A method for removing residual contamination from amplicon sequencing products and its application

Abstract

This application relates to the field of gene sequencing technology, and in particular to a method for removing residual contamination from amplicon sequencing products and its application. The method includes: adding DNA spikes to the first-step PCR reaction system for constructing a sequencing library of a sample to be tested, followed by first-step PCR amplification using a first multiplex primer to obtain a first amplification product; adding a second-step PCR reaction system and a second PCR amplification primer to the first amplification product, and performing a second-step PCR amplification to obtain a sequencing library; the DNA spikes are designed and synthesized based on the sequences of genomic marker sites of the target organism in the sample to be tested; the first-step PCR reaction system includes dUTP and UDG; the second-step PCR reaction system includes dUTP; by adding DNA spikes to the first-step PCR reaction system, the amplification of residual contamination from the amplification products is reduced; and by adding dUTP to each PCR reaction system and adding UDG to the first-step PCR reaction system, uracil-containing PCR products can be degraded, thus removing residual contamination from the amplification products.

Description

Technical Field

[0001] This application relates to the field of gene sequencing technology, and in particular to a method for removing residual contamination from amplicon sequencing products and its application. Background Technology

[0002] Currently, amplicon sequencing, a type of next-generation sequencing, is widely used in research and clinical diagnosis for detecting microorganisms. The core of amplicon sequencing lies in the construction of the sequencing library. The general process involves first amplifying the target gene using PCR, and then adding sample barcodes and sequencing adapters to the PCR products via PCR or ligation reactions to enable parallel high-throughput sequencing of multiple samples. During library construction, the PCR reaction setup and amplification product purification steps inevitably require opening the reaction tubes, allowing amplification products to easily diffuse into the air, leading to residual contamination. Amplicon sequencing detects multiple marker sites on the target organism's genome using multiplex PCR in each reaction, making it both accurate and sensitive for pathogen detection, but also highly sensitive to residual amplification product contamination. If residual amplification products from previous batches are present during library construction, it will compromise the accuracy of the final test results, leading to false positives. The presence of residual amplification product contamination will also increase the difficulty of detecting low concentrations of the target organism, resulting in false negatives.

[0003] Therefore, how to provide a method to remove residual contamination from amplicon sequencing products in order to reduce contamination during the amplicon sequencing library construction stage is a technical problem that urgently needs to be solved. Summary of the Invention

[0004] This application provides a method for removing residual contamination from amplicon sequencing products, thereby solving the technical problem of residual contamination from amplicon sequencing products in the prior art.

[0005] This application provides a method for removing residual contamination from amplicon sequencing products, the method comprising:

[0006] DNA Spike-ins were added to the first step PCR reaction system for constructing the sequencing library of the sample to be tested, and then the first step PCR amplification was performed with the first multiplex amplification primer to obtain the first amplification product; the second step PCR reaction system and the second PCR amplification primer were added to the first amplification product, and the second step PCR amplification was performed to obtain the sequencing library with residual contamination of the amplification product removed.

[0007] The DNA Spike-ins are designed and synthesized based on the sequences of genomic marker sites of the target organism in the sample to be tested;

[0008] The first step PCR reaction system includes dUTP and uracil DNA glycosylation enzyme;

[0009] The second step of the PCR reaction system includes dUTP.

[0010] Furthermore, the DNA Spike-ins fragment includes a primer region and an amplification region. The primer region is the same as the primer region of the genomic marker site, and the base composition of the amplification region is the same as that of the genomic marker site but the arrangement is different.

[0011] Furthermore, the copy number of the DNA Spike-ins per reaction is ≥10,000.

[0012] Furthermore, the first step PCR reaction system also includes dTTP.

[0013] Furthermore, the second-step PCR reaction system also includes dTTP.

[0014] Furthermore, the first multiplex amplification primer is used to amplify the genomic marker sites and DNA spikes.

