Probe mixing ratio design method and mixed probe set and sequencing library construction method

Abstract

A probe mixing ratio design method and a mixed probe set and sequencing library construction method, the probe mixing ratio design method includes: an estimation step, including providing a probe set, estimating the mixing ratio of the probe set according to the chip size of each probe set and the sequencing depth requirement; a pre-sequencing step, including setting the pre-mixing ratio according to the estimated mixing ratio, mixing the probe set according to the pre-mixing ratio to obtain a pre-mixed probe, hybridizing the pre-mixed probe with a sub-library, and performing pre-sequencing to obtain a probe mixing ratio that meets the expectations. The present invention can design probes for multiple different target areas according to product requirements, and can prepare mixed probes according to sequencing depth requirements, so as to achieve the purpose of simultaneously performing different sequencing depth detection for different target areas, with low cost; the probe ratio can also be adjusted according to changes in product targets, or detection of different probe combinations can be performed, with high flexibility.

Description

Technical Field

[0001] The present invention relates to the technical field of gene sequencing, and in particular to a method for designing a probe mixing ratio and a method for constructing a mixed probe set and a sequencing library. Background Art

[0002] In high-throughput sequencing of tumors, there are three common detection types: SNV, InDel and SV. SNV, or single nucleotide variations, is a type of variation caused by a single base change, including missense mutation, nonsense mutation and synonymous mutation. InDel, including insertion and deletion, refers to the insertion or deletion of a small fragment sequence at a certain position in the genome, and its length is usually ≤50bp. SV, or structural variation, refers to the sequence changes and positional relationship changes of a longer length on the genome, including insertion or deletion of long fragment sequences of more than 50bp in length (Big InDel), tandem repeat, chromosome inversion, sequence translocation within or between chromosomes, copy number variation (CNV) and more complex mosaic variation. Among them, copy number variation (CNV) is an important part of genomic structural variation. It generally refers to the copy number amplification or deletion of large genomic fragments with a length of more than 1kb. The number and complexity of CNVs are the main causes of many solid tumors, and are also important diagnostic and prognostic indicators. The detection principle of CNV is as follows: According to the size of CNV fragments, it can usually be divided into three levels. The first is the conventional CNV at the exon level or gene level, and the fragment size of its CNV variation is usually at the Kb level; the second is the larger CNV fragments associated with genomic scars, including LOH (Loss of Heterozygosity), TAI (TelomericAllelic Imbalance) and LST (Large-scale Sate Transition), and the fragment size of its CNV variation is usually at the Mb level; the third is the amplification or deletion of the entire chromosome level, which will lead to chromosomal instability in tumor cells, usually caused by unequal separation of chromosomes.

[0003] At the same sequencing depth, the larger the CNV fragment, the easier it is to observe in the sequencing data. Therefore, different sequencing depths need to be set according to different detection purposes. For example, in order to more comprehensively evaluate the homologous recombination deficiency status of patients, it is necessary to use high-depth target region targeted capture sequencing technology to detect various types of variations in HRR (homologous recombination repair) related genes such as BRCA1 / 2, while using low-depth target region targeted capture sequencing technology to detect larger CNV fragments associated with genomic scars (LOH, TAI and LST).

[0004] In the prior art, if differentiated depth detection of different target areas is to be achieved, detection is mainly performed through the following technical solutions:

[0005] A) Product packaging: By executing different experimental processes, targeted capture and high-throughput sequencing of different target areas are performed, so as to achieve the purpose of high-throughput detection of different target areas with different sequencing depths. This method has high production costs and requires more manpower and material resources.

[0006] B) Capture probe optimization: According to the product design concept, when designing probes, the probes are encrypted for target areas with high depth, while the number of probes can be halved for target areas with low depth. The main disadvantage of this design method is that the encryption or halving of probes based on theoretical calculations may not meet the detection requirements in actual application. At this time, it is necessary to remedy the situation by increasing the amount of sequencing data or re-synthesizing the probes.

[0007] Therefore, the existing differentiated depth detection methods for different target areas have defects such as poor flexibility and high detection cost. Summary of the invention

[0008] According to the first aspect, in one embodiment, a probe mixing ratio design method is provided, comprising:

[0009] The estimation step includes providing at least two probe sets for capturing target regions in the library to be tested, each probe set capturing a different target region, and estimating the mixing ratio of the probe sets according to the chip size of each probe set and the sequencing depth requirements of different target regions in the library to be tested;

[0010] The pre-sequencing step includes setting at least one pre-mixing ratio according to the estimated mixing ratio, mixing the at least two probe sets for capturing the target region in the library to be tested according to the pre-mixing ratio to obtain at least one pre-mixed probe, each pre-mixing ratio corresponds to one pre-mixed probe, hybridizing the pre-mixed probes of various pre-mixing ratios with sub-libraries derived from the library to be tested, performing pre-sequencing, obtaining sequencing depths and ratios of different target regions, and obtaining an expected probe mixing ratio according to the sequencing depths and ratios of the different target regions.

[0011] According to the second aspect, in one embodiment, a mixed probe set is provided, comprising at least two probe sets for capturing target regions in a library to be tested, wherein the mixing ratio of the at least two probe sets for capturing target regions in a library to be tested is the probe mixing ratio obtained by the probe mixing ratio design method of the first aspect.

[0012] According to the second aspect, in one embodiment, a kit is provided, comprising the mixed probe set described in the second aspect.

[0013] According to the fourth aspect, in one embodiment, a method for constructing a sequencing library is provided, comprising:

[0014] The mixed probe set described in the second aspect is mixed with the library to be tested to obtain a library after probe capture.

[0015] According to the probe mixing ratio design method and the mixed probe set and sequencing library construction method of the above-mentioned embodiment, probes for multiple different target areas can be designed according to product requirements, and mixed probes can be prepared according to sequencing depth requirements, so as to achieve the purpose of simultaneously performing different sequencing depth detections on different target areas, which is reflected in the low cost feature of the present invention; the probe ratio can also be adjusted according to changes in product targets, so as to achieve new product performance, or perform detection of different probe combinations, which is reflected in the high flexibility of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 A diagram showing the design process of a hybrid probe ratio according to an embodiment of the present invention.

