Method for constructing linkage analysis library based on single sperm or polar body

Abstract

The invention discloses a method for constructing a linkage analysis library based on single sperms or polar bodies. The method comprises the following steps: splitting a single sperm or a polar body to prepare a split single sperm or polar body; and carrying out multiple PCR amplification, terminal repair, linker connection and PCR enrichment and purification to prepare the target library. The method at least has the advantages that single cell whole genome amplification does not need to be carried out, library construction can be directly carried out on a polar body or a single sperm, the detection period is shortened, and the detection cost is reduced.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of molecular biology detection, and in particular to a method for constructing a linkage analysis library based on a single sperm or a polar body. BACKGROUND

[0002] A genetic disease refers to a disease caused by a change in the structure or function of a gene or genome. A monogenic genetic disease is a disease caused by a mutation in a specific gene. These gene mutations can be inherited by offspring, causing the offspring to suffer from the same disease or symptoms. Due to its genetic characteristics, most monogenic diseases are teratogenic, disabling, and lethal, and there is a lack of targeted treatment and drugs in clinical practice, and the treatment cost is high. With the rapid development of molecular genetic diagnosis technology, to prevent or prevent birth defects caused by this, preimplantation genetic diagnosis technology provides a feasibility for blocking the vertical transmission of pathogenic genes and preventing the birth of children with genetic diseases.

[0003] Preimplantation genetic testing for monogenic (PGT-M) selects normal embryos for implantation by performing genetic testing on the embryos, thereby avoiding harm to pregnant women and families caused by selective abortion. In order to ensure the accuracy of the test results and avoid diagnostic ambiguity caused by factors such as allele drop-out (ADO) and gene recombination, direct detection of gene pathogenic variants combined with NGS-based genetic polymorphic site (STR or SNP) linkage analysis technology is currently widely used in clinical practice. In the case of incomplete family members, polar bodies or single sperm carrying pathogenic mutations can be used as the basis for linkage analysis.

[0004] Currently, linkage analysis using polar bodies or single sperm carrying pathogenic mutations requires single-cell whole-genome amplification (MDA technology, DOP-PCR technology, and MALBAC technology), quality inspection of single amplification products, and then multiple PCR library construction and sequencing.

[0005] However, the entire experimental process is complicated and time-consuming, and single-cell whole-genome amplification not only has high detection costs, but if amplification errors occur, they will be amplified indefinitely, resulting in inaccurate test results. At the same time, due to the extremely low amount of single sperm / polar body samples, single-cell whole-genome amplification will result in a large amount of non-specific amplification and amplification bias, resulting in a decrease in the coverage of the target genome or target region, causing greater deviations in the sequencing depth of different polymorphic sites in multiple PCR amplification, affecting uniformity and accuracy, and thus affecting the results of linkage analysis experiments. At the same time, due to the influence of sequence GC content, high GC regions often have poor coverage during single-cell whole-genome amplification, making subsequent detection difficult or impossible.

[0006] Therefore, a new library construction method based on single sperm / polar body linkage analysis is urgently needed to solve the current problems. Summary of the Invention

[0007] Based on this, it is necessary to provide at least a method for constructing a linkage analysis library based on a single sperm or polar body.

[0008] In a first aspect of the present application, a method for constructing a linkage analysis library based on a single sperm or polar body is provided, comprising the following steps:

[0009] Single sperm or polar bodies are lysed to prepare lysed single sperm or polar bodies; multiple PCR amplification, end repair, adapter ligation, PCR enrichment, and purification are performed to prepare the target library.

[0010] In some embodiments, the single sperm or polar body is lysed by alkaline lysis or enzymatic lysis.

[0011] In some embodiments, the alkaline lysis conditions are: lysis temperature 56° C. to 65° C., incubation 10 min to 60 min.

[0012] In some embodiments, the alkaline lysis conditions are: lysis temperature 65° C., incubation 10 min to 20 min.

[0013] In some embodiments, the step of performing multiplex PCR amplification after lysis comprises:

[0014] The lysed single sperm or polar body is mixed with a buffer, dNTP, a SNP site amplification primer pool within the range of 1M to 2M upstream and downstream of the target gene mutation site, and a DNA polymerase to form an amplification system, and the amplification system is amplified to prepare an amplification product.