[0015] Furthermore, the ends of the forward and reverse primers of the second PCR amplification primers carry sample tags, and the ends of the forward and reverse primers of the second PCR amplification primers also carry sequencing adapters.

[0016] Furthermore, the first step of PCR amplification, the second step of PCR amplification, and the purification are performed under physically isolated conditions; and / or,

[0017] The extraction of nucleic acid from the sample to be tested, the first step of PCR amplification, and the second step of PCR amplification are all performed using filter tip methods.

[0018] Furthermore, the concentration of the sequencing library is ≥5 ng / μL.

[0019] This application provides an application of the method, the application including: using the method in the amplicon sequencing detection process of SARS-CoV-2, the amplicon sequencing detection process of human metapneumovirus, and the amplicon sequencing detection process of EB virus.

[0020] The technical solutions provided in this application have the following advantages compared with the prior art:

[0021] The method for removing residual contamination from amplicon sequencing products provided in this application embodiment addresses the issue that while the DNA Spike-ins sequences differ significantly from the original sequences, the detection primers remain the same. Therefore, adding a sufficient number of DNA Spike-ins to the first-step PCR reaction system in amplicon sequencing reduces the risk of amplicon contamination through competitive amplification and ensures that samples containing extremely low or no viral concentrations can generate enough material for sequencing and analysis. Adding dUTP to both the first-step and second-step PCR reactions ensures that both amplification products contain uracil. Adding uracil DNA glycosyltransferase (UDG) to the first-step PCR reaction system effectively degrades uracil-containing amplification products, thereby removing this residual contamination. Through these steps, residual contamination from amplicon sequencing products can be effectively removed. Attached Figure Description

[0022] The accompanying drawings, which are incorporated in and form part of this specification, illustrate embodiments consistent with this application and, together with the description, serve to explain the principles of this application.

[0023] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, for those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1 A flowchart illustrating the method provided in the embodiments of this application; Figure 2 A schematic diagram showing the comparison of signal values ​​detected in sterile, enzyme-free water negative samples using pipette tips with and without filters in ordinary and standardized laboratories, respectively, as provided in the embodiments of this application;

[0025] Figure 3 A comparison of the concentrations of high-throughput sequencing libraries prepared from DNA Spike-ins with different copy numbers provided in the embodiments of this application;

[0026] Figure 4 A graph showing the comparison of signal values ​​detected in 9 consecutive tests over 3 days in NTC and standard samples, provided for an embodiment of this application;

[0027] Figure 5 This is a schematic diagram comparing signal values ​​after adding different concentrations of UDG, provided in an embodiment of this application.

[0028] Figure 6 This is a schematic diagram illustrating the results of ccAMP-Seq detection of SARS-CoV-2 in 62 clinical samples, provided in an embodiment of this application. Figure 6 Figure A shows the signal-to-noise ratio of SARS-CoV-2 detected by ccAMP-Seq in 62 clinical samples; Figure 6 B shows the number of effective detection sites of SARS-CoV-2 detected by ccAMP-Seq in 62 clinical samples; Figure 6 C is a schematic diagram showing the results of ccAMP-Seq in detecting SARS-CoV-2 in 62 clinical samples. Detailed Implementation

[0029] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0030] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this application can be purchased from the market or prepared by existing methods.

[0031] like Figure 1 As shown in the embodiments of this application, a method for removing residual contamination from amplicon sequencing products is provided, the method comprising:

[0032] DNA Spike-ins were added to the first step PCR reaction system for constructing the sequencing library of the sample to be tested, and then the first step PCR amplification was performed with the first multiplex amplification primer to obtain the first amplification product; the second step PCR reaction system and the second PCR amplification primer were added to the first amplification product, and the second step PCR amplification was performed to obtain the sequencing library with residual contamination of the amplification product removed.

[0033] The DNA Spike-ins are designed and synthesized based on the sequences of genomic marker sites of the target organism;

[0034] The first step PCR reaction system includes dUTP and uracil DNA glycosylation enzyme (UDG);

[0035] The second step of the PCR reaction system includes dUTP.