[0017] Figure 2 This is the QC analysis diagram of Example 1.

[0018] Figure 3 This is a depth ratio diagram of two probe areas of mixed probe C based on 53 blood cells in Example 1.

[0019] Figure 4 This is a statistical result diagram of the capture efficiency of mixed probe C based on 53 blood cells in Example 1.

[0020] Figure 5This is the Fold80 statistical result of the mixed probe C based on 53 blood cells in Example 1.

[0021] Figure 6 This is a depth ratio diagram of two probe areas of mixed probe B based on 53 blood cells in Example 3.

[0022] Figure 7 This is a statistical result diagram of the capture efficiency of mixed probe B based on 53 blood cells in Example 3.

[0023] Figure 8 This is the Fold80 statistical result of mixed probe B based on 53 blood cells in Example 3. DETAILED DESCRIPTION

[0024] The present invention is further described in detail below by specific embodiments in conjunction with the accompanying drawings. Wherein similar elements in different embodiments adopt associated similar element numbers. In the following embodiments, many detailed descriptions are for making the present application better understood. However, those skilled in the art can easily recognize that some features can be omitted in different situations, or can be replaced by other elements, materials, methods. In some cases, some operations related to the present application are not shown or described in the specification, this is to avoid the core part of the present application being overwhelmed by too much description, and for those skilled in the art, it is not necessary to describe these related operations in detail, and they can fully understand the related operations according to the description in the specification and the general technical knowledge in the art.

[0025] In addition, the features, operations or characteristics described in the specification can be combined in any appropriate manner to form various implementations. At the same time, the steps or actions in the method description can also be interchanged or adjusted in a manner that is obvious to those skilled in the art. Therefore, the various sequences in the specification and the drawings are only for the purpose of clearly describing a certain embodiment and are not meant to be a required sequence, unless otherwise specified that a certain sequence must be followed.

[0026] The serial numbers assigned to the components herein, such as "first", "second", etc., are only used to distinguish the objects described and do not have any order or technical meaning. The "connection" or "coupling" mentioned in this application include direct and indirect connections (couplings) unless otherwise specified.

[0027] Explanation of terms

[0028] CNV: Copy number variations, gene copy number variation.

[0029] LOH: Loss of heterozygosity, refers to the loss of one of the two alleles (or part of the nucleotide fragments) located at the same gene locus on a pair of homologous chromosomes, while it still exists on the paired chromosome.

[0030] TAI: Telomeric Allelic Imbalance, telomeric allelic imbalance.

[0031] LST: Large-scale state Transition, large-scale migration.

[0032] SNV: single nucleotide variation.

[0033] Indel: insertion or deletion.

[0034] In the prior art, when designing a probe, differentiated depth detection of different areas is usually achieved through methods such as probe encryption. This method is relatively limited, and the encryption design is a theoretical value, which may not be consistent with the actual measurement expectations. In addition, once the product demand changes, the probe must be redesigned, which is costly.

[0035] According to the first aspect, in one embodiment, a probe mixing ratio design method is provided, comprising:

[0036] The estimation step includes providing at least two probe sets for capturing target regions in the library to be tested, each probe set capturing a different target region, and estimating the mixing ratio of the probe sets according to the chip size of each probe set and the sequencing depth requirements of different target regions in the library to be tested;

[0037] The pre-sequencing step includes setting at least one pre-mixing ratio according to the estimated mixing ratio, mixing at least two probe sets for capturing the target region in the library to be tested according to the pre-mixing ratio to obtain at least one pre-mixed probe, each pre-mixing ratio corresponds to one pre-mixed probe, and hybridizing the pre-mixed probes of various pre-mixing ratios with sub-libraries derived from the library to be tested, respectively, to perform pre-sequencing, to obtain sequencing depths and ratios of different target regions, and to select a probe mixing ratio that meets expectations according to the sequencing depths and ratios of different target regions.

[0038] In one embodiment, probes for multiple different target areas can be designed according to product requirements, and mixed probes can be prepared according to sequencing depth requirements, so as to achieve the purpose of simultaneously performing different sequencing depth detections on different target areas, which is reflected in the low cost feature of the present invention; the probe ratio can also be adjusted according to changes in product targets, so as to achieve new product performance, or perform detection of different probe combinations, which is reflected in the high flexibility of the present invention.

[0039] In one embodiment, the probes can be purchased from a company that synthesizes the probes.

[0040] In one embodiment, the pre-estimation step further includes diluting at least one probe set to obtain a diluted probe set for mixing in the pre-mixing ratio in the pre-sequencing step. For example, two probe sets are provided, including a first probe set and a second probe set, the first probe set is diluted, and the second probe set is not diluted, then, in the pre-sequencing step, the diluted first probe set is mixed with the undiluted second probe set in the pre-mixing ratio for pre-sequencing.

[0041] In one embodiment, the pre-sequencing step further includes diluting the premixed probes, and then hybridizing the sub-library derived from the library to be tested with the diluted premixed probes.

[0042] In one embodiment, the diluent used for dilution can be a probe dissolving solution provided with the probe, which can be provided by the manufacturer of the synthetic probe or prepared by the user.

[0043] In one embodiment, at least two probe sets for capturing target regions in the library to be tested include at least one of the following probe combinations:

[0044] 1) comprising a first probe set and a second probe set, wherein the first probe set is used to detect at least one of single nucleotide variation, insertion or deletion, and gene copy number variation of homologous recombination repair genes, and the second probe set is used to detect genomic scars;

[0045] 2) comprising a third probe set, a fourth probe set, and a fifth probe set, wherein the third probe set is used to detect whole exome sequencing data (WES), the fourth probe set is used to detect microsatellite instability (MSI), and the fifth probe set is used to detect fusion genes.

[0046] This is just an exemplary list, and other probe combinations are also possible.