[0015] In some embodiments, the amplification system further includes a PCR enhancer.

[0016] In some embodiments, the PCR enhancer comprises one or more of betaine, glycerol, dimethyl sulfoxide, bovine serum albumin, tetramethylammonium chloride, Tween 20, and Tween 40.

[0017] In some embodiments, the adaptor ligation product is purified after adaptor ligation.

[0018] In some embodiments, the adapter ligation product is purified using purification magnetic beads.

[0019] In some embodiments, the adapter ligation product is purified using 1X to 1.5X purification magnetic beads.

[0020] In some embodiments, the purification method is magnetic bead purification.

[0021] In some embodiments, purification is performed using 1X to 1.5X purification magnetic beads.

[0022] In some embodiments, the platform used to sequence the target library includes one or more of a BGI platform, an Illumina platform, and a Proton platform.

[0023] In some embodiments, the method does not rely on a reference sample, which includes a proband.

[0024] The method for constructing a linkage analysis library based on a single sperm or polar body in one embodiment of the present application has at least the following advantages:

[0025] 1) No single-cell whole genome amplification is required, and libraries can be constructed directly from polar bodies or single sperm, shortening the testing cycle and reducing testing costs;

[0026] 2) It can reduce the impact of PCR amplification errors on the accuracy of test results;

[0027] 3) Reduce nonspecific amplification and amplification bias, thereby reducing the problem of excessive coverage deviation of the target genome or target region;

[0028] 4) Improve coverage, capture efficiency, and uniformity; multiplex PCR panel design principles and amplification systems can be adjusted to target high GC content and amplification uniformity, regardless of the coverage of single-cell whole-genome amplification. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the technical solutions in the implementation methods and examples of this application and to more completely understand the application and its beneficial effects, the following briefly introduces the drawings required for the description of the implementation methods or examples. Obviously, the drawings described below are only some embodiments of the present application. For those skilled in the art, other drawings can be obtained based on these drawings without creative work. It should also be noted that the drawings are all drawn in a simplified form and are only used to conveniently and clearly assist in explaining the present application.

[0030] Figure 1 This is a flow chart of an implementation scheme in one embodiment of the present application (Effect Example 1).

[0031] Figure 2 This is a flow chart of an implementation scheme in one embodiment of the present application (Effect Example 2). DETAILED DESCRIPTION

[0032] To facilitate understanding of the present application, a more comprehensive description of the present application will be provided below with reference to the accompanying drawings. The accompanying drawings illustrate preferred embodiments of the present application. However, the present application may be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a more thorough and comprehensive understanding of the disclosure of the present application.

[0033] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this application pertains. The terms used herein in the specification of this application are for the purpose of describing specific embodiments only and are not intended to limit this application. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0034] In this application, unless otherwise specified, "one or more" refers to any one of the listed items or any combination of the listed items. Similarly, "one or more" and other similar expressions that refer to "one or more" are also understood in the same way unless otherwise specified.

[0035] The terms "combination thereof", "any combination thereof", "any combination thereof" and the like used in this application include all suitable combinations of any two or more of the listed items.

[0036] In this application, the word "suitable" in "suitable combination", "suitable method", "any suitable method", etc. shall be based on the ability to implement the technical solution of this application, solve the technical problems of this application, and achieve the expected technical effects of this application.

[0037] In this application, the terms "further," "further," "particularly," "for example," "such as," "example," and "for example" are used for descriptive purposes to indicate that the preceding and following technical solutions are related in terms of the content covered, but should not be construed as limiting the preceding technical solution or the scope of protection of this application. In this application, unless otherwise specified, A (such as B) means that B is a non-limiting example of A, and it is understood that A is not limited to B.

[0038] In this application, the terms "optionally," "optional," and "optional" mean optional, that is, they refer to either option selected from the two parallel options of "yes" or "no." If multiple "options" appear in a technical solution, unless otherwise specified and there are no contradictions or mutual constraints, each "optional" is independent. Unless otherwise specified, the descriptions "optionally include," "optionally include," etc. in this application, taking "optionally include" as an example, mean "may include or not include."