[0036] In some alternative implementations, the DNA Spike-ins fragment includes a primer region and an amplification region.

[0037] The primer region is the same as the primer region of the genomic marker site, and the base composition of the amplification region is the same as that of the genomic marker site but the arrangement is different.

[0038] In this embodiment, the specific composition of the DNA Spike-ins fragment is defined, and the corresponding characteristics of the primer region and amplification region are defined. This can further ensure that the derived fragment meets the requirements for use of DNA Spike-ins. Based on the significant difference between the nucleotide sequence and the original sequence of the SARS-CoV-2 to be tested, on the one hand, the risk of amplicon contamination can be reduced through competitive amplification, and on the other hand, it can ensure that samples containing extremely low or no viral concentrations can generate sufficient material for sequencing and analysis, thereby removing amplicon contamination present in amplicon sequencing.

[0039] In some alternative implementations, the DNA Spike-ins have a copy number ≥10,000 per reaction.

[0040] In this embodiment of the application, the specific copy number of DNA Spike-ins is limited to ensure a sufficient number of DNA Spike-ins, thereby further reducing amplicon contamination in amplicon sequencing.

[0041] In some alternative implementations, the first-step PCR reaction system may further include dTTP.

[0042] In this embodiment, the specific reagents in the first-step PCR reaction system are defined, which can effectively reduce residual contamination in the sample to be tested while ensuring the amplification efficiency of the multiplex PCR system, thereby further reducing amplicon contamination in amplicon sequencing.

[0043] In the first step of the PCR reaction system, dTTP and dUTP can be used together in a ratio of 2:1. Alternatively, dUTP can be used alone in the first step of the PCR reaction system. The specific usage conditions depend on the actual situation of the target organism's marker site being effectively amplified by the PCR system.

[0044] In some optional implementations, the second-step PCR reaction system also includes dTTP.

[0045] In this embodiment of the application, the specific reagents in the second-step PCR reaction system are specified, which can further reduce the residual contamination in the products amplified by the second round of PCR amplification.

[0046] In the second-step PCR reaction mixture, dTTP and dUTP can be used together in a ratio of 2:1. Alternatively, dUTP can be used alone in the second-step PCR reaction mixture. The specific usage conditions depend on the actual situation of effectively amplifying the marker sites of the target organism in the PCR system.

[0047] In some alternative implementations, the first multiplex amplification primers are used to amplify the genomic marker sites and DNA spikes.

[0048] In this embodiment of the application, the specific target fragment to be amplified by the first multiplex amplification primer is defined, which can ensure that the preset marker sites and DNA spikes of the target organism's genome are amplified simultaneously. This ensures that even samples with low concentrations of the target organism can have a sufficient number of sequencing libraries, and reduces the amplification of contaminants through competitive amplification, thereby achieving highly sensitive detection of the target organism in the sample to be tested.

[0049] In some optional embodiments, the ends of the forward and reverse primers of the second PCR amplification primers carry sample tags, and the ends of the forward and reverse primers of the second PCR amplification primers also carry sequencing adapters.

[0050] In this embodiment of the application, the composition of the terminal sequences of the forward and reverse primers of the second PCR amplification primers is specified, which can further ensure that the second amplification product after the second PCR amplification contains sample tags and sequencing adapters, thereby satisfying the requirement for parallel sequencing of multiple samples in the subsequent high-throughput sequencing stage.

[0051] In some optional implementations, the first-step PCR amplification, the second-step PCR amplification, and the purification are performed in a physically isolated environment; and / or,

[0052] The extraction of nucleic acid from the sample to be tested, the first step of PCR amplification, and the second step of PCR amplification are all performed using filter tip methods.

[0053] In this embodiment, the environment for PCR amplification and purification of amplification products is defined, as well as the sampling method for PCR amplification and extraction of nucleic acid from the sample to be tested. This can reduce contamination during the manual operation stage through physical isolation, thereby further reducing contamination in amplicon sequencing.