[0047] In one embodiment, the second probe set is used to detect CNV fragments associated with genomic scars.

[0048] In one embodiment, the CNV fragment associated with the genomic scar includes at least one of Loss of Heterozygosity (LOH), Telomeric Allelic Imbalance (TAI), and Large-scale Sate Transition (LST).

[0049] In one embodiment, the premixed probe is hybridized with the sub-library derived from the library to be tested according to the same hybridization base as the library to be tested. The hybridization base can be 1 hybrid (also known as single hybrid), 2 hybrids or multiple hybrids. In one embodiment, 1, 2 or more sub-libraries are hybridized with the probe after being mixed in proportion, and the hybridization mode of the premixed probe and the sub-library derived from the library to be tested is consistent with the hybridization mode of the subsequent mixed probe and the library to be tested. For example, the subsequent test library x (test library 1 is 8 hybrid 1), the test library y (test library y is 8 hybrid 1) and the test library z (test library z is 5 hybrid 1) are mixed and hybridized with the premixed probe a. Then, in the pre-sequencing step, the sub-library of the test library x, the sub-library of the test library y, and the sub-library of the test library z are mixed and hybridized with the premixed probe a, so that the hybridization base of the sub-library in the pre-sequencing step is consistent with the hybridization base when the subsequent test library is sequenced.

[0050] In one embodiment, the expected probe mixing ratio refers to a probe premix ratio that meets the sequencing depth ratio requirements of different target regions.

[0051] In one embodiment, the premix ratio includes volume ratios of different probe sets.

[0052] In one embodiment, the desired probe mixing ratio includes volume ratios of different probe sets.

[0053] According to the second aspect, in one embodiment, a mixed probe set is provided, comprising at least two probe sets for capturing target regions in a library to be tested, wherein the mixed probe set is obtained by mixing at least two probe sets for capturing target regions in a library to be tested according to a probe mixing ratio obtained by the probe mixing ratio design method of the first aspect.

[0054] In one embodiment, the mixed probe set comprises at least one of the following probe combinations:

[0055] 1) comprising a first probe set and a second probe set, wherein the first probe set is used to detect at least one of single nucleotide variation, insertion or deletion, and gene copy number variation of homologous recombination repair genes, and the second probe set is used to detect genomic scars;

[0056] 2) comprising a third probe set, a fourth probe set, and a fifth probe set, wherein the third probe set is used to detect whole exome sequencing data, the fourth probe set is used to detect microsatellite instability, and the fifth probe set is used to detect fusion genes.

[0057] In one embodiment, the volume ratio of the first probe set to the second probe set is 1:2.

[0058] In one embodiment, before the first probe set and the second probe set are mixed, the concentration of the first probe set is 4.14 ng / μL, and the concentration of the second probe set is 24.8 ng / μL.

[0059] In one embodiment, a diluent is further added to the mixed system, and the ratio of the volume of the added diluent to the volume of the first probe set is 1:1.

[0060] According to the third aspect, in one embodiment, a kit is provided, comprising the mixed probe set of the second aspect.

[0061] In one embodiment, the kit can be used to detect genomic scars and HRR-related gene mutations, thereby realizing the detection of genomic instability and homologous recombination repair gene mutations.

[0062] In one embodiment, the kit can be used to perform HRD Score analysis and BRCA1 / 2 mutation analysis.

[0063] In one embodiment, the kit may further include reagents such as a buffer for diluting the probe.

[0064] In one embodiment, the kit may further include other reagents required for library construction.

[0065] According to the fourth aspect, in one embodiment, a method for constructing a sequencing library is provided, comprising: reacting the mixed probe set of the second aspect with a library to be tested to obtain a library after probe capture.

[0066] In one embodiment, the library to be tested comprises nucleic acid molecules that have been subjected to fragmentation, end repair, end "A" addition reaction, adapter ligation, and PCR amplification in sequence.

[0067] In one embodiment, the nucleic acid molecule comprises DNA.

[0068] In one embodiment, the method further includes performing magnetic bead hybridization, elution, PCR amplification, and purification on the library after probe capture to obtain a library that can be used for sequencing.

[0069] In one embodiment, the core point of the present invention is that probes for multiple different target regions can be designed according to product requirements, and mixed probes can be prepared according to sequencing depth requirements, so as to achieve the purpose of simultaneously performing different sequencing depth tests on different target regions, which is reflected in the low cost feature of the present invention; the probe ratio can also be adjusted according to changes in product targets, so as to achieve new product performance, or perform tests in different probe combinations, which is reflected in the high flexibility of the present invention. For example, probes for N target regions can be designed in total, and there can be N kinds of probe combinations.

[0070] In one embodiment, the design process of the mixed probe ratio is as follows: Figure 1 As shown, according to the product design content, the following evaluations and tests are carried out:

[0071] 1. Confirm the detection range, mutation type and frequency, design different probes, or select appropriate combinations from existing probes.

[0072] 2. Evaluate the performance of each probe separately and confirm the required average sequencing depth of the samples based on the type and frequency of variants to be detected.

[0073] 3. According to the principle of synthetic probes, select the same detection method for concentration determination, and make a preliminary estimate and dilution of the premix ratio based on the chip size of the premixed probe and the depth of the target area.

[0074] 4. According to the preliminarily estimated ratio, multiple gradients (for example, 2 to 4) are set above and below it, and preliminary experimental tests are performed using the same sub-library and hybridization base.

[0075] 5. Evaluate the sequencing depth and ratio obtained in the preliminary experiment, and select the expected probe mixing scheme for subsequent stability testing; if the above scheme does not meet the expected results, the concentration gradient can be refined according to the measured data until the most expected probe mixing scheme is selected.

[0076] 6. According to the probe mixing scheme determined above, the mixed probes are prepared by different experimenters on different days to conduct inter-batch stability tests; the mixed probes prepared by the same person on the same day are used for intra-batch stability tests.

