence. If the read segment has a segment that matches the second reference sequence and the segment length is greater than or equal to 7bp, then the read segment is indicated to be the undetected read segment. The first reference sequence comprises at least a portion of the specific sequence or its complementary sequence, and the second reference sequence is (S0)p, where p is an integer from 2 to 100; Optionally, the error rate of the comparison is set to be less than or equal to 0.1 or 0.2; Optionally, for the sequencing data corresponding to a given short tandem repeat sequence generated by the first sequencing primer, step 12 includes: Step 122: Determine the number of the penetration test segments and the number of the optional untested penetration test segments; Step 124: Based on the number of the test-through segments, determine the number of motif sequences contained in the short tandem repeat sequence; Optionally, step 124 includes: Step 1242: Based on the number of test-breakthrough reads, determine the proportion of the test-breakthrough reads in the sequencing data corresponding to the given short tandem repeat sequence generated by the first sequencing primer; Step 1244: Based on the ratio, determine the number of motif sequences contained in the short tandem repeat sequence; Optionally, step 1244 includes, Based on the ratio, the number of motif sequences contained in the short tandem repeat sequence is determined according to a predetermined ratio-motif number standard relationship; Optionally, for the sequencing data corresponding to a given short tandem repeat sequence generated by the second sequencing primer, step 12 includes: Step 126: Based on the second sequencing primer corresponding to the test-break read and the sequence information of the test-break read, determine the sequence of the short tandem repeat sequence. 6. A method for determining short tandem repeat sequences, characterized in that it comprises: (i) Obtain sequencing data of the short tandem repeat sequence, wherein the sequencing data is obtained by the method according to any one of claims 1 to 5; (ii) The sequencing data is aligned with a reference sequence to classify the plurality of reads into test-through reads and non-test-through reads, wherein the test-through reads contain at least a portion of the specific sequence or a complementary sequence of the specific sequence, and the non-test-through reads do not contain the non-specific sequence or a complementary sequence of the non-specific sequence; and (iii) Determine the short tandem repeat sequence based on the measured and unmeasured segments. 7. The method according to claim 6, characterized in that, in step (ii), the measured penetration reading segment and the unmeasured penetration reading segment are determined by the following method: (ii-1) The read segment is compared with the first reference sequence, wherein the read segment has a segment that matches the first reference sequence and the segment length is not less than 10bp, which is an indication that the read segment is a test-through read segment; (ii-2) The read segment is compared with the second reference sequence, wherein the read segment has a segment that matches the second reference sequence and the segment length is greater than or equal to 7bp, which is an indication that the read segment is an undetected read segment; The first reference sequence comprises at least a portion of the specific sequence or its complementary sequence; the second reference sequence is (S0)p, where p is an integer from 2 to 100; Optionally, the error rate of the comparison is set to be less than or equal to 0.1 or 0.2; Optionally, for the sequencing data generated by the given first sequencing primer, step (iii) further includes: (iii-1) Determine the number of the penetration test segments and the number of optional non-penetration test segments; (iii-2) Based on the number of the test-through segments, determine the number of the motif sequences in the short tandem repeat sequence; Optionally, step (iii-2) further includes: (iii-2-1) Based on the number of test-through reads, determine the proportion of the test-through reads in the sequencing data corresponding to a given short tandem repeat sequence generated by the first sequencing primer; (iii-2-2) Based on the ratio, determine the number of motif sequences contained in the short tandem repeat sequence; Optionally, in step (iii-2-2), based on the ratio, the number of motif sequences contained in the short tandem repeat sequence is determined according to a predetermined ratio-motif sequence number standard relationship; Optionally, step (iii) further includes, for the sequencing data corresponding to a given short tandem repeat sequence generated by the second sequencing primer: (iii-a) Based on the second sequencing primer corresponding to the test-break read and the sequence information of the test-break read, determine the number of motif sequences contained in the short tandem repeat sequence. 8. A chip, characterized in that it comprises: A substrate, having a surface; The first sequencing primer is fixed on the surface and has the following nucleotide sequence: (S0)x， Wherein, S0 represents the motif sequence of the short tandem repeat sequence or its complementary sequence. x is not less than 1. 