[0054] In some alternative implementations, the concentration of the sequencing library is ≥5 ng / μL.

[0055] In this embodiment of the application, the specific concentration of the sequencing library is limited to ensure that there are a sufficient number of test samples in the high-throughput sequencing stage, thereby ensuring the accuracy of the sequencing stage.

[0056] The present application is further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the application. Experimental methods in the following embodiments that do not specify specific conditions are generally determined according to national standards. If there is no corresponding national standard, then general international standards, conventional conditions, or conditions recommended by the manufacturer are followed.

[0057] Example 1

[0058] An amplicon sequencing detection workflow for SARS-CoV-2, AMP-Seq, was constructed.

[0059] Detection of SARS-CoV-2 in samples using a two-step PCR method, such as Figure 1 As shown, the first step involves PCR amplification using ultramultiplex amplification primers to detect 164 markers on the SARS-CoV-2 genome. After the first step PCR product is purified, it is used as a template for the second step PCR amplification. The primers used are sample-specific primers with tags and sequencing adapters at the ends to distinguish the samples. The purified second step PCR product is the sequencing library. After quality control of the sequencing library, sequencing can be performed.

[0060] Example 2

[0061] Comparing Example 2 with Example 1, the difference between Example 2 and Example 1 is as follows:

[0062] The effectiveness of using cartridge-based pipette tips was demonstrated in AMP-Seq: Negative samples composed of sterile, enzyme-free water were tested using the AMP-Seq testing procedure in both physically isolated (standardized laboratory) and non-physically isolated (general laboratory) laboratories. Pipettes were used with or without cartridges, and five technical replicates were performed for each condition. The contamination level (hereinafter referred to as signal value) in each sample was assessed by the proportion of SARS-CoV-2 sequencing sequences detected in each sample to the total number of sequencing sequences in that sample. The results are shown in Table 1 and... Figure 2 As shown.

[0063] Table 1. Signal values ​​detected in sterile, enzyme-free, water-negative samples using pipette tips with and without filters in ordinary and standardized laboratories.

[0064]

[0065] The significance test was performed using the rank-sum test, and P < 0.05 was considered significant.

[0066] As shown in Table 1 and Figure 2The results showed that the average signal value of samples tested using filter tips in a standardized laboratory was 0.43%, significantly lower than the average signal value of 1.12% for samples tested without filter tips in a standardized laboratory, and the average signal values ​​of 0.97% and 1.28% for samples tested with and without filter tips in a general laboratory, respectively. This indicates that using filter tips in combination with pipetting in a standardized laboratory can effectively reduce the level of contamination.

[0067] Example 3

[0068] Comparing Example 3 and Example 2, the differences between Example 3 and Example 2 are as follows:

[0069] Seventeen SARS-CoV-2 marker sites were designed and synthesized, as shown in Table 2, to form DNA Spike-ins. Subsequently, 100,000, 50,000, 10,000, and 5,000 copies of DNA Spike-ins were added to sterile, enzyme-free, and water-negative samples, respectively, with each condition tested 12 times. Figure 3 As shown, the library concentration constructed from a negative sample containing 10,000 copies of DNA Spike-ins can stably reach the minimum library concentration required for high-throughput sequencing, which is approximately 5 ng / μL. Therefore, a concentration of 10,000 copies per reaction is considered a suitable concentration of DNA Spike-ins.

[0070] Table 2. Information on DNA Spike-ins

[0071]

[0072]

[0073]

[0074] In Table 2, the underlined parts are the forward primer region and the reverse primer region, respectively, and the ununderlined part is the amplification region.