[0077] 7. Strictly follow the SOP and intra-batch and inter-batch experimental design to evaluate the stability of the system, including but not limited to the commonly used probe performance evaluation indicators in NGS, such as experimental success rate, sequencing depth and its ratio, capture efficiency and coverage uniformity. The evaluation criteria shall be based on the detection requirements of the product design.

[0078] 8. Based on the above test results, the final confirmed probe mixing ratio is obtained, and the mixed probe can be used for methodological evaluation later.

[0079] Example 1

[0080] This example designs and confirms the ratio of mixed probes.

[0081] According to the product design requirements, two probes with different target capture areas are designed respectively: the capture interval size of probe 1 is 0.71Mb, which is used to detect HRR-related gene mutations, including more than 3% (including 3%) variations of SNV and InDel and gene-based CNV detection; the capture interval size of probe 2 is 15.38Mb, which is used to detect genomic scars. After calculation, HRDScore can be obtained, including three large-fragment CNVs of LOH, TAI and LST. According to the probe performance evaluation, the sequencing depth of probe 1 should reach more than 900X to detect more than 3% variations, and the sequencing depth of probe 2 should reach more than 150X to detect three large-fragment CNVs, that is, the sequencing depth ratio of the two target regions (the target region of probe 1 and the target region of probe 2) should be about 5 to 7.

[0082] Probes 1 and 2 were synthesized by Roche Diagnostics (Shanghai) Co., Ltd.

[0083] Part of the capture region of probe 1 is as follows:

[0084] Table 1

[0085]

[0086]

[0087]

[0088] Part of the capture region of probe 2 is as follows:

[0089] Table 2

[0090]

[0091]

[0092]

[0093] The probe concentration was detected using Qubit dsDNA HS Assay kit 2.0 Fluorometer. The concentration of probe 1 was 4.14 ng / μL. Probe 2 had a large chip range and a high total concentration. The probe dissolution buffer (hereinafter referred to as RB) provided with the probe was used to dilute its concentration to about 6 to 7 times that of probe 1. The final dilution factor was 1 / 16, and the diluted probe concentration was 24.8 ng / μL.

[0094] The experiment was carried out according to Roche hybridization elution SOP, where the amount of probe added to the hybridization system was 4 μL, and the mixed probe was prepared according to the proportions in the following table:

[0095] Table 3

[0096] Mixed probe number Mixed probe preparation method A Probe 1 (1X) 2 μL + Probe 2 (1 / 16) 2 μL B Probe 1 (1X) 2μL + Probe 2 (1 / 16) 1μL + 1μL RB C Probe 1 (1X) 1μL + Probe 2 (1 / 16) 2μL + 1μL RB

[0097] Using sublibraries of 8 cell line samples, the above 3 probes with different ratios were hybridized respectively. The experiments were carried out strictly according to the standard SOP, and QC analysis was performed on the offline data.

[0098] The analysis results are as follows Figure 2 As shown in the figure, the results show that the probe preparation according to the ratio of "probe 1 (1X) 1μL + probe 2 (1 / 16) 2μL + 1μL RB" can achieve the expected goal, that is, mixed probe C meets the sequencing depth ratio requirements. Therefore, according to the above ratio, different personnel prepared 3 different batches of mixed probes (named mixed probe C-1, mixed probe C-2, and mixed probe C-3) on different 3 days for subsequent testing.

[0099] 53 blood cell samples were selected (one blood cell sample was taken from each subject), and stability tests and mixed probe stability tests prepared between batches were performed according to the probe ratio determined above. A total of 6 8-hybrid 1 (numbered as library 1 to 6) and 1 5-hybrid 1 (numbered as library 7) hybridization elution experiments were performed on the sublibraries of the 53 blood cell samples, among which library 1 and library 2 were hybridized with mixed probe C-1, library 3 and library 4 were hybridized with mixed probe C-2, and library 5 to library 7 were hybridized with mixed probe C-3. The sequencing depth ratios of the 53 blood cells were counted, and the results are as follows: Figure 3 As shown, the results showed that the sequencing depth ratio of mixed probes in different batches and the same batch ranged from 5.85 to 6.92, with a median of 6.46 and an average of 6.45. There were no abnormal values, which met the expected requirements.

[0100] At the same time, the capture efficiency of the hybrid probe and Fold 80 were also counted. The statistical results are as follows: Figure 4 , Figure 5As shown in the figure, it can be seen that the mixed probes of different batches and the same batch have good stability, and the sequencing depth and various QC indicators are in line with expectations and are stable.

[0101] Fold80 represents the average depth / (the depth at which more than 80% of the area is covered). The larger the Fold80, the worse the uniformity, and the smaller the Fold80, the better the uniformity.

[0102] Therefore, after the above tests and evaluations, this embodiment finally achieved the simultaneous detection of 3% variation of HRR gene and HRD Score detection in the same experimental process, saving material costs and labor costs.

[0103] Example 2

[0104] This embodiment provides a kit for comprehensive evaluation of genomic instability and homologous recombination repair gene mutation detection

[0105] (1) Sample selection

[0106] Three commercial cell line samples (purchased from Nanjing Kebai Biotechnology Co., Ltd.) were randomly selected, and the combination probe designed in Example 1 was used to simultaneously detect different sequencing depths of different target regions, and HRD Score analysis and BRCA1 / 2 mutation analysis were performed respectively to verify the example.

[0107] Table 4

[0108] Sample name HCC38 MX-1 MDA-MB-415

[0109] (2) Sample fragmentation

[0110] The kit is compatible with 100-400ng DNA samples. Use ultrapure water (NF water) that does not contain nuclease to make up the volume to 75μL. Transfer the liquid to the shearing tube and place it in the nucleic acid shearing instrument (Bioruptor pico). The shearing instrument is precooled to 4℃, and the shearing parameters are shown in the table below.

[0111] Table 5

[0112] Target fragment size (bp) Interrupt Parameters Number of cycles 200 Interrupt 30s / Stop 30s 16

[0113] (3) End repair

[0114] 1) Take out the final buffer in advance and thaw it at room temperature, vortex and centrifuge briefly. Take out the enzyme reagent before use.

