Quality control primer group and quality control method for human-derived sample

Abstract

The invention provides a quality control primer group and a quality control method for a human-derived sample, and belongs to the technical field of gene sequencing, and the quality control primer group comprises a human-derived DNA quality control primer designed based on conserved genes of GJB2MD, GJB3MD, IVSM and 12SD, a human-derived RNA quality control primer designed based on conserved and highly-expressed housekeeping genes of GAPDH, ACTB and PARP2, and a sex quality control primer designed based on genes of SRY142, DYS14134, YSR240 and YSR186 on a Y chromosome. According to the scheme provided by the invention, multi-dimensional and full-process monitoring on the human-derived sample can be realized, manual operation errors such as reagent failure, reagent addition error, sample confusion and cross contamination in the existing sequencing process are reduced, and the reliability of a detected result is effectively improved.

Description

Technical Field

[0001] This invention relates to the field of gene sequencing technology, and more specifically to quality control primer sets and quality control methods for human samples. Background Technology

[0002] Gene sequencing technology originated in the 1970s and has undergone several generations of technological advancements. The first generation, Sanger sequencing, based on the dideoxy method and developed in 1977, is still widely used. Subsequently, second-generation sequencing technologies such as Solexa from Illumina and SOLID from ABI emerged. Second-generation sequencing represents a fundamental change from traditional sequencing technologies, employing a sequencing-as-synthesis principle, allowing for the generation of millions of nucleic acid molecular sequences in a single run. While second-generation sequencing offers significant improvements in throughput and cost compared to traditional methods, its drawbacks, such as short read lengths and complex sequence assembly, remain apparent. In recent years, third-generation sequencing based on the single-molecule readout principle—nanopore sequencing—has gained popularity. Compared to other traditional sequencing technologies, nanopore sequencing eliminates the need for enzymes and fluorescent labels, avoids errors introduced by PCR amplification, is simple and stable to operate, and has lower sequencing costs, truly achieving real-time and rapid sequencing.

[0003] Pathogen detection is a crucial step in the diagnosis and treatment of infectious diseases. Traditional pathogen detection methods, such as culture methods, immunological detection, and molecular biological detection, have limitations such as low sensitivity, long processing times, and inability to detect unknown pathogens. With the development of genomics technology, high-throughput sequencing technology has provided new technical means for the identification of pathogens. Nanopore sequencing technology, as a representative of third-generation sequencing technology, has shown great application potential in the field of pathogen detection due to its advantages such as long read lengths, real-time processing, and portability. By improving detection sensitivity, shortening diagnostic time, and reducing detection costs, this technology provides new solutions for the diagnosis and treatment of infectious diseases.

[0004] For pathogen-targeted genome sequencing, a single chip can simultaneously detect multiple samples. This means that a single experiment can complete pathogen detection for multiple clinical samples, shortening the time from sample to result, saving manpower, and reducing errors. However, nanopore sequencing experiments are lengthy and complex. Processing multiple samples simultaneously can lead to problems such as incorrect reagent addition, sample contamination, failed nucleic acid extraction, and nucleic acid degradation. These issues can all result in erroneous pathogen detection results and misdiagnosis of the disease. Therefore, setting up reasonable quality control is a crucial and necessary step to monitor for any abnormalities throughout the entire testing process. Summary of the Invention

[0005] In view of this, the present invention proposes a quality control primer set and quality control method for human samples, wherein the quality control primer set includes human DNA quality control primers, human RNA quality control primers and sex quality control primers, which can monitor the sequencing process from different dimensions, thereby solving the technical problem that the existing sequencing quality control is not reasonable and comprehensive enough.

[0006] The specific technical solution of the present invention is as follows: In a first aspect, the present invention provides a quality control primer set for human samples, comprising the following: based on GJB2MD , GJB3MD , IVSM , 12SD Quality control primers for human DNA with conserved gene design; based on GAPDH , ACTB , PARP2 Human RNA quality control primers designed for conserved and highly expressed housekeeping genes; Based on the Y chromosome SRY142 , DYS14134 , YSR240 , YSR186 Genetically designed sex control primers.