[0075] Serial dilutions of SARS-CoV-2 nucleic acid standards:

[0076] According to the SARS-CoV-2 concentration indicated in the product instructions of the purchased SARS-CoV-2 nucleic acid standards, nucleic acid standards containing ~1, 10, and 100 copies of SARS-CoV-2 were prepared by dilution. The corresponding Ct values, measured by qRT-PCR, were uncertain, 37, and 33, respectively. Then, 10,000 copies of DNA spike-ins were added to each sample. Non-target control groups (NTCs) were also set up, consisting of sterile enzyme-free water, the reverse transcription product of sterile enzyme-free water (hereinafter referred to as reverse transcription control), and sterile enzyme-free water containing 10,000 copies of DNA spike-ins (hereinafter referred to as DNA spike-insNTC). The technique was repeated three times daily for three consecutive days to evaluate the contamination levels in different negative controls, the repeatability of the technique, and the sensitivity of the detection. The results are shown in Table 3 and... Figure 4 As shown.

[0077] Table 3 shows the signal values ​​detected in 9 consecutive tests over 3 days in NTC and standard samples.

[0078]

[0079]

[0080] The significance test was performed using the rank-sum test, and a p-value < 0.005 was considered significant.

[0081] From Table 3 and Figure 4 The results showed that DNA spike-ins NTC consistently had the lowest signal values ​​in the samples tested each day over three consecutive days. In all tests, the signal value of DNA spike-ins (average 0.05%) was significantly lower than that of sterile enzyme-free water NTC (average 1.14%) and the signal value of each sample containing 1 copy of SARS-CoV-2 (average 0.28%). In contrast, the signal value of sterile enzyme-free water NTC was significantly higher than that of each sample containing 1 copy of SARS-CoV-2, but lower than that of each sample containing 10 copies of SARS-CoV-2 (average 2.37%).

[0082] The results above indicate that using NTCs with DNA spike-ins significantly reduced contamination levels in the negative control compared to using NTCs without DNA spike-ins, resulting in at least a 10-fold increase in the sensitivity of AMP-Seq.

[0083] Example 4

[0084] Comparing Example 4 with Example 3, the differences between Example 4 and Example 3 are as follows:

[0085] The effectiveness of adding the dUTP / UDG system before each PCR in removing residual amplicon contamination from samples:

[0086] The dUTP / UDG system was incorporated into the AMP-Seq process. dUTP / UDG was added in the first-step PCR amplification, and dUTP was added in the second-step PCR amplification. To obtain the optimal dUTP concentration without affecting the efficiency of multiplex amplification, dUTP concentrations at ratios of 1:1, 2:1, and 3:1 were added in the first-step multiplex PCR step, and tests were performed on a standard containing 100 copies of SARS-CoV-2.

[0087] The results showed that the quality control of the sample library with dUTP added at a concentration ratio of 3:1 to dTTP was not up to standard. Therefore, the optimal dUTP concentration for the multiplex PCR system was determined to be dUTP:dTTP = 2:1 for each reaction.

[0088] Using a library prepared from a sample with added dUTP:dTTP = 2:1 as a template, amplicon contamination was simulated to determine the appropriate UDG concentration for each reaction. The specific steps were as follows: two experimental conditions were set, with 0.5 U and 1 U of UDG added, and each experimental condition was repeated twice. The results are shown in Table 4. Figure 5 As shown.

[0089] Table 4. Signal values ​​after adding different concentrations of UDG

[0090] Should UDG be added? Signal value (%) Simulated amplicon contamination UDG- 67.05 Simulated amplicon contamination UDG- 53.78 Simulated amplicon contamination UDG- 26.92 Simulated amplicon contamination UDG- 40.72 Simulated amplicon contamination 0.5U UDG+ 0.16 Simulated amplicon contamination 0.5U UDG+ 0.16 Simulated amplicon contamination 1U UDG+ 0.08 Simulated amplicon contamination 1U UDG+ 0.05

[0091] From Table 4 and Figure 5 The results showed that under experimental conditions with the addition of 0.5 U and 1 U of UDG, the average signal values ​​of the samples decreased to 0.16% and 0.07%, respectively, significantly lower than the 47.12% of the samples without UDG. By comparing the number of sequencing sequences at co-detected marker sites in samples containing and without UDG, the addition of UDG reduced the number of sequencing fragments at MNP sites by 22 to 199,016 times.