[0115] 2) Add the components according to the table below, pipette to mix well and place on ice.

[0116] Table 6

[0117] End repair components Each reaction volume (μL) Fragmented DNA 75 End-repair buffer 14 Endothelin 11 Total volume 100

[0118] 3) The end repair program is set on the PCR instrument as follows. The end repair reaction system is placed in the PCR instrument for reaction. The repair program is shown in the following table.

[0119] Table 7

[0120] End repair procedure (heat cover 50℃) 20℃,30min 4℃, Hold

[0121] 4) After the end repair process is completed, perform 1.5 times magnetic bead purification (magnetic plate purification, manual purification or automated purification is possible). Add 150 μL of magnetic beads A, and finally dissolve back in a 0.2 mL tube with 28.1 μL of DNA dissolving solution.

[0122] (4) Add 'A' at the end

[0123] 1) Take out the 'A' buffer and thaw it at room temperature, vortex and centrifuge briefly. Take out the enzyme reagent before use.

[0124] 2) Add the components according to the table below, pipette to mix well and place on ice.

[0125] Table 8

[0126] Add 'A' component Each reaction volume (μL) Unpurified DNA 28 Add 'A' buffer 5 Add 'A' enzyme 2 Total volume 35

[0127] 3) Set up the Add 'A' program on the PCR instrument as follows and place the Add 'A' reaction system in the PCR instrument.

[0128] Table 9

[0129] Add 'A' program (heat cover 50℃) 37℃, 30min 4℃, Hold

[0130] 4) After the addition of 'A', proceed directly to the next step without purification.

[0131] (5) Connector connection

[0132] 1) Take out the adapter ligation buffer and thaw the adapter at room temperature, vortex and briefly centrifuge. Take out the enzyme reagent before use.

[0133] 2) Add the components according to the table below, pipette to mix, and place on an ice tray.

[0134] Table 10

[0135] Connector components Each reaction volume (μL) Previous step product 35 Ligation Buffer 18 Ligase 2 Connector x 5 Total volume 60

[0136] The corresponding relationship between the sample and the connector number is as follows:

[0137] Table 11

[0138] Sample name Connector Name HCC38 Connector 1 MX-1 Connector 2 MDA-MB-415 Connector 3

[0139] The linker sequence is as follows:

[0140] Table 12

[0141]

[0142]

[0143] 3) Set up the adapter connection program on the PCR instrument as follows, and place the adapter connection reaction system in the PCR instrument.

[0144] Table 13

[0145] Connector connection procedure (heat cover 50℃) 23℃,20min 4℃, Hold

[0146] 4) After the adapter connection procedure is completed, the magnetic beads A are purified (plate pure, manual purification or automated purification is possible). 30 μL of nuclease-free water and 50 μL of magnetic beads A are added, and finally 41 μL of DNA dissolution solution is used to dissolve and transfer to a new 0.2 mL PCR tube.

[0147] (6) Library amplification

[0148] 1) Take out the label primers and PCR reaction solution and thaw them at 4°C. Vortex and briefly centrifuge them and place them in an ice box.

[0149] 2) Prepare the reaction system according to the table below and store it in an ice box.

[0150] Table 14

[0151] Library amplification components Each reaction volume (μL) Previous step: DNA purification 40 PCR reaction solution 50 Index Primer x 10 Total volume 100

[0152] 3) Set the program on the PCR instrument as follows, place the reaction system in the previous step into the PCR instrument, and start the reaction.

[0153] Table 15

[0154]

[0155] The corresponding relationship between samples and adapter primers is as follows:

[0156] Table 16

[0157] Sample name Connector Name Primer name HCC38 Connector 1 Primer 1 MX-1 Connector 2 Primer 2 MDA-MB-415 Connector 3 Primer 3

[0158] The primer sequences are as follows:

[0159] Table 17

[0160]

[0161] 4) After the PCR program is completed, 100 μL of magnetic beads A are added for purification (plate purification, manual purification or automated purification is possible). Finally, 32 μL of nuclease-free water is used to dissolve the purified DNA in a 0.2 mL PCR tube for storage.

[0162] (7) Library Pooling

[0163] Library mixing was performed based on the Qubit results, as shown in the following table:

[0164] Table 18

[0165] Sample name Library input amount (ng) HCC38 500 MX-1 500 MDA-MB-415 500

[0166] (8) Hybridization library preparation

[0167] 1) Take out the required amount of magnetic beads B in advance and equilibrate them at room temperature for at least 30 minutes.

[0168] 2) Take 20 μL of hybridization reagent 1 into a new 1.5 mL centrifuge tube and add the hybridization substrate prepared in the previous step.

[0169] 3) Add 130 μL of magnetic beads B and vortex for 10 seconds to mix thoroughly.

[0170] 4) Let stand at room temperature for 10 minutes. Centrifuge briefly and place on a magnetic rack for 1 minute until the liquid becomes clear.

[0171] 5) Carefully aspirate and discard the supernatant, add 190 μL 80% ethanol, and let stand for 30 seconds until the liquid becomes clear.

[0172] 6) Carefully aspirate and discard the supernatant. Keep the centrifuge tube on the magnetic rack, open the tube cover, and let it stand at room temperature for 5 minutes to air dry.

[0173] 7) Add 2 μL of hybridization reagent 2 and 11.4 μL of nuclease-free water to each tube, remove from the magnetic stand, and vortex thoroughly to mix.

[0174] (9) Chip hybridization

[0175] 1) Take out hybridization reagent 3 and hybridization reagent 4 in advance and thaw them at room temperature; take out the HRR+HRD mixed probe prepared in Example 1 from the -20°C refrigerator and thaw it on ice. Prepare the hybridization premix according to the following table.

[0176] Table 19

[0177] Hybridization Master Mix Components Each reaction volume (μL) Hybridization reagent 3 28 Hybridization reagent 4 12 Nuclease-free water 3 Total volume 43

[0178] The above components can be pre-mixed together and stored and named "hybridization solution".