[0007] Preferably, the above-mentioned quality control primer set specifically includes the following primers: Human DNA quality control primers: GJB2MD-F / R primers with sequences as shown in SEQ ID NO.1-2, GJB3MD-F / R primers with sequences as shown in SEQ ID NO.3-4, IVSM-F / R primers with sequences as shown in SEQ ID NO.5-6, and 12SDF-F / R primers with sequences as shown in SEQ ID NO.7-8; Human RNA quality control primers: GAPDH-inF / R with sequences as shown in SEQ ID NO. 9-10, ACTB-inF / R with sequences as shown in SEQ ID NO. 11-12, and PARP2-inF / R with sequences as shown in SEQ ID NO. 13-14; Sex control primers: SRY142-F2 / R2 with sequences as shown in SEQ ID NO.15-16, DYS14134-F2 / R2 with sequences as shown in SEQ ID NO.16-17, YSR240-F / R with sequences as shown in SEQ ID NO.18-19, and YSR186-F / R with sequences as shown in SEQ ID NO.20-21.

[0008] When using the above-mentioned quality control primer set for sequencing quality control, human DNA quality control primers, human RNA quality control primers, and sex quality control primers can be used individually or in combination. Combination use can monitor the sequencing process from multiple dimensions at once, thereby improving the reliability of sequencing data.

[0009] Secondly, the present invention provides a kit containing the above-mentioned quality control primer set, which may further include multiplex PCR premix, negative control, etc. Specifically, the multiplex PCR premix may include PCR buffer, MgCl2, dNTPs and Taq enzyme, and the negative control may be nuclease-free water.

[0010] Thirdly, this invention provides the application of the above-mentioned quality control primer set in the sequencing analysis of human samples, wherein the sequencing can be next-generation sequencing or nanopore sequencing, and the human samples include human blood, plasma, serum, urine, tissue, saliva, oral swabs, nasal swabs, bronchoalveolar lavage fluid, etc.

[0011] Preferably, in the above applications, the sequencing analysis is a sequencing analysis of pathogenic microorganisms in human samples.

[0012] Fourthly, the present invention provides a quality control method for nanopore targeted sequencing of human samples, comprising the following steps: S1. Extract nucleic acid from human samples (if RNA is extracted, it needs to be reverse transcribed) to obtain the nucleic acid sample to be tested; S2. Construct a multiplex amplification system containing a quality control primer set, a multiplex amplification primer set, and the nucleic acid sample to be tested, amplify it, purify it, and ligate it with a sequencing adapter; S3. Sequencing and analyzing the data generated from sequencing.

[0013] In the above quality control method, multiplex amplification primer sets are used to target and amplify the target sequence. By adjusting the input amounts of quality control primer sets and multiplex amplification primer sets in the multiplex amplification system, the proportion of human sequences in the final amplification product can be controlled, avoiding excessive content that could affect the detection of the target sequence. Preferably, the ratio of quality control primer sets to multiplex amplification primer sets is 1:5.

[0014] In the aforementioned quality control methods, when human genomic DNA is present in the sample, the target corresponding to the human DNA quality control primer can be detected, thus enabling the assessment of whether any uncontrolled processes, such as nucleic acid extraction and amplification, have occurred in the detection system. When human RNA is present in the sample and RNA detection is performed, the target corresponding to the human RNA quality control primer can be detected, thus determining whether reverse transcription is normal. Furthermore, it will not be detected if reverse transcription is not performed during sequencing. If abnormal detection occurs during the DNA process, it suggests that the sample is likely contaminated, and the test results may be unreliable. The target corresponding to the sex quality control primer will be detected only in male samples; it will not be detected in female samples. Therefore, it can be used to determine the sex of the sample being tested. When multiple samples of different sexes are tested simultaneously, the sex quality control primer can be used to determine if sample confusion exists. Additionally, if a sex internal standard is detected in a female sample, it may indicate contamination during the testing process, and the test results may be unreliable.