[0092] Therefore, the above results indicate that the dUTP / UDG system, composed of dUTP:dTTP = 2:1 and 1U of UDG, can effectively remove residual contamination from amplification products. The AMP-Seq system using standard laboratory filter tips, DNASpike-ins with a fixed copy number, and the AMP-Seq system with the added dUTP / UDG is named ccAMP-Seq.

[0093] Example 6

[0094] Comparing Example 6 and Example 5, the differences between Example 6 and Example 5 are as follows:

[0095] Applications of ccAMP-Seq in the highly sensitive and accurate detection of clinical samples:

[0096] ccAMP-Seq was used to detect SARS-CoV-2 nucleic acid standards at 1, 10, and 100 copies per reaction, as well as 62 clinical specimens from COVID-19 patients, including 38 pharyngeal swabs, 21 plasma samples, and 3 whole blood samples, with two negative controls. Of the 62 clinical specimens, 53 were positive samples confirmed by qRT-PCR, and 9 were negative samples identified by qRT-PCR from early-stage confirmed patients. The total sequence number obtained by ccAMP-Seq in the two negative controls was 4.8 × 10⁻⁶. 6 and 3.6×10 6 The number of sequences matched to SARS-CoV-2 were 0 and 1 respectively, indicating that ccAMP-Seq almost completely removed all contaminants (such as...). Figure 6 (As shown in A and 6B), while the signal-to-noise ratio and the number of effective detection sites for 62 clinical samples were 3.8 × 10⁻⁶. 2 ~4.3×10 8 (like Figure 6 (as shown in A) and 22–164 (as shown in A) Figure 6 As shown in B), the method described in CN114672591B (patent number: ZL202210026905.0) was used to determine whether a sample contained SARS-CoV-2 by utilizing the number of detected marker sites and the signal-to-noise ratio. The signal index is the ratio of the number of SARS-CoV-2 virus sequences detected in the sample to the number of DNA spike-ins sequences, and the noise index refers to the maximum value of the signal index in the negative control. All qRT-PCR positive samples were identified as SARS-CoV-2 nucleic acid positive by ccAMP-Seq, and 7 out of 9 qRT-PCR negative samples were also confirmed as positive (e.g., ...). Figure 6 (As shown in C), this indicates that there is a risk of false negatives in negative samples detected by qRT-PCR, and indirectly shows that ccAMP-Seq can be applied to various forms of clinical specimens, and that its risk of false negatives in SARS-CoV-2 detection is low. Since ccAMP-Seq almost completely eliminates residual amplification contamination, and the signal-to-noise ratio and number of sites detected in 1 copy of SARS-CoV-2 nucleic acid standard per reaction are much greater than in negative samples, it indicates that ccAMP-Seq can accurately detect positive samples with low viral concentrations, and the risk of false positives is also reduced.

[0097] Example 7

[0098] Comparing Example 7 and Example 6, the differences between Example 7 and Example 6 are as follows:

[0099] Application of ccAMP-Seq in the detection of human metapneumovirus:

[0100] ccAMP-Seq was used to detect 24 clinical specimens confirmed by qRT-PCR. Twelve were positive for human metapneumovirus (HMV) nucleic acid, and 12 were negative. ccAMP-Seq detected 11 marker sites on the HMV genome. The amplification regions of the DNA spike-ins were identical to sequences 1-7 used in the SARS-CoV-2 detection, except that the primer regions were replaced with primer sequences for the 11 HMV marker sites. The primer sequences for amplifying HMV and the method for determining the conclusion are shown in the patent (application number: CN202111329194.6). The number of detected marker sites and the signal-to-noise ratio were used to determine whether the sample contained HMV. The signal index is the ratio of the number of HMV sequences detected in the sample to the number of DNA spike-ins sequences, and the noise index refers to the maximum signal index in the negative control. The results are shown in Table 5.

[0101] Table 5 shows the number of marker sites and signal-to-noise ratio of human metapneumovirus detected in the samples.

