A standard substance for calibrating a high-throughput sequencer and a preparation method thereof

Abstract

The application belongs to the field of gene sequencing, and relates to a standard substance for calibrating a high-throughput sequencer, a preparation method and application thereof. 97 DNA fragments of 320 bp in length are selected from a genome. The DNA fragments are optimized through the principle of chirality, reverse design, GC content homogenization, structural stability control, series modularization integration, and are added with tags and primers at two ends. The candidate fragment library with a length of 456 bp is prepared through gene fragment synthesis, cloning and identification, and target fragment amplification and purification. After correction, the above-mentioned fragments are mixed according to the correction ratio to obtain a multi-fragment DNA mixed standard substance. Nucleic acid quantification, sequencing, GC content calculation, uniformity test and stability research are carried out on the standard substance, and value assignment and uncertainty evaluation are completed, so that the metrological traceability is ensured. The high-throughput sequencer calibration standard substance containing 97 456 bp fragments is successfully developed, and the measurement characteristics are systematically confirmed.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology, specifically relating to a standard substance for high-throughput sequencer calibration, its preparation method, and its application. Background Technology

[0002] With the development of next-generation sequencing (NGS), also known as high-throughput sequencing or "next-generation" sequencing technology, the cost of sequencing has decreased far faster than Moore's Law, ushering in an era of high-throughput and low-cost sequencing. It can sequence hundreds of thousands to millions of DNA molecules in parallel at one time, which can be said to be the core of the epoch-making revolution of first-generation sequencing technology and is the most widely used sequencing technology today.

[0003] Gene sequencing typically involves six steps: sample preparation, DNA library construction, flow cell sequencing reaction, bridge PCR amplification and denaturation, sequencing, and data analysis. Obtaining accurate sequencing results requires strict adherence to each step. For example, sample quality must be assessed before library preparation; during library preparation, quality control measures are necessary to evaluate the quality of the prepared library. Low library purity leads to low cluster generation, and insufficient adapter sequences result in sequence loss. When analyzing the sequencing data, the lack of standardized data analysis procedures means the process is tailored to the specific type of sequence variation being detected. High-GC-content gene regions are difficult to enrich, and captured fragments may be difficult to sequence using NGS platforms, making it challenging to obtain the first exon sequence information for many genes. Furthermore, the gene sequences currently used for sequencing are incomplete, complicating NGS read alignment. Simultaneously, due to technological limitations and industry development, a series of unavoidable problems stemming from the drawbacks of NGS technology are intensifying, all of which will affect sequencing results to varying degrees.

[0004] For example, there is the "Preparation Method of Takara 16S rRNA Quantitative Standard Material Based on Artificially Synthesized 16S Variable Region Sequence". This method uses 12 artificially synthesized 16S variable region sequences, mixed at preset copy numbers, for quantitative calibration in microbiome sequencing. Its drawbacks include: highly specific application scenarios with extremely poor versatility; single sequence characteristics, making it impossible to comprehensively assess sequencing bias; weak metrological traceability; fixed universal primers at both ends of the sequence, lacking comprehensive adaptability to sequencing platforms; and limited performance evaluation dimensions, focusing only on quantitative accuracy.

[0005] Another example is "metagenomic composite standard material based on artificially synthesized multi-pathogen RNA sequences." This method uses artificially synthesized multi-pathogen RNA sequences to simulate a clinical sample background for sensitivity and specificity verification in metagenomic sequencing, covering multiple species and multiple targets. Its drawbacks include limited sequence features and complexity, making it unable to simulate whole-genome scenarios; and limited platform applicability.

[0006] Currently, the main drawback of high-throughput sequencing is:

[0007] 1. Quality control deficiencies and technical bottlenecks in library preparation: Library preparation is a critical starting step in next-generation sequencing, and this step has significant quality control and technical shortcomings. On the one hand, low library purity directly leads to insufficient cluster generation, and missing adapter sequences cause sequence information loss. These problems all stem from imperfect quality control measures during preparation, directly affecting the basic quality of sequencing. On the other hand, gene sequence regions with high GC content are difficult to enrich. Even if capture is completed, the corresponding fragments are difficult to adapt to the NGS platform for effective sequencing, resulting in the inability to obtain the first exon sequence information of many genes, thus limiting the integrity and effectiveness of sequencing data from the source.