[0039] The terms "comprise", "contain", and "include", as used herein, are synonymous with each other, are inclusive or open-ended and do not exclude additional, unrecited members, or features. Members or features, for example, materials or components, structures, elements, instruments, etc.; non-limiting examples of members or features also like actions, conditions, timing, states, etc.

[0040] In the present application, the technical features or technical solutions described in open language include closed technical features or technical solutions composed of listed contents, and also include open technical features or technical solutions containing listed contents.

[0041] In the present application, the exemplary description involving "in some embodiments (or examples)", "in an embodiment (or example)", etc. can cover but is not limited to the following meanings: these solutions can be combined with other solutions in a suitable manner to form new technical solutions.

[0042] In the present application, in "first aspect", "second aspect", "third aspect", "fourth aspect", etc., the terms "first", "second", "third", "fourth", etc. are only for descriptive purposes, and cannot be understood as indicating or implying relative importance or quantity, nor can it be understood as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first", "second", "third", "fourth", etc. only serve the purpose of non-exhaustive enumeration description, and should be understood as not constituting a closed limitation on the quantity.

[0043] In the present application, with respect to the numerical interval (i.e. numerical range), if no special instructions are given, the distribution of the optional values in the numerical interval is considered to be continuous, and includes both numerical endpoints (i.e. minimum value and maximum value) of the numerical interval, and every value between the two numerical endpoints. If no special instructions are given, when the numerical interval only points to the integers in the numerical interval, including the two endpoint integers of the numerical range and every integer between the two endpoints, it is equivalent to directly listing each integer. When multiple numerical ranges are provided to describe a feature or characteristic, these numerical ranges can be combined. In other words, unless otherwise indicated, the numerical ranges disclosed herein should be understood to include any and all sub-ranges therein. The "numerical value" in the numerical interval can be any quantitative value, such as a number, a percentage, a ratio, etc. The "numerical interval" is allowed to include numerical interval types such as percentage interval, ratio interval, and value interval in a broad sense.

[0044] In the present application, unless otherwise explicitly stated herein, the execution of the steps involved in the method flow has no strict order limitation, and can be executed in other orders than described. Moreover, any step can include multiple sub-steps or multiple stages, which do not necessarily be executed at the same time, but can be executed at different times, and the execution order is not necessarily sequential, but can be executed alternately or simultaneously with other steps or sub-steps or stages of other steps.

[0045] Without performing single-cell whole genome amplification, the multiple PCR library construction is directly performed after lysis, which reduces the detection cost and shortens the detection period; effectively reduces the amplification bias and error rate, improves the coverage, uniformity and capture efficiency, and further improves the accuracy of detection; the multiple PCR panel design can be adjusted accordingly, which better ensures the uniformity, rather than the amplification bias caused by whole genome amplification, making the multiple PCR panel design more difficult or difficult to meet the requirements; due to the GC content of the sequence, the coverage of single-cell whole genome amplification in high GC region is usually poor, which causes difficulties or inability to detect in subsequent detection, therefore, without performing single-cell whole genome amplification, the amplification system can be adjusted for targeted enrichment in high GC target region during multiple PCR amplification, thereby improving the coverage; it has universality and wide application range; it is simple and efficient to operate, suitable for routine clinical work, and easy to popularize.

[0046] In the first aspect of the present application, a method for constructing a linkage analysis library based on a single sperm or a polar body is provided, which comprises the following steps:

[0047] The single sperm or the polar body is lysed to prepare the lysed single sperm or the polar body; multiple PCR amplification, end repair, adapter ligation and PCR enrichment, purification are performed to prepare the target library.

[0048] In some embodiments, the lysis of the single sperm or the polar body is alkaline lysis or enzyme lysis.

[0049] In some embodiments, the alkaline lysis conditions are as follows: lysis temperature 56-65℃, incubation for 10-60min. Illustratively, the lysis temperature is 56℃, 57℃, 58℃, 59℃, 60℃, 61℃, 62℃, 63℃, 64℃, 65℃, or any value or range between any two values. The incubation time can be illustratively 10min, 15min, 20min, 25min, 30min, 35min, 40min, 45min, 50min, 55min, 60min, or any value or range between any two values. In some embodiments, the incubation time is 10-20min.