9. The chip according to claim 8, wherein the first sequencing primer has the following structure: L-(S0)x, Wherein, L represents a linker group, used to immobilize the first sequencing primer on the chip; Optionally, x is 3–20 or 3–10; Optionally, x is an integer; Optionally, the system further includes a second sequencing primer, which is immobilized on the surface, and the second sequencing primer has the following nucleotide sequence: S1-(S0)y, where, S1 is the specific sequence or a sequence corresponding to the specific sequence, and S0 is the motif sequence of the short tandem repeat sequence or its complementary sequence. The specific sequence can indicate the position of the short tandem repeat sequence on the reference sequence. y is greater than or equal to 0; Optionally, the second sequencing primer connected to the surface has the following structure: L-S1-(S0)y, wherein the second sequencing primer is immobilized on the surface by means of L; Optionally, S1 is derived from at least one of the following: (a) The upstream sequence of the short tandem repeat sequence; (b) the downstream sequence of the short tandem repeat sequence; and (c)(a) or (b) complementary sequences; Optionally, for a given short tandem repeat sequence, the chip carries multiple second sequencing primers, each of which has a different y value; Optionally, the value of y is determined based on the following formula: y = a + k × d, where, a is a first predetermined constant, and a is an integer. d can be multiple integers in the range of 0 to 100. k is a second predetermined constant; Optionally, in the scenario of using the chip to perform sequencing-by-synthesis to determine the short tandem repeat sequence, k is determined based on the read length of the sequencing-by-synthesis and/or the length of the motif sequence; Optionally, k is determined based on the following formula: in, b represents the read length of the sequencing-by-synthesis reaction. t is the length of the motif sequence; Optionally, a is an integer not greater than k, and/or d is a plurality of consecutive integers in the range of 0 to 10; Optionally, the L does not contain oligonucleotides; Optionally, the L comprises an oligonucleotide; Optionally, the L comprises at least one of polynucleotides, polyamino acids, polyethylene glycol, and polyethylene glycol-polyamino acid copolymers; Optionally, the chip is used in a single-molecule sequencing platform. 10. A chip, characterized in that it comprises: A substrate, having a surface; The second sequencing primer is immobilized on the surface and has the following nucleotide sequence: S1-(S0)y， Wherein, S1 is the specific sequence or a sequence corresponding to the specific sequence, and S0 is the motif sequence of the short tandem repeat sequence or its complementary sequence. The specific sequence can indicate the position of the short tandem repeat sequence on the reference sequence. y is greater than or equal to 0. 11. The chip according to claim 10, wherein S1 is derived from at least one of the following: (a) The upstream sequence of the short tandem repeat sequence; (b) the downstream sequence of the short tandem repeat sequence; and (c)(a) or (b) complementary sequences; Optionally, for a given short tandem repeat sequence, the chip carries multiple second sequencing primers, each of which has a different y value; Optionally, the value of y is determined based on the following formula: y = a + k × d, where, a is a first predetermined constant, and a is an integer. d can be multiple integers in the range of 0 to 100. k is a second predetermined constant; Optionally, in the scenario of using the chip to perform sequencing-by-synthesis to determine the short tandem repeat sequence, k is determined based on the read length of the sequencing-by-synthesis reaction and/or the length of the motif sequence; Optionally, k is determined based on the following formula: in, b represents the read length of the sequencing-by-synthesis reaction. t is the length of the motif sequence; Optionally, a is an integer not greater than k, and/or d is a plurality of consecutive integers in the range of 0 to 10; Optionally, the chip further includes a first sequencing primer connected to the surface, and the first sequencing primer has the following nucleotide sequence: (S0)x, where, S0 represents the motif sequence of the short tandem repeat sequence or its complementary sequence. x is not less than 1; Optionally, the first sequencing primer connected to the surface has the following structure: L-(S0)x, wherein the first sequencing primer is immobilized on the chip using the L; Optionally, x is 3–20 or 3–10; Optionally, x is an integer; Optionally, the L does not contain oligonucleotides; Optionally, the L comprises an oligonucleotide; Optionally, the L comprises at least one of polynucleotides, polyamino acids, polyethylene glycol, and polyethylene glycol-polyamino acid copolymers. 12. A reagent kit, characterized in that it comprises: (1) the first sequencing primer and/or the second sequencing primer as described in any one of claims 1-7; or (2) The chip as described in any one of claims 8-11. 13. A method for identifying an individual, characterized in that it comprises: The method according to any one of claims 1-7 determines the sequence of a plurality of given short tandem repeat sequences in a test sample, the test sample comprising nucleic acids; Based on the sequence, the origin of the test sample can be determined from one or more individuals.