[0008] 2. Incomplete gene sequence coverage affecting read alignment: Current second-generation sequencing uses gene sequences with incomplete coverage, which directly hinders the read alignment of sequencing data. Due to insufficient integrity of the reference sequence, reads obtained from sequencing are prone to mismatch, incorrect matching, or low matching efficiency during alignment. This not only reduces the effective utilization of data but may also lead to deviations in subsequent analysis results such as sequence variation detection and gene localization. In addition, combined with the limitations of the technology itself and the stage of industry development, the inherent drawbacks of NGS technology continue to become prominent, ultimately having a negative impact on the accuracy and completeness of sequencing results to varying degrees from multiple dimensions.

[0009] The existing standard materials suffer from the problem of "simulation failure": they cannot simulate the physical and biochemical states of real samples, resulting in distorted quality control signals.

[0010] Most existing synthetic or cloning standard materials are "pure" DNA solutions. Their physical form (naked DNA) and template structure (short fragments or circular plasmids) are fundamentally different from the linear DNA fragments in real-world / clinical samples that bind to heterogeneous matrices and originate from complex genomes. This "state gap" prevents them from accurately reflecting the actual behavior of DNA extraction efficiency and initial PCR templates, causing quality control conclusions based on them to be disconnected from the performance of real samples, rendering quality control ineffective.

[0011] 3. The problem of "single evaluation dimension" of existing quality control tools: They are limited to basic qualitative assessment and cannot achieve quantitative and multi-dimensional performance analysis.

[0012] Existing methods primarily answer the question of "whether or not it is detected," but fail to address the questions of "whether it is good or bad" and "why it is bad." Key dimensions in the sequencing process that directly impact data quality and biological conclusions, such as quantitative linearity, detection limits for low abundance, amplification / sequencing bias towards high-GC or complex sequences, and the ability to classify highly homologous sequences, lack corresponding, known "standard answers" for calibration and evaluation.

[0013] 4. The lack of a shareable "performance benchmarking platform" in the industry: This leads to inaccurate methodological comparisons and hinders technological progress and standardization.

[0014] The lack of a universally accepted reference material with a clearly defined composition, traceable values, and a standardized challenge set (i.e., a set of standardized samples or variant sites designed to "test" detection / analysis procedures based on known real results) makes it impossible to objectively and fairly compare the performance of different laboratories, commercial reagent kits, and bioinformatics procedures. This has led to a fragmented technological ecosystem, making it difficult to mutually recognize and integrate research results, and slowing down the process of methodological optimization and standardization across the industry.

[0015] 5. The problem of "inaccessibility of preparation" of high-fidelity standard materials: traditional routes are costly and complex, and cannot meet the demand for universal access.

[0016] Standard materials that can partially reflect the above-mentioned complexity rely heavily on in vivo biological culture and complex processes for preparation. They have inherent limitations such as large batch-to-batch variations, long preparation cycles, biosafety risks, and extremely high costs, and cannot be used as routine consumables to support the growing scientific research and clinical diagnostic applications with stringent data quality requirements. Summary of the Invention

[0017] The purpose of this invention is to overcome the shortcomings of the prior art and provide a standard substance for calibration of high-throughput sequencers based on a total synthesis strategy and its preparation method.

[0018] This invention aims to construct a complete system for comprehensive calibration, quality control, and internal control of high-throughput sequencing platforms. This system includes physical DNA standard materials and a dedicated digital sequence reference database. Its design follows a progressive process from basic sequence screening and core sequence optimization to full-function library simulation, ultimately forming a multi-level system that combines fragment-level precision (i.e., in the reference materials / standard library, key parameters such as sequence, length, structure, and molar ratio of each DNA fragment are precisely designed and accurately determined, enabling traceability and quality control at the single-fragment level) and fragment structure.

[0019] To achieve the above objectives, the present invention employs the following technical solution:

[0020] This invention provides a method for preparing standard materials for high-throughput sequencer calibration, comprising:

[0021] Step a) describes the design, synthesis, and absolute quantification of DNA simulation materials used for high-throughput sequencer calibration.

[0022] Step b) Perform a corrective mixing to obtain a mixed standard substance, which is the standard substance for high-throughput sequencer calibration.