[0050] Without wishing to be bound by any theory, it is found that the success rate of amplification is higher when the lysis temperature is 65°C than 56°C. The term "success rate" can be understood as the percentage of the number of samples with specific bands after amplification divided by the number of all samples subjected to amplification.

[0051] In some embodiments, the conditions for alkaline lysis are a lysis temperature of 65°C and incubation for 10-20 min. In further embodiments, the conditions for alkaline lysis are a lysis temperature of 65°C and incubation for 20 min.

[0052] In some embodiments, the conditions for lysis are a lysis temperature of 65°C and incubation for 10 min.

[0053] In some embodiments, the conditions for lysis are a lysis temperature of 56°C and incubation for 60 min.

[0054] The kit used for alkaline lysis can exemplarily be REPLI-g Single Cell Kit, which can also be replaced by other manufacturers' single-cell amplification kits.

[0055] In some embodiments, the step of performing multiplex PCR amplification after lysis comprises:

[0056] The lysed single sperm or polar body is mixed with a buffer, dNTP, a SNP site amplification primer pool in the range of 1M-2M upstream and downstream of the target gene variation site, and a DNA polymerase to form an amplification system, and the amplification system is subjected to amplification to prepare an amplification product.

[0057] The reagent used for performing multiplex PCR amplification can be a conventional kit in the art, including but not limited to KAPA2G Fast Multiplex Mix, etc., which can also be replaced by other manufacturers' multiplex PCR kits.

[0058] The method for performing amplification on the amplification system can be a conventional method known and used by those skilled in the art. Exemplarily, the primer pool used can comprise at least 100 pairs of primers, which can be designed by a special software or technical platform (including but not limited to Huada AToplex, Aigtekang MultipSeq, Thermo Ampliseq, etc.).

[0059] In some embodiments, the amplification system further comprises a PCR enhancer. Without wishing to be bound by any theory, it is believed that the role of the PCR enhancer at least includes the PCR enhancer. The available PCR enhancer includes but is not limited to betaine, glycerol, dimethyl sulfoxide, bovine serum albumin, tetramethylammonium chloride, Tween 20, Tween 40, etc.

[0060] In some embodiments, the adapter ligation product is purified after adapter ligation. Exemplarily, the adapter ligation product is purified using purification magnetic beads.

[0061] In some embodiments, the adapter ligation product is purified using 1X to 1.5X purification magnetic beads (understood as 1X to 1.5X volume), for example, 1X, 1.1X, 1.2X, 1.3X, 1.4X, 1.5X, etc.

[0062] In some embodiments, the purification method after PCR enrichment can also be a conventional purification method in the art. For example, magnetic bead purification. In some embodiments, the adapter ligation product is purified using 1X to 1.5X purification magnetic beads (understood as 1X to 1.5X the volume). Exemplary methods include 1X, 1.1X, 1.2X, 1.3X, 1.4X, 1.5X, etc.

[0063] In some embodiments, the platform used to sequence the target library includes one or more of a BGI platform, an Illumina platform, and a Proton platform.

[0064] In some embodiments, the method does not rely on a reference sample. The reference sample includes but is not limited to a proband.

[0065] Some examples are provided below.

[0066] The embodiments of the present application will be described in detail below with reference to the examples. It should be understood that these examples are intended to illustrate the present application only and are not intended to limit the scope of the present application. The experimental methods for which the conditions are not specified in the following examples are preferably referred to the guidance provided in the present application, and may also be based on the experimental manuals or conventional conditions in this area, or on the conditions recommended by the manufacturer, or with reference to experimental methods known in the art.

[0067] Example 1

[0068] 1. Cell Lysis

[0069] Single sperm or polar body lysis was performed using the REPLI-g Single Cell Kit.

[0070] 1. Prepare denaturation buffer according to Table 1 below:

[0071] Table 1

[0072] Components Volume (μL) Buffer DLB 33 DTT,1M 3 Total volume 36

[0073] 2. Centrifuge the PCR tube containing the single sperm / polar body sample for 10 seconds. If the volume is less than 4 μL, add phosphate buffer to make up to 4 μL.

[0074] 3. Add 3 μL of prepared denaturing buffer, carefully tap the tube wall to mix, and centrifuge for 10 seconds to prevent the sample from sticking to the tube wall.