[0102]

[0103]

[0104] / : indicates that it is not applicable.

[0105] As shown in Table 5, no human metapneumovirus (HMV) sequences were detected in the three negative controls, indicating a noise index of 0, which demonstrates that the method completely eliminated contamination. The results were evaluated: the ccAMP-Seq results of the 12 positive samples were completely consistent with the qRT-PCR results; two of the 12 negative samples (marked in bold in Table 5) were positive for ccAMP-Seq, and the qRT-PCR retest also confirmed the results. This demonstrates that the method of this application is also applicable to the detection of HMV, effectively reducing amplicon sequencing contamination and improving detection accuracy.

[0106] Example 8

[0107] Comparing Example 8 and Example 7, the differences between Example 8 and Example 7 are as follows:

[0108] Application of ccAMP-Seq detection in 23 clinical samples:

[0109] ccAMP-Seq was used to detect 23 clinical samples. qRT-PCR analysis revealed 13 positive cases of EBV nucleic acid and 10 negative cases. ccAMP-Seq detected 15 marker sites on the EBV genome. The amplification regions of the DNA spike-ins were identical to sequences 1-15 used for SARS-CoV-2 detection, with the primer regions replaced by primer sequences for the 15 EBV marker sites. The primer sequences for the 15 EBV marker sites and the conclusion determination method are shown in the patent (application number: CN202110962190.5). The presence of human metapneumovirus (HMV) in the sample was determined using the number of detected marker sites and the signal-to-noise ratio. The signal index was the ratio of the number of EBV sequences detected in the sample to the number of sequences detected by the DNA spike-ins, and the noise index was the maximum signal index in the negative control. The detection results are shown in Table 6.

[0110] Table 6. Number of marker sites and signal-to-noise ratio of EB virus.

[0111]

[0112]

[0113] / : indicates that it is not applicable.

[0114] The results showed that no EB virus sequences were detected in the three negative controls, meaning the EB virus noise index was 0, indicating that the method completely removed contamination. The test results for positive samples were completely consistent with those of qRT-PCR. Of the 10 samples that tested negative by qRT-PCR, only 3 were confirmed to be EB nucleic acid negative, while the other 7 had indeterminate results but were later confirmed to be EB virus positive in validation, indicating a low risk of false negatives in ccAMP-Seq test reports.

[0115] One or more technical solutions in the embodiments of this application have at least the following technical effects or advantages:

[0116] (1) The method for removing residual contamination of amplicon sequencing products provided in this application is designed by derivatizing fragments targeting 17 SARS-CoV-2 marker sites, so that DNA Spike-ins have significant nucleotide differences from the original sequence, but the detection primers are the same. Therefore, before library preparation, a sufficient number of DNA Spike-ins are added to the sample. On the one hand, contamination can be reduced through competitive amplification, and on the other hand, it can ensure that samples containing extremely low or no concentration of virus can produce enough material for sequencing and analysis.

[0117] (2) The method for removing residual contamination of amplicon sequencing products provided in this application embodiment is applicable to various commonly used amplicon library construction processes. The method provided in this application embodiment is based on two-step PCR amplification to construct an amplicon library. When configuring the first step PCR amplification reaction, dUTP with a concentration ratio of 2:1 to dTTP and 1U of UDG are added to form a mixed reaction system. Before the first step PCR reaction, the system is incubated at 25°C for 10 minutes. Then the first step PCR reaction is performed normally. In the second step PCR reaction system, dUTP with a concentration ratio of 2:1 to dTTP is also added to ensure that the products of both rounds of PCR carry dUTP.

[0118] (3) The method for removing residual contamination of amplicon sequencing products provided in the application embodiment is also applicable to the amplicon library construction process of one-step multiplex PCR amplification and one-step ligation reaction. When this method is used, the first step operation is the same as in the application embodiment. After completion, no further modifications are required. The second step ligation reaction is then performed directly to add sample tags and sequencing adapters.