[0179] 2) Take 43 μL of the prepared hybridization reaction solution and add it to the 13.4 μL of hybridization substrate (with magnetic beads) in the previous step. Mix thoroughly, let stand at room temperature for 2 minutes, and place on a magnetic rack until the liquid becomes clear.

[0180] 3) Take out the hybridization probe and melt it on ice. Take out new PCR tubes corresponding to the number of hybridization reactions, mark them with the library number of this hybridization, and add 4 μL of probe to each tube.

[0181] 4) Transfer 56.4 μL of supernatant from step 2) to the PCR tube containing 4 μL of HRR+HRD mixed probe in step 3). Vortex for 10 seconds to mix thoroughly. Check mark.

[0182] 5) After brief centrifugation, place on PCR and run the hybridization reaction program in the table below.

[0183] Table 20

[0184] temperature time 95℃ 5min 55℃ 16～20h 55℃ hold

[0185] The thermal cover of the PCR instrument should be set to 105°C.

[0186] (10) Preparation before elution

[0187] 1) Preparation of hybridization elution buffer

[0188] Adjust the thermostatic metal bath to 55°C in advance, and prepare 100 μL 1*Wash Buffer I and 400 μL 1*Stringent Wash Buffer for each hybridization reaction for preheating.

[0189] Table 21

[0190]

[0191] 2) Prepare hybridization magnetic beads

[0192] a) Prepare 1X Magnetic Bead Wash Buffer C according to the table below.

[0193] Table 22

[0194]

[0195] b) Take out magnetic beads C and equilibrate at room temperature for 30 minutes. Vortex for 15 seconds before use to mix thoroughly.

[0196] c) Take new 1.5 mL centrifuge tubes and add 50 μL of magnetic beads C to each tube. Place on a magnetic rack for 1 min until the liquid becomes clear, then carefully aspirate and discard the supernatant.

[0197] d) Add 100 μL of 1X Magnetic Bead Wash Solution C to each tube. Remove from the magnetic rack and mix thoroughly. Place the centrifuge tube back on the magnetic rack and let it stand for at least 1 minute until the liquid is clear. Carefully aspirate and discard the supernatant. Repeat this step once for a total of 2 washes.

[0198] e) Add 50 μL of 1X Magnetic Bead Wash Solution C to each tube. Remove from the magnetic stand and vortex for 10 seconds to mix thoroughly. Transfer to a new 1.5 mL centrifuge tube.

[0199] f) Place the centrifuge tube on a magnetic rack and let it stand for at least 1 minute until the liquid becomes clear. Carefully aspirate and discard the supernatant.

[0200] g) Proceed to the next step immediately. Avoid leaving the beads too dry for too long.

[0201] 3) Hybridization library combined with magnetic beads C

[0202] After hybridization, continue to maintain the hybridization mixture at 55°C. After the magnetic beads C are washed, quickly transfer the hybridization reaction mixture of step (9) to the magnetic beads C prepared in the previous step, cover the tube, vortex for 10 seconds to mix thoroughly, and place it on a PCR instrument and incubate at 55°C for 15 minutes (heat cover 105°C).

[0203] (11) Elution process

[0204] a) After 15 minutes of incubation, remove the hybridization mixture and 1X Elution Buffer 1 from the PCR instrument.

[0205] b) Add 100 μL of 1X Elution Buffer 1 preheated to 55°C to each centrifuge tube containing 60.4 μL of hybridization mixture-magnetic beads C. Vortex for 10 seconds to mix thoroughly. Place on a magnetic rack until the liquid is clear, and carefully aspirate and discard the supernatant.

[0206] c) Add 200 μL of 1X Elution Buffer S preheated to 55°C to each tube. After removing from the magnetic rack, vortex for 10 seconds to mix thoroughly, and return to the PCR instrument to incubate at 55°C for 5 minutes (heat cover 105°C). After removing, place on the magnetic rack until the liquid is clear, carefully aspirate and discard the supernatant. Repeat this step once.

[0207] d) Add 200 μL of 1X Elution Buffer 1 (room temperature) to each tube and vortex for 10 seconds to mix thoroughly. Let stand at room temperature for 1 minute, centrifuge briefly, and place on a magnetic rack until the liquid is clear. Carefully aspirate and discard the supernatant.

[0208] e) Add 200 μL of 1X Elution Buffer 2 to each tube and vortex for 10 seconds to mix thoroughly. Centrifuge briefly and transfer to a new 1.5 mL centrifuge tube. Let stand at room temperature for 1 minute and place on a magnetic rack until the liquid becomes clear. Carefully aspirate and discard the supernatant.

[0209] f) Add 200 μL of 1X Elution Buffer 3 to each tube and vortex for 10 seconds to mix thoroughly. Let stand at room temperature for 1 minute, centrifuge briefly, and place on a magnetic rack until the liquid clarifies. Carefully aspirate and discard the supernatant.

[0210] g) Add 20 μL of PCR-grade water / nuclease-free water to each tube and mix thoroughly with a pipette. After a short centrifugation, transfer all the liquid to a new PCR tube (containing magnetic beads).

[0211] (12) Capture library PCR reaction and purification

[0212] 1) Prepare the post-capture PCR reaction mixture according to the following ratio and perform the reaction.

[0213] Table 23

[0214]

[0215]

[0216] 2) PCR product magnetic bead purification

[0217] Preparation: Equilibrate magnetic beads B at room temperature for 30 minutes in advance, and vortex for 15 seconds to mix thoroughly for later use.

[0218] Purify with 1.0x magnetic beads (50 μL) and finally dissolve in 32 μL ddHO 2 In O.

[0219] The primer sequences are listed in the table below.

[0220] Table 24

[0221] name Sequence (5'-3') 5' modification Flowcell primers F(10μM) GAACGACATGGCTACGA Phosphorylation Flowcell primers R (10μM) TGTGAGCCAAGGAGTTG -

[0222] (13) Sequencing and data analysis

[0223] The library constructed in the previous step was sequenced on the BGI MGISEQ-2000 sequencer after passing the electrophoresis test. The constructed single-stranded circular DNA library was prepared for DNA nanoball preparation and sequenced on the MGISEQ-2000. The sequencing process was strictly carried out in accordance with the standard operating procedures of the MGISEQ-2000.