[0075] Incubate at 465°C for 20 min (use 105°C for the thermal cover of the PCR instrument).

[0076] Add 3 μL Stop Solution, gently tap the tube to mix, centrifuge for 10 seconds, and place on ice.

[0077] 2. Multiplex PCR Amplification

[0078] Multiplex PCR amplification was performed using the KAPA2G Fast Multiplex PCR Kit.

[0079] 1. Prepare a new 0.2 mL PCR tube on ice according to Table 2 below:

[0080] Table 2

[0081] Reagents Volume (μL) KAPA2G Fast MμLtiplex Mix 12.5 Primer pool 1.5 Previous step cleavage product 10 PCR Enhancer (31% (w / w) Glycerol) 1 Total volume 25

[0082] 2. Vortex to mix, centrifuge briefly, place the PCR tube in the PCR machine, and set the PCR program according to Table 3 below:

[0083] Table 3

[0084]

[0085]

[0086] 3. Library Construction

[0087] The single sperm multiplex PCR product library was constructed using the Ion Plus Fragment Library Kit.

[0088] 3.1 End repair

[0089] 1. Prepare a new 0.2 mL PCR tube and prepare the end repair reaction mixture on ice according to Table 4 below:

[0090] Table 4

[0091] Components Volume (μL) Multiplex PCR amplification products 25 5X End Repair Buffer 10 End Repair Enzyme 0.5 Nuclease-free water 14.5 Total 50

[0092] 2. Vortex mix and centrifuge briefly.

[0093] 3. Place the PCR tube on the PCR instrument and react at 20℃ for 30 minutes.

[0094] 3.2 Connector connection

[0095] Add reagents in new 0.2 mL PCR tubes in order according to Table 5 below. The ligation reaction needs to be prepared on ice.

[0096] Table 5

[0097] Components Volume (μL) End-repair product purified in the previous step 32 10×Ligase Buffe 5 Universal connector 1 Specific linker 1 Nuclease-free water 10 DNALigase 1 Total 50

[0098] Note: The universal adapter is a DNA universal sequence;

[0099] The specific adapter is a universal sequence containing 8 different barcode base sequences.

[0100] 1. Vortex mix, and centrifuge briefly.

[0101] 2. Place the PCR tube on the PCR machine, and react at 20°C for 30 min.

[0102] 3. Purification of ligation product

[0103] 1. Add 65 μL (1.3X the volume) of purified magnetic beads to the ligation reaction. Vortex mix 8-10 times to suspend the DNA and magnetic beads, and transfer to a 1.5 mL centrifuge tube. Let stand at room temperature for 5 min.

[0104] 2. Centrifuge briefly, and place the 1.5 mL centrifuge tube on a magnetic stand. Let stand for 2-5 min until the solution is clear. Carefully aspirate and discard the supernatant without disturbing the magnetic beads.

[0105] 3. Add 200 μL of freshly prepared 70% ethanol, and gently spin the 1.5 mL centrifuge tube once to move the magnetic beads to the wall. Let stand at room temperature for 30 sec, and discard the supernatant.

[0106] 4. Repeat Step 3.

[0107] 5. Centrifuge briefly, and place the 1.5 mL centrifuge tube on a magnetic stand. Aspirate the residual liquid, and let stand at room temperature for 2-5 min to evaporate the ethanol without cracking the magnetic beads.

[0108] 6. Remove the 1.5 mL centrifuge tube from the magnetic stand, and add 18 μL of Low TE. Vortex mix 5-10 times to completely resuspend the magnetic beads, and let stand at room temperature for 5 min.

[0109] 7. Centrifuge briefly, and place the centrifuge tube on a magnetic stand for about 2-5 min to separate the magnetic beads from the supernatant. Aspirate 15 μL of the supernatant to a new centrifuge tube.

[0110] 3.4 PCR enrichment

[0111] 1. Take a new 0.2 mL PCR tube, and prepare the PCR enrichment reaction on ice according to Table 6 below:

[0112] Table 6

[0113] Components Volume (μL) Adapter ligation product after purification in the previous step 15 Platium PCR Supermix High Fidelity 47.5 Library Amplification Primer Mix 2.5 Total volume 65

[0114] 2. Vortexed, spun down, and placed in a PCR machine to run the following program:

[0115] Table 7

[0116]

[0117]

[0118] 3.5 Purification of enriched product

[0119] 1. Add 84.5 μΐ^ (1.3X sample volume) of clean magnetic beads to the enriched product. Vortex 8-10 times to mix the DNA and magnetic beads suspension well and transfer to a 1.5 mL centrifuge tube. Let it sit at room temperature for 5 min.