[0015] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention develops a multi-dimensional, end-to-end quality control system comprised of human DNA quality control, sex quality control, and human RNA quality control. This system covers multiple key steps in sequencing experiments, reducing human error, ensuring experimental accuracy and stability, and improving data reliability and reproducibility. Simultaneously, it enables problem tracing and troubleshooting; when data anomalies occur, this system helps researchers quickly pinpoint the root cause, saving significant investigation time and costs. Furthermore, it meets compliance requirements in high-standard fields. In regulated areas such as clinical diagnostics and drug development, comprehensive quality control processes are mandatory, and this invention provides a specific and actionable solution to meet these requirements. Detailed Implementation

[0016] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains; the terminology used herein is for the purpose of describing particular implementations only and is not intended to limit the invention; the terms “comprising” and “having”, and any variations thereof, in the specification and claims of this invention are intended to cover non-exclusive inclusion.

[0017] While nanopore sequencing technology offers unique advantages such as real-time processing, long read lengths, and direct sequencing, its core technology relies on measuring the current changes caused by DNA / RNA strands passing through nanopores to identify bases. Furthermore, the pathogen detection process based on nanopore sequencing involves complex sample handling procedures, including cell disruption, lysis, nucleic acid extraction, reverse transcription, amplification, purification, and ligation of sequencing adapters. Therefore, this process is susceptible to interference from various factors, necessitating higher and more comprehensive quality control requirements. Currently, general quality control primarily relies on library concentration measurement, using fluorescence methods such as Qubit to determine successful nucleic acid amplification and ensure sufficient high-molecular-weight nucleic acids for subsequent sequencing. Additionally, during real-time nanopore sequencing, data yield and quality can be assessed by monitoring indicators such as the number of active wells, data output rate, and read length distribution. However, these quality control methods only determine whether the final library product meets sequencing requirements and remain unaffected by issues such as sample confusion, extraction reagent failure, reverse transcription reagent failure, or cross-contamination.

[0018] To address the limitations of existing nanopore sequencing technologies for pathogen detection in terms of quality control, this invention proposes a quality control scheme for human-derived samples, including human DNA quality control, human RNA quality control, and gender quality control. Human DNA quality control uses a human DNA internal standard, which detects the presence of human genomic DNA in the sample. Its significance lies in accurately determining whether the sample is of human origin and whether the nucleic acid extraction reagent is ineffective, avoiding the limitations of Qubit quality control. Qubit measures the total amount of all double-stranded DNA in the sample, including host DNA, contaminant DNA, and degraded short DNA fragments. These short fragments or contaminant DNA cannot be used for nanopore library construction but are included in the total concentration by Qubit. Human RNA quality control uses a human RNA internal standard, which detects the presence of human RNA in the sample during RNA reverse transcription testing. This can be used to monitor the ineffectiveness of RNA reverse transcription reagents throughout the experimental process. Unlike DNA sequencing, RNA sequencing is more complex, involving a reverse transcription step to convert RNA into cDNA, and the efficiency of this step is crucial. Since the sequence of the human RNA internal standard is known and present in every extracted sample, it should theoretically occupy a constant proportion in the sequencing data of each sample. Therefore, if the proportion of human RNA internal standard readings in a sample is abnormally low, it indicates low reverse transcription efficiency, which may indicate operational errors or reagent problems. Sex control, on the other hand, is a sex internal standard amplified on the Y chromosome. It is detected only in male samples and not in female samples. It can be used to identify the sample and verify the sample label against its biological sex, preventing sample confusion and human error. In large research projects, clinical diagnostics, or forensic science, a large number of samples are usually processed simultaneously. From sampling, DNA extraction, library construction to sequencing, there are many steps involved. Human error can lead to mislabeling or misplacement of sample labels. Therefore, by introducing sex control into sequencing, the experimental results are compared with the sample's sex information, providing rapid, low-cost, and reliable cross-validation of sample identity, preventing sample confusion or contamination, and ensuring the accuracy and reliability of the data.

[0019] Specifically, for different quality control directions in the present invention, this embodiment provides a quality control primer set for human samples, which includes the following: based on GJB2MD , GJB3MD , IVSM , 12SD Quality control primers for human DNA with conserved gene design; based on GAPDH , ACTB , PARP2 Human RNA quality control primers designed for conserved and highly expressed housekeeping genes; Based on the Y chromosome SRY142 , DYS14134 , YSR240 , YSR186 Genetically designed sex control primers.