[0023] The DNA simulation design for high-throughput sequencer calibration standard materials in step a) includes:

[0024] (I) Reference sequence screening and basic database design: N fragments of length M are randomly selected from different chromosomes of the human reference genome using bioinformatics methods to construct the basic database of the design; preferably, the human reference genome is version GRCh38.p14; preferably, 50-200 fragments of length 200-400bp are selected, more preferably, 97 fragments of length 320bp are selected as the basic database of the design;

[0025] (II) Optimization of the basic database and design of the sequencing reference database SMRD: Based on the basic database design, the sequences therein are optimized using bioinformatics methods to obtain N optimized reference unit sequences, and / or the optimized reference unit sequences are modularly integrated and further assembled into two integrated long reference sequences, named Integrated Reference Sequence 1 and Integrated Reference Sequence 2, abbreviated as RIS1 and RIS2. The designed reference database SMRD includes optimized reference unit sequences, and / or two integrated reference sequences RIS1 and RIS2 assembled from optimized reference unit sequences; preferably, the optimized reference unit sequences are obtained by using chiral principle reverse design, GC content homogenization, and structural stability control to eliminate potential secondary structure interference; preferably, the sequences are modularly integrated based on the principles of base balance, sequence complexity, and physical distribution uniformity.

[0026] (III) Sequencing adapter design: The sequencing adapter includes a P5 / P7 adapter, an index or barcode sequence, and a sequencing primer binding region. Preferably, the sequencing primer binding region includes R1 SP / R2 SP. Preferably, the sequencing adapter is ligated using methods such as DNA fragmentation, end repair, A-tailing, adapter ligation, fragment screening, and PCR amplification.

[0027] (IV) Sequencing Standard Material Library Database (SMLD) Design: Add a universal adapter sequence containing some or all of the elements in (III) to both ends of all the reference unit sequences of the sequencing standard material reference database optimized in step (II) to obtain the designed sequencing standard material library database (SMLD).

[0028] (V) The design of the preparation of the standard material for the calibration of the high-throughput sequencer includes (1) designing a method for obtaining and verifying high-precision DNA samples of the N optimized design benchmark unit sequences respectively; (2) designing the signal ratio of the high-precision DNA samples of the N optimized design benchmark unit sequences after purification, i.e., the design ratio or "target observation ratio".

[0029] Step a) involves the synthesis and absolute quantification of standard materials for high-throughput sequencer calibration, including:

[0030] (I) High-fidelity synthesis: Using gene synthesis technology, the complete DNA fragments of the N optimized design reference unit sequences are obtained and verified according to the method designed in claim 2(V). Sequencing is performed to ensure sequence accuracy, and purity verification is performed to ensure the purity of the synthesized sample. Preferably, all DNA fragments can be chemically synthesized. Preferably, some DNA fragments can be obtained using "PCR amplification + in vitro splicing," i.e., the target region and its flanking sequences are first PCR amplified from the genomic DNA of a standard strain, and then these fragments are in vitro spliced ​​with a synthesized "challenge sequence variant" to construct the required simulation template. Preferably, the in vitro splicing can be achieved using overlap extension PCR or Gibson assembly technology. Preferably, the "challenge sequence variant" is a high-fidelity DNA fragment. GC sequences, homologous sequences, and artificial chimeras are preferred. The "challenge sequence variant" is a chimera containing conserved regions of both bacteria and fungi. Preferably, the preparation via gene synthesis technology involves synthesizing the N optimized baseline unit sequences through sequence fragment synthesis, constructing a vector to obtain a strain containing the baseline unit sequence fragment, sequencing the strain, preserving the correctly sequenced monoclonal strains, enriching the correctly sequenced monoclonal strains by shaking, extracting plasmids, amplifying the target fragment using the extracted plasmid as a template, performing agarose gel electrophoresis on the product, excising and recovering the target fragment, purifying it using a gel recovery method, and finally sequencing the purified product to identify the final N DNA samples with accurate sequence information.

[0031] (II) Absolute Quantification:

[0032] Each synthesized fragment is absolutely quantified to determine its copy number concentration. Preferably, absolute quantification is performed using a digital PCR-based method, i.e., ddPCR. More preferably, quantification is performed using "molecular counting based on next-generation sequencing" combined with an external reference standard of known concentration.