[0120] 2. Spin down the 1.5 mL centrifuge tube and place it on a magnetic stand. Let it sit for 2-5 min until the solution is clear. Carefully pipette out and discard the supernatant without disturbing the magnetic beads.

[0121] 3. Add 200 μΐ^ of freshly prepared 70% ethanol. Gently spin the 1.5 mL centrifuge tube once to move the magnetic beads to the wall of the tube. Let it sit at room temperature for 30 sec and discard the supernatant.

[0122] 4. Repeat step 3.

[0123] 5. Spin down the 1.5 mL centrifuge tube and pipette out the residual liquid. Let it sit at room temperature for 2-5 min to evaporate the ethanol without cracking the magnetic beads.

[0124] 6. Remove the 1.5 mL centrifuge tube from the magnetic stand and add 20 μΐ^ of Low TE. Vortex 5-10 times to resuspend the magnetic beads completely. Let it sit at room temperature for 5 min.

[0125] 7. Spin down the 1.5 mL centrifuge tube and place it on a magnetic stand for about 2-5 min to separate the magnetic beads from the supernatant. Pipette out 18 μΐ^ of the supernatant to a new centrifuge tube.

[0126] Example 1:

[0127] The main steps of the detection method were optimized and the conditions were selected as follows:

[0128] A semen sample was collected and sent to Shandong Beikang Medical Testing Co., Ltd. for experiments according to the experimental protocol.

[0129] Most of the human sperm DNA is bound to protamine, only a small part is bound to histone, and the density of sperm head chromatin is very high, so whether the lysis efficiency can be improved by adjusting the lysis time and high temperature treatment to reduce the density of chromatin.

[0130] Single sperm sorting:

[0131] Sperm was isolated from semen sample by density gradient centrifugation, and the isolated sperm was washed in sperm washing medium (Vitrolife, Sweden). The motile sperm was collected by swim-up method in G-IVF (Vitrolife, Sweden) containing 10% human serum albumin. A small amount of motile sperm was transferred to a 30 μL phosphate buffer saline (PBS) mineral oil droplet containing 10% serum replacement supplement (Irvine Scientific, USA), and the sperm was first found under an inverted microscope at 100 times, then converted to 200 times, adjusted to a clear state, and an appropriate amount of PBS was sucked into another culture dish. The micro-needle was inserted into the mouth of the pipette, and the micro-needle was first sucked a small amount of liquid in the culture dish by using the siphon effect. The micro-needle was inserted into the culture dish by observing through the microscope, and the microscope was focused on a single sperm in the liquid layer. A single sperm (sperm with complete head and tail) was sucked, and then the single sperm was transferred to a sterile PCR tube, and the droplet was about 2 μL.

[0132] The experimental scheme can be referred to Figure 1 .

[0133] 1.1 Lysis

[0134] The sorted single sperm was subjected to single sperm whole genome amplification using REPLI-g Single Cell Kit according to the scheme in Table 8 below:

[0135] Table 8

[0136]

[0137] The single amplification products of different schemes were subjected to quality inspection using two pairs of specific primers, i.e. PCR amplification using single amplification products as templates, and the PCR amplification products were analyzed by 2% agarose gel electrophoresis. The electrophoresis results are shown in Table 9:

[0138] Table 9

[0139]

[0140]

[0141] The two primer-specific bands in each single expansion product were simultaneously detected to be qualified for quality inspection, i.e. successful amplification, and the successful amplification rate was 67% at 65°C for 10 min, 83% at 65°C for 20 min, and 50% at 56°C for 1 h.

[0142] The qualified samples were selected, and Ion Plus Fragmnet Library Kit was used for single sperm multiplex PCR library construction, and the constructed library was sequenced by using DA8600. The sequencing quality control results are shown in Table 10.