[0119] (4) The SARS-CoV-2 amplicon sequencing method provided in this application shows that the constructed ccAMP-Seq can be applied to various forms of clinical specimens, and its SARS-CoV-2 test report has a low risk of false negatives.

[0120] Various embodiments of this application may exist in the form of a range; it should be understood that the description in the form of a range is merely for convenience and brevity and should not be construed as a hard limitation on the scope of this application; therefore, it should be considered that the range description has specifically disclosed all possible sub-ranges and single numerical values ​​within that range. For example, it should be considered that the range description from 1 to 6 has specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., and single numbers within the range, such as 1, 2, 3, 4, 5, and 6, regardless of the range. Furthermore, whenever a numerical range is referred to herein, it means including any referenced number (fraction or integer) within the referred range.

[0121] In this application, unless otherwise stated, directional terms such as "upper" and "lower" specifically refer to the orientation shown in the accompanying drawings. Furthermore, in the description of this application, the terms "comprising," "including," etc., mean "including but not limited to."

[0122] In this document, relational terms such as "first" and "second" are used merely to distinguish one entity or operation from another, without necessarily requiring or implying any actual relationship or order between these entities or operations. In this document, "and / or" describes the association between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A alone, A and B simultaneously, or B alone. A and B can be singular or plural. In this document, "at least one" means one or more, and "more than" means two or more. "At least one," "at least one of the following," or similar expressions refer to any combination of these items, including any combination of single or plural items. For example, "at least one of a, b, or c," or "at least one of a, b, and c," can both represent: a, b, c, ab (i.e., a and b), ac, bc, or abc, where a, b, and c can be single or multiple.

[0123] The above description is merely a specific embodiment of this application, enabling those skilled in the art to understand or implement this application. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of this application. Therefore, this application is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features claimed herein.

Claims

1. A method for removing residual contamination from amplicon sequencing products, characterized in that, The method includes: DNA Spike-ins were added to the first step PCR reaction system for constructing the sequencing library of the sample to be tested, and then the first step PCR amplification was performed with the first multiplex amplification primer to obtain the first amplification product; the second step PCR reaction system and the second PCR amplification primer were added to the first amplification product, and the second step PCR amplification was performed to obtain the sequencing library with residual contamination of the amplification product removed. The DNA Spike-ins are designed and synthesized based on the sequences of genomic marker sites of the target organism in the test sample; The first step PCR reaction system includes dUTP and uracil DNA glycosylation enzyme; The second step of the PCR reaction system includes dUTP; The DNA Spike-ins fragment includes a primer region and an amplification region. The primer region is the same as the primer region of the genomic marker site, and the base composition of the amplification region is the same as that of the genomic marker site but the arrangement is different. The copy number of the DNA Spike-ins per reaction is ≥10000; The first step PCR reaction system also includes dTTP; The second step of the PCR reaction system also includes dTTP.

2. The method according to claim 1, characterized in that, The first multiplex amplification primer is used to amplify the genomic marker sites and DNA spikes.

3. The method according to claim 1, characterized in that, The forward and reverse primers of the second PCR amplification primers each carry a sample tag at their ends, and the forward and reverse primers of the second PCR amplification primers each carry a sequencing adapter at their ends.

4. The method according to claim 1, characterized in that, The first step of PCR amplification, the second step of PCR amplification, and purification are performed in physically isolated environments; and / or, The extraction of nucleic acid from the sample to be tested, the first step of PCR amplification, and the second step of PCR amplification are all performed using filter tip methods.

5. The method according to claim 1, characterized in that, The concentration of the sequencing library is ≥5 ng / µL.

6. An application of the method as described in any one of claims 1-5, characterized in that, The applications include: using the method described in any one of claims 1-5 for non-diagnostic and non-therapeutic purposes in amplicon sequencing detection process for SARS-CoV-2, amplicon sequencing detection process for human metapneumovirus, and amplicon sequencing detection process for EB virus.

Images