[0020] When designing quality control primers for human DNA, highly conserved regions of the aforementioned genes are first screened out, and primers are then designed based on these regions. Additionally, the genes corresponding to the human DNA quality control primers are deafness-related genes, so in addition to human DNA quality control, the presence of deafness mutations in the samples can be simultaneously identified. When designing sex-specific quality control primers, sex-specific regions on the Y chromosome (i.e., regions present only in male samples) are first screened out, and primers are then designed based on these regions. When designing quality control primers for human RNA, RNA quality control primers are unique. Both the F-terminal and R-terminal primers must be located at the junction of two exons in the transcriptome gene sequence and contain one or more CDS regions. The purpose of this design is that when only genomic DNA is present, each designed primer will align to two different regions, leading to primer mismatch and initiation of amplification. Only when RNA reverse transcription is present will the designed primers perfectly match the cDNA produced by RNA reverse transcription, allowing for the amplification of the target product. In addition, the amplification sequence length of the above-mentioned quality control primers is preferably 100bp to 500bp.

[0021] In some embodiments of the present invention, the quality control primer set includes the quality control primers shown in Table 1.

[0022] Table 1 Quality control primer sequence information

[0023] The quality control primer set provided by this invention can be used for quality control of third-generation nanopore sequencing and second-generation high-throughput genome sequencing. The quality control detection data indicators are shown in Table 2.

[0024] Table 2

[0025] Understandably, for both human DNA quality control and human RNA quality control, it is not required that all corresponding targets be completely detected, especially internal and external standards Homo05-07, because RNA expression in the human body varies over time, and RNA in clinical samples also suffers from severe degradation. Therefore, it is sufficient to detect at least one of them.

[0026] The following are some specific embodiments. It should be noted that the embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0027] The clinical samples used in these examples are swab or sputum samples collected from individuals; negative samples are sampling solutions that are not collected. Where specific techniques or conditions are not specified in these examples, they should be performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0028] Example 1 This example provides a detection kit for quality control sequencing of human samples, which includes the following reagents: All quality control primers are shown in Table 1; Multiplex PCR premix consists of PCR buffer, MgCl2, dNTPs, and hot-start Taq enzyme. Negative control product, i.e., nuclease-free water.

[0029] Example 2 Taking nanopore targeted sequencing technology as an example, this example demonstrates a method for quality control sequencing using the detection kit provided in Example 1, including the following steps: (1) Nucleic acid extraction from human samples.

[0030] Because nanopore targeted sequencing technology specifically amplifies the target fragment during the process, there is no need to remove the host nucleic acid during extraction in this case. Therefore, the nucleic acid extracted using a conventional commercial automated extractor can meet the requirements for subsequent library construction and sequencing. Specifically, in this case, the NAS48 from Mindray Bio-Medical Electronics was used to extract nucleic acid from the clinical sample to obtain the nucleic acid sample to be tested.

[0031] (2) Reverse transcription.

[0032] The RNA samples to be tested were transcribed into cDNA using a reverse transcription kit. The reaction procedure is shown in Table 3.

[0033] Table 3 Reverse transcription reaction procedure

[0034] (3) Multiple amplification.

[0035] Synthesize the 22 primers shown in Table 1, and mix them in equal volume ratios of three different categories to obtain a quality control primer mixture (or add only one type of quality control primer). Use the cDNA obtained in step (2) or the extracted DNA as a template, and perform multiplex PCR amplification according to the amplification system (Table 4). The amplification reaction procedure is shown in Table 5.

[0036] Table 4 Multiplex PCR amplification system

[0037] Table 5. Multiplex PCR amplification reaction procedure

[0038] (4) Purification of amplification products.