[0143] Table 10

[0144]

[0145] Note: Raw_reads represents the number of sequencing reads; Mapped_reads represents the number of aligned reads; mean_DP represents the average sequencing depth; coverage represents the coverage; capture_rate represents the capture efficiency.

[0146] According to the coverage (coverage) and capture efficiency (capture_rate) scheme one and scheme two are better than scheme three and scheme two is slightly better than scheme one.

[0147] 1.2 Density

[0148] The separated single sperm was subjected to single sperm whole genome amplification by using REPLI-g Single Cell Kit according to the following scheme.

[0149] Table 11

[0150]

[0151]

[0152] The single expansion products of different schemes were subjected to quality inspection by using two pairs of specific primers, i.e. PCR amplification with single expansion product as template, and the PCR amplification products were analyzed by using 2% agarose gel electrophoresis. The electrophoresis results are shown in Table 12:

[0153] Table 12

[0154]

[0155]

[0156] Whether it is 56℃ for 1 hour, 65℃ for 10 minutes, or 65℃ for 20 minutes, the success rate of single amplification after adding the stop solution at 95℃ for 15 minutes is much lower than that without pretreatment. At the same time, the success rate of single amplification by the lysis method at 65℃ for 20 minutes is higher than that at 56℃ for 1 hour and 65℃ for 10 minutes.

[0157] According to the above experimental results, the success rate of single sperm lysis quality inspection is higher when it is lysed at 65℃ for 20 minutes. Therefore, lysis at 65℃ for 20 minutes is preferred.

[0158] Effect Example 2

[0159] A sample of semen was collected and sent to Shandong Beikang Medical Laboratory Co., Ltd. under room temperature for experiments according to the experimental plan.

[0160] Single sperm retrieval:

[0161] Sperm were isolated from semen samples by density gradient centrifugation and washed in sperm washing medium (Vitrolife, Sweden). Motile sperm were collected by the swim-up method in G-IVF (Vitrolife, Sweden) containing 10% human serum albumin. A small number of motile sperm were transferred to a 30 μL drop of phosphate buffered saline (PBS) mineral oil supplemented with 10% serum replacement supplement (Irvine Scientific, USA). The sperm were first identified under an inverted microscope at 100x magnification, then magnified at 200x to achieve clarity. An appropriate amount of PBS was pipetted into another culture dish. A microscopic needle was inserted into a mouth pipette and, using the siphon effect, a small amount of liquid from the culture dish was aspirated. The needle was then inserted into the culture dish under microscopic observation. The microscope was focused on the single sperm layer, and a single sperm (with its head and tail intact) was aspirated. The single sperm was then transferred to a sterile PCR tube in a drop of approximately 2 μL.

[0162] The experimental protocol can be found in Figure 2 .

[0163] Multiplex PCR amplification was performed using the KAPA2G Fast MμLtiplex PCR Kit, and the multiplex PCR products were purified using 1.5X magnetic beads using the conventional method. The single sperm multiplex PCR product library was constructed using the Ion Plus Fragment Library Kit.

[0164] 2.1 Product concentration

[0165] The single-cell whole-gene amplification products, multiplex PCR amplification products (purified by conventional methods), and libraries were quantified using Qubit. The quantitative results are shown in Table 13 below:

[0166] Table 13

[0167]

[0168] Note: S1-S10 are the selected single sperm samples that have passed the quality inspection.

[0169] 2.2 Next-generation sequencing quality control results

[0170] The constructed library was sequenced on the Proton platform DA8600. The sequencing quality control results are shown in Table 14 below:

[0171] Table 14

[0172]

[0173]

[0174] Note: Raw_reads indicates the number of sequencing reads; Mapped_reads indicates the number of aligned reads; mean_DP indicates the average sequencing depth; coverage indicates the coverage; capture_rate indicates the capture efficiency; Frac_30X indicates the proportion of sequencing depth greater than 30X; Frac_100X indicates the proportion of sequencing depth greater than 100X.

[0175] 2.3 Results Analysis

[0176] Based on the annotation results, the analysis results are summarized in the following table.

[0177] Table 15

[0178]

[0179] According to the quality control results, the average coverage, average capture efficiency, and the proportion of polymorphic sites with a sequencing depth greater than 100X of this application were 0.994, 0.677, and 0.95, respectively, while the conventional method was 0.99, 0.649, and 0.917, respectively. Therefore, the coverage, capture efficiency, and proportion of polymorphic sites with a sequencing depth greater than 100X of the method of this application are generally better than those of the conventional method.