[0224] After low-quality data filtering, comparison, and deduplication, the offline data was subjected to quality control analysis, and the average sample depths of the HRR gene target region and the HRD target region were counted, as shown in the following table. According to product design, the average sample depth of the HRD Score analysis area must be ≥150X, and the average sample depth of the HRR gene mutation analysis area must be ≥900X. The three samples in the embodiment all meet the depth requirements.

[0225] Table 25

[0226]

[0227] The HRR gene BRCA1 / 2 gene variation analysis was analyzed, and the homologous recombination defect score (HRD Score) was obtained by measuring heterozygosity (LOH), telomeric allele imbalance (TAI) and large fragment migration (LST). The genomic instability of the test sample can be evaluated from two perspectives: BRCA1 / 2 gene and HRD Score.

[0228] Table 26

[0229]

[0230] Example 3

[0231] This embodiment provides mixed probe ratio design and confirmation.

[0232] According to the product design requirements, two probes with different target capture regions are designed respectively: the capture interval size of probe 1 is 0.71Mb, which is used to detect HRR-related genes, including SNV and InDel with more than 1% (including 1%) variation; the capture interval size of probe 2 is 15.38Mb, which is used to detect genomic scars. After calculation, the HRD Score can be obtained, including three large-fragment CNVs: LOH, TAI and LST. According to the probe performance evaluation, the sequencing depth of probe 1 should reach more than 2700X to detect more than 1% variation, and the sequencing depth of probe 2 should reach more than 150X to detect three large-fragment CNVs, that is, the sequencing depth ratio of the two target regions (the target region of probe 1 and the target region of probe 2) should be about 17 to 20.

[0233] Combination Figure 2 , mixed probe B met the expected requirements. Therefore, on three different days, different personnel prepared three different batches of mixed probe B (named mixed probe B-1, mixed probe B-2, and mixed probe B-3) for subsequent testing.

[0234] 53 blood cell samples were selected to perform stability tests of the probe ratios determined above and stability tests of mixed probes prepared between batches. A total of 6 8-hybrid 1 (numbered as libraries 1 to 6) and 1 5-hybrid 1 (numbered as library 7) hybridization elution experiments were performed on the sublibraries of the 53 blood cell samples, where library 1 and library 2 were hybridized with mixed probe B-1, library 3 and library 4 were hybridized with mixed probe B-2, and library 5 to library 7 were hybridized with mixed probe B-3. The sequencing depth ratios of the 53 blood cells were counted, and the results are as follows: Figure 6 As shown, the results showed that the ratio of mixed probe sequencing depth in different batches and the same batch ranged from 17.33 to 21.47, the median was 19.70, the average was 19.52, there were no abnormal values, and it met the expected requirements.

[0235] At the same time, the capture efficiency of the hybrid probe and Fold 80 were also counted. The statistical results are as follows: Figure 7 , Figure 8 As shown in the figure, it can be seen that the mixed probes of different batches and the same batch have good stability, and the sequencing depth and various QC indicators are in line with expectations and are stable.

[0236] Fold80 represents the average depth / (the depth at which more than 80% of the area is covered). The larger the Fold80, the worse the uniformity, and the smaller the Fold80, the better the uniformity.

[0237] Therefore, after the above tests and evaluations, this embodiment finally achieved the simultaneous detection of 1% variation of HRR gene and HRD Score detection in the same experimental process, saving material costs and labor costs.

[0238] In one embodiment, the present invention can achieve different sequencing depth requirements for different target regions in the same experimental system, and has the characteristics of high flexibility and low cost.

[0239] In one embodiment, the present invention can be applied to a comprehensive solution for genomic scar detection and homologous recombination repair gene mutation detection.

[0240] In one embodiment, the present invention can simultaneously perform differentiated depth detection of different target areas according to the target depths required by different target areas, thereby achieving the purpose of saving detection material costs and labor costs.

[0241] In one embodiment, different probes may be used separately, or may be flexibly adjusted as the target depth of the product design changes.

[0242] In one embodiment, the present invention is not limited to differential depth detection of different variant types, and differential depth detection of the same variant type of different genes can also be performed based on the importance of the gene.