[0039] Prepare 75% ethanol freshly with nuclease-free water. Remove the magnetic beads from 2-8°C 30 minutes in advance and allow them to equilibrate to room temperature. Before use, vortex or invert the magnetic beads to mix thoroughly. Add 22 μL of magnetic beads to the amplification product obtained in step (3), vortex to mix thoroughly, and incubate at room temperature for 5 minutes to allow DNA to bind to the magnetic beads. Centrifuge briefly, place the sample on a magnetic rack and aspirate for about 1 minute. After the solution becomes clear, carefully remove the supernatant. Keep the sample on the magnetic rack at all times, add 150 μL of freshly prepared 75% ethanol to rinse the magnetic beads, and carefully remove the supernatant. Repeat the previous step, rinsing twice in total. Centrifuge briefly, place the sample on a magnetic rack and aspirate, and carefully remove all the supernatant. Add 20 μL of nuclease-free water, vortex to mix thoroughly, and let stand at room temperature for 5 minutes. Centrifuge briefly, place the sample on a magnetic rack and aspirate for about 1 minute. After the solution becomes clear, carefully transfer the supernatant to a new octet. In a fume hood, add 20 μL (1 times the total volume) of purification magnetic beads to the purified product, vortex to mix, briefly centrifuge, and let stand at room temperature for 5 minutes; then place on a magnetic rack and magnetize for 1 minute, discarding all supernatant. Add 150 μL of 75% ethanol and discard the supernatant; repeat the steps once. Briefly centrifuge, discard all waste liquid with a 10 μL pipette, and air dry at room temperature until the magnetic beads are matte. Add 17 μL of eluent, vortex to mix, briefly centrifuge, let stand at room temperature for 5 minutes, and then place on a magnetic rack and magnetize for 1 minute to obtain the purified product.

[0040] (5) Amplify the tag sequence.

[0041] Take 16 μL of the purified product obtained in the previous step and add it to 24 μL of buffer B (Mingde, catalog number 2202000060). Vortex to mix, centrifuge briefly, and amplify according to Table 6. This amplification system uses universal primer sequences to further amplify the amplification product, and tag sequences can be added to both ends of the product to distinguish different samples.

[0042] Table 6 Second Round Amplification Reaction Procedure

[0043] (6) Purification.

[0044] Equilibrate the purified magnetic beads at room temperature for at least 30 minutes, then vortex to mix. Add 40 μL of the second-round amplification product to the purified magnetic beads, vortex to mix, briefly centrifuge, and let stand at room temperature for 5 minutes. Place on a magnetic rack and aspirate for 1 minute, then discard the supernatant. Add 150 μL of freshly prepared 75% ethanol, and discard the supernatant. Repeat the above steps. Briefly centrifuge, discard all waste liquid with a 10 μL pipette, and air dry at room temperature for 1 minute, observing that the magnetic beads are matte. Add 10 μL of elution buffer, vortex to mix, briefly centrifuge, and let stand at room temperature for 5 minutes. Place on a magnetic rack and aspirate for 1 minute to obtain 9 μL of supernatant, which is the purified second-round amplification product.

[0045] (7) Hybrid library.

[0046] Based on the concentration, multiple libraries were mixed in equal amounts into the same tube of an eight-tube bundle, vortexed to mix, and then centrifuged briefly.

[0047] (8) Connect the sequencing adapter and run the sequencing machine.

[0048] The prepared products were sequenced using the nanopore sequencer according to the official sequencing protocol. The sequencing data were simultaneously analyzed using the Mindray Pathogenic Microorganism Analysis Software; for specific analysis procedures, please refer to the software's user manual.

[0049] Example 3 This example demonstrates human DNA quality control performed according to the method described in Example 2. Sequencing and analysis were performed on 8 clinical samples and 8 negative samples. The results are shown in Table 7: All clinical samples tested positive for the internal and external standards Homo01-04, indicating the presence of human DNA in the clinical samples. The negative samples, lacking human DNA, did not test positive for Homo01-04. These results demonstrate that the human DNA quality control primers provided by this invention can achieve DNA quality control for human samples.

[0050] Table 7. Results of Human DNA Quality Control

[0051] Example 4 This example demonstrates human RNA quality control according to the method shown in Example 2.

[0052] Eight clinical samples were set up in the experimental group, a blank control group (containing no samples), and a negative sample (not undergoing RNA reverse transcription). The results were compared after performing the same experimental procedure. The results are shown in Table 8. All clinical samples tested positive for the internal and external standards Homo05-07, proving that the reverse transcription process was performed correctly. Furthermore, the blank control group and negative samples did not test positive for the internal and external standards Homo05-08, demonstrating from two different perspectives that there was no RNA environmental or procedural contamination during the experiment. These experimental results confirm the effectiveness of human RNA quality control.