[0180] According to the analysis of the results, it was found that:

[0181] (1) Mutation site

[0182] The conventional method has a large deviation in the sequencing depth of variant sites, some are greater than 100X, some are less than 100X, and some are less than 30X. However, the sequencing depth of variant sites in the method of the present application is greater than 100X, and the overall uniformity is better.

[0183] (2) Heterozygous sites

[0184] Single sperm is homozygous for haploid polymorphic sites. Due to PCR amplification errors and other reasons, the heterozygosity rate of conventional methods is 2.2% to 18.44%, while the heterozygosity rate of the present application is 0.55% to 3.85%. Therefore, the present application has a lower error rate and a higher accuracy.

[0185] (3) The total number of sites and sequencing depth are greater than 100X the number of sites

[0186] The total number of sites captured in this application is higher than that of conventional methods as a whole, and the sequencing depth is greater than 100X the number of sites in this application is much higher than that of conventional methods.

[0187] In summary, the method of this application is better than conventional methods in terms of coverage, capture efficiency, proportion of polymorphic sites with sequencing depth greater than 100X, and uniformity, which can effectively reduce amplification bias and improve detection accuracy.

[0188] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0189] The above-described embodiments merely represent several implementation methods of the present application. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art could make various modifications and improvements without departing from the spirit of the present application, all of which fall within the scope of protection of the present application. Therefore, the scope of protection of the patent in this application shall be determined by the appended claims, and the specification and drawings shall serve to interpret the claims.

Claims

1. A method for constructing a linkage analysis library based on a single sperm or polar body, characterized in that: It includes the following steps: Single sperm or polar bodies are lysed to prepare lysed single sperm or polar bodies; multiple PCR amplification, end repair, adapter ligation, PCR enrichment, and purification are performed to prepare the target library.

2. The method for constructing a linkage analysis library based on a single sperm or polar body according to claim 1, wherein: The method for lysing single sperm or polar body is alkaline lysis or enzymatic lysis.

3. The method for constructing a linkage analysis library based on a single sperm or polar body according to claim 2, wherein: The alkaline lysis conditions are: lysis temperature 56° C. to 65° C., and incubation 10 min to 60 min.

4. The method for constructing a linkage analysis library based on a single sperm or polar body according to claim 3, wherein: The alkaline lysis conditions are: lysis temperature 65° C., incubation 10 min to 20 min.

5. The method for constructing a linkage analysis library based on a single sperm or polar body according to claim 1, wherein: The steps for multiplex PCR amplification after lysis include: The lysed single sperm or polar body is mixed with a buffer, dNTP, a SNP site amplification primer pool within the range of 1M to 2M upstream and downstream of the target gene mutation site, and a DNA polymerase to form an amplification system, and the amplification system is amplified to prepare an amplification product.

6. The method for constructing a linkage analysis library based on a single sperm or polar body according to claim 5, wherein: The amplification system also includes a PCR enhancer; Optionally, the PCR enhancer includes one or more of betaine, glycerol, dimethyl sulfoxide, bovine serum albumin, tetramethylammonium chloride, Tween 20 and Tween 40.

7. The method for constructing a linkage analysis library based on a single sperm or polar body according to any one of claims 1 to 6, wherein: Purifying the adapter ligation product after adapter ligation; Optionally, purifying the linker ligation product using purification magnetic beads; Further optionally, the linker ligation product is purified using 1X to 1.5X purification magnetic beads.

8. The method for constructing a linkage analysis library based on a single sperm or polar body according to any one of claims 1 to 6, wherein: The purification method is magnetic bead purification; Optionally, purification is performed using 1X to 1.5X purification magnetic beads.

9. The method for constructing a linkage analysis library based on a single sperm or polar body according to any one of claims 1 to 6, wherein: The platform used for sequencing the target library includes one or more of the BGI platform, the Illumina platform, and the Proton platform.

10. The method for constructing a linkage analysis library based on a single sperm or polar body according to any one of claims 1 to 6, wherein: The method described does not rely on reference samples; The reference sample includes a proband.

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