[0243] The above specific examples are used to illustrate the present invention, which is only used to help understand the present invention and is not intended to limit the present invention. For those skilled in the art, according to the concept of the present invention, some simple deductions, modifications or substitutions can be made. SEQUENCE LISTING <110> Shenzhen BGI Co., Ltd. <120> Probe mixing ratio design method and mixed probe set and sequencing library construction method <130> 21I32743 <160> 19 <170> PatentIn version 3.3 <210> 1 <211> 32 <212> DNA <213> Artificial sequence <400> 1 gaacgacatg gctacgatcc gacttacgta ct 32 <210> 2 <211> 32 <212> DNA <213> Artificial sequence <400> 2 gaacgacatg gctacgatcc gactttgcat gt 32 <210> 3 <211> 38 <212> DNA <213> Artificial sequence <400> 3 gtacgtaagt cggaggccaa gcggtcttag gaagacaa 38 <210> 4 <211> 38 <212> DNA <213> Artificial sequence <400> 4 catgcaaagt cggaggccaa gcggtcttag gaagacaa 38 <210> 5 <211> 32 <212> DNA <213> Artificial sequence <400> 5 gaacgacatg gctacgatcc gacttgaacg tt 32 <210> 6 <211> 32 <212> DNA <213> Artificial sequence <400> 6 gaacgacatg gctacgatcc gacttcttgc at 32 <210> 7 <211> 38 <212> DNA <213> Artificial sequence <400> 7 acgttcaagt cggaggccaa gcggtcttag gaagacaa 38 <210> 8 <211> 38 <212> DNA <213> Artificial sequence <400> 8 tgcaagaagt cggaggccaa gcggtcttag gaagacaa 38 <210> 9 <211> 32 <212> DNA <213> Artificial sequence <400> 9 gaacgacatg gctacgatcc gacttacacg tt 32 <210> 10 <211> 32 <212> DNA <213> Artificial sequence <400> 10 gaacgacatg gctacgatcc gactttgtgc at 32 <210> 11 <211> 38 <212> DNA <213> Artificial sequence <400> 11 acgtgtaagt cggaggccaa gcggtcttag gaagacaa 38 <210> 12 <211> 38 <212> DNA <213> Artificial sequence <400> 12 tgcacaaagt cggaggccaa gcggtcttag gaagacaa 38 <210> 13 <211> 38 <212> DNA <213> Artificial sequence <400> 13 tggtagaagt cggaggccaa gcggtcttag gaagacaa 38 <210> 14 <211> twenty one <212> DNA <213> Artificial sequence <400> 14 gaacgacatg gctacgatcc g 21 <210> 15 <211> 51 <212> DNA <213> Artificial sequence <400> 15 tgtgagccaa ggagttgatc ggacctattg tcttcctaag accgcttggc c 51 <210> 16 <211> twenty one <212> DNA <213> Artificial sequence <400> 16 gaacgacatg gctacgatcc g 21 <210> 17 <211> 51 <212> DNA <213> Artificial sequence <400> 17 tgtgagccaa ggagttggat tccgtccttg tcttcctaag accgcttggc c 51 <210> 18 <211> twenty one <212> DNA <213> Artificial sequence <400> 18 gaacgacatg gctacgatcc g 21 <210> 19 <211> 51 <212> DNA <213> Artificial sequence <400> 19 tgtgagccaa ggagttgcgg cattaagttg tcttcctaag accgcttggc c 51

Claims

1. A probe mixing ratio design method, characterized in that: include: The estimation step includes providing at least two probe sets for capturing target regions in the library to be tested, each probe set capturing a different target region, and estimating the mixing ratio of the probe sets according to the chip size of each probe set and the sequencing depth requirements of different target regions in the library to be tested; The step of predicting sequencing includes setting at least one premixing ratio according to the estimated mixing ratio, mixing the at least two probe sets for capturing the target region in the library to be tested according to the premixing ratio to obtain at least one premixed probe, each premixing ratio corresponds to one premixed probe, hybridizing the premixed probes of various premixing ratios with sub-libraries derived from the library to be tested, performing predicting sequencing, obtaining sequencing depths and ratios of different target regions, and obtaining an expected probe mixing ratio according to the sequencing depths and ratios of the different target regions; The estimation and pre-testing includes the following steps: 1) Confirm the detection range, variant type and frequency, design different probes, or select appropriate combinations from existing probes; 2) Evaluate the performance of each probe separately and determine the required average sequencing depth of the sample based on the type and frequency of the target variants; 3) According to the principle of synthetic probes, select the same detection method for concentration determination, and make a preliminary estimate and dilution of the premix ratio based on the chip size of the premixed probe and the depth of the target area; 4) Based on the initial estimated ratio, set multiple gradients above and below it, and conduct preliminary experimental tests using the same sub-library and hybridization base number; 5) Evaluate the sequencing depth and ratio obtained in the preliminary experiment, and select the probe mixing scheme that meets the expectations for subsequent stability testing; if the above scheme does not meet the expected results, refine the concentration gradient according to the measured data until the probe mixing scheme that best meets the expectations is selected; 6) According to the probe mixing scheme determined in step 5), mixed probes are prepared, and inter-batch stability tests and intra-batch stability tests are performed respectively; 7) Conduct system stability assessment, and the evaluation criteria shall be based on the testing requirements of product design; 8) Comprehensive test results to obtain the final confirmed probe mixing ratio.

2. The probe mixing ratio design method according to claim 1, characterized in that: The estimation step also includes diluting at least one probe set to obtain a diluted probe set for mixing according to the premixing ratio in the sequencing step.

3. The probe mixing ratio design method according to claim 1, characterized in that: The at least two probe sets for capturing the target region in the library to be tested include at least one of the following probe combinations: 1) comprising a first probe set and a second probe set, wherein the first probe set is used to detect at least one of single nucleotide variation, insertion or deletion, and gene copy number variation of homologous recombination repair genes, and the second probe set is used to detect genomic scars; 2) comprising a third probe set, a fourth probe set, and a fifth probe set, wherein the third probe set is used to detect whole exome sequencing data, the fourth probe set is used to detect microsatellite instability, and the fifth probe set is used to detect fusion genes.

4. The probe mixing ratio design method according to claim 3, characterized in that: The second probe set is used to detect CNV fragments associated with genomic scars; The CNV fragment associated with the genomic scar includes at least one of loss of heterozygosity, telomeric allele imbalance, and large fragment migration.

5. The probe mixing ratio design method according to claim 1, characterized in that: The premixed probes are hybridized with the sublibrary derived from the library to be tested according to the same hybridization base as the library to be tested.

6. The probe mixing ratio design method according to claim 1, characterized in that: The expected probe mixing ratio refers to the probe premixing ratio that meets the sequencing depth ratio requirements of different target regions; The premix ratio includes the volume ratio of different probe sets; The expected probe mixing ratio includes the volume ratio of different probe sets; The pre-sequencing step also includes diluting the premixed probes, and then hybridizing the sub-library derived from the library to be tested with the diluted premixed probes.

7. A method for constructing a sequencing library, characterized in that: include: The probe mixing ratio is obtained by the probe mixing ratio design method as described in any one of claims 1 to 6, and the obtained mixed probe set is mixed with the library to be tested to obtain the library after probe capture.

8. The method for constructing a sequencing library according to claim 7, wherein: The library to be tested comprises nucleic acid molecules that have been subjected to fragmentation, end repair, end "A" reaction, adapter ligation, and PCR amplification in sequence; The nucleic acid molecule comprises DNA; It also includes magnetic bead hybridization, elution, PCR amplification, and purification of the library after probe capture to obtain a library that can be used for sequencing.

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