[0053] Table 8. Quality control results of human RNA

[0054] Example 5 In this example, gender quality control was performed according to the method described in Example 2. Sequencing and analysis were performed on 8 male and 8 female clinical samples. The results are shown in Table 9. Y chromosome targets were detected in all male clinical samples, while no Y chromosome was detected in the female samples, consistent with the gender detection results. This result indicates that the gender quality control primers and method provided by this invention can achieve quality control of human-derived samples.

[0055] Table 9. Gender Quality Control Results

[0056] Example 6 Referring to the method shown in Example 2, this example uses three quality control systems simultaneously to test samples with different concentration gradients of human genomic DNA. The samples used in this example are a series of samples obtained by gradient dilution of two clinical samples (male and female).

[0057] The test results are shown in Table 10. All three quality control measures functioned effectively and could detect the corresponding targets. Moreover, the percentage of reads was not high and did not significantly affect the detection performance of pathogens.

[0058] Table 10

[0059] In summary, the DNA, RNA, and sex control primers developed in this invention for human samples can effectively monitor the nanopore sequencing process from different dimensions, collectively improving the reliability, reproducibility, and interpretability of sequencing data. Especially for fields with extremely high requirements for data accuracy, such as clinical diagnostics and precision scientific research, they can provide essential quality assurance.

[0060] It should be noted that the present invention is not limited to the above-described embodiments. The above embodiments are merely examples, and any embodiments that have the same structure and perform the same effects as the technical concept within the scope of the present invention are included within the scope of the present invention. Furthermore, various modifications that can be conceived by those skilled in the art to the embodiments, and other ways of constructing by combining some of the constituent elements of the embodiments, without departing from the spirit of the present invention, are also included within the scope of the present invention.

Claims

1. A quality control primer set for human samples, characterized in that, Including the following: based on GJB2MD , GJB3MD , IVSM , 12SD Quality control primers for human DNA with conserved gene design; based on GAPDH , ACTB , PARP2 Human RNA quality control primers designed for conserved and highly expressed housekeeping genes; Based on the Y chromosome SRY142 , DYS14134 , YSR240 , YSR186 Genetically designed sex control primers.

2. The quality control primer set according to claim 1, characterized in that, Includes the following primers: The sequences of primers are as shown in SEQ ID NO. 1-2 (GJB2MD-F / R), SEQ ID NO. 3-4 (GJB3MD-F / R), SEQ ID NO. 5-6 (IVSM-F / R), SEQ ID NO. 7-8 (12SDF-F / R); SEQ ID NO. 9-10 (GAPDH-inF / R), SEQ ID NO. 11-12 (ACTB-inF / R), SEQ ID NO. 13-14 (PARP2-inF / R); SEQ ID NO. 15-16 (SRY142-F2 / R2), SEQ ID NO. 16-17 (DYS14134-F2 / R2), SEQ ID NO. 18-19 (YSR240-F / R), and SEQ ID NO. 20-21 (YSR186-F / R).

3. A reagent kit, characterized in that, Includes the quality control primer set as described in claim 2.

4. The application according to claim 3, characterized in that, It also includes a multiplex PCR premix, which comprises PCR buffer, MgCl2, dNTPs, and Taq enzyme.

5. The application of the quality control primer set as described in claim 2 in the sequencing analysis of human samples.

6. The application according to claim 5, characterized in that, The sequencing is either next-generation sequencing or nanopore sequencing.

7. The application according to claim 5, characterized in that, The sequencing analysis is a sequencing analysis of pathogenic microorganisms in human-derived samples.

8. The application according to claim 5, characterized in that, The human-derived samples include human blood, plasma, serum, urine, tissue, saliva, oral swabs, and bronchoalveolar lavage fluid.

9. A quality control method for nanopore targeted sequencing of human samples, characterized in that, Includes the following steps: S1. Extract nucleic acid from human samples to obtain nucleic acid samples to be tested; S2. Construct a multiplex amplification system containing the quality control primer set, multiplex amplification primer set, and nucleic acid sample to be tested as shown in claim 2, amplify, purify, and ligate sequencing adapters; S3. Sequencing and analyzing the data generated from sequencing.

10. The quality control method according to claim 9, characterized in that, The ratio of the quality control primer set to the multiplex amplification primer set is 1:5.

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