[0033] Specifically, in step a), the high-throughput sequencer calibration standard material DNA simulation design, synthesis, and absolute quantification select DNA sequences according to gradient difference ratios.

[0034] Step b) includes:

[0035] (I) Establishment and application of the calibration model: Based on historical data or pre-experiments, a deviation prediction model is established. This model quantifies the signal intensity deviation coefficient that different DNA sequences in the sequencing standard reference database may produce in a specific amplification or sequencing process due to differences in GC content, length and corresponding primer and probe efficiency of the target DNA sequence. According to the design ratio in step a)(V), the deviation prediction model is used to back-calculate and obtain the "corrected mixing ratio" required for each component to achieve this design ratio, i.e., the actual input ratio.

[0036] (II) Precision Hybrid Execution in a Stabilized Environment

[0037] A multi-component DNA stabilization diluent is prepared, comprising Tris-HCl, EDTA, vector nucleic acid, and a surfactant, preferably EDTA. Specifically, a TE buffer at pH 8.0 is used, i.e., 10 mM Tris-HCl, 1 mM EDTA. The DNA stock solutions, as determined by absolute quantification in step a), are precisely mixed with the above stabilization diluent according to the calculated "corrected mixing ratio". Preferably, the "corrected mixing ratio" is expressed in absolute copy number.

[0038] Preferably, the corrective mixing adopts "iterative feedback mixing", that is, preliminary mixing is first carried out and a small-scale prediction sequence is performed. Based on the deviation between the prediction sequence result and the design value, the correction coefficient is calculated, and the proportion of the mixture is adjusted a second time. This process is repeated until the optimal match is achieved.

[0039] Preferably, the DNA stock solutions obtained in step a)(II) are bound to an inert carrier material and then eluted; more preferably, the inert carrier material is selected from inert inorganic microspheres, artificially simulated complex matrices, inactivated real background substances, and gel embedding systems; preferably, the artificially simulated complex matrix is ​​selected from synthetic particles made of proteins, polysaccharides, lipids, and humic acids mixed in a specific ratio; preferably, the inactivated real background substances are selected from... Standard soil, saliva, or fecal sample powders that have been completely inactivated and have had their background DNA removed by radiation irradiation; the gel embedding system is used to embed synthetic DNA fragments in microspheres to simulate the spatial confinement effect of DNA in biological membranes or tissues; the hydrogel is preferably made of agarose or polyacrylamide.

[0040] (III) Formation of the final product

[0041] After being thoroughly mixed, the solution is dispensed and stored at low temperature to obtain the standard material for high-throughput sequencer calibration.

[0042] The method further includes:

[0043] Step c): Comprehensive performance verification, including:

[0044] (I) Multidimensional sequencing validation:

[0045] Using the high-throughput sequencer calibration standard material obtained in step b)(III), repeat sequencing is performed on at least two mainstream high-throughput sequencing platforms. Preferably, the mainstream high-throughput sequencing platforms are Illumina MiSeq and NovaSeq.

[0046] (II) Generate a "Performance Evaluation Report":

[0047] Bioinformatics analysis of sequencing data not only verifies the detection rate but also systematically outputs key performance indicators.

[0048] Preferably, the key performance indicators are:

[0049] Quantitative accuracy: the correlation coefficient (R²) between observed abundance and designed abundance. 2 ) and deviation;

[0050] Process challenge assessment: Evaluate the coverage uniformity and classification resolution of high GC sequences and homologous sequences;

[0051] Cross-platform comparability: Comparing the differences in analytical results of the same standard substance from different sequencing platforms.

[0052] In this step, “RNA standard material based on in vitro transcription” replaces “standard material DNA simulation design” in step a). That is, a DNA template containing the target sequence is first synthesized, and then the corresponding RNA mixture is generated through in vitro transcription, and then the RNA sample is simulated.

[0053] The present invention therefore provides a set of standard materials for high-throughput sequencer calibration, which contains 97 nucleic acid sequences shown in SEQ ID NO: 1-SEQ ID NO: 97, preferably, the mixing ratio of SEQ ID NO: 1-SEQ ID NO: 97 is equal.

[0054] Furthermore, the present invention provides an article comprising the set of standard substances for calibrating the high-throughput sequencer.

[0055] This invention also provides the application of the aforementioned collection or products of standard substances for high-throughput sequencer calibration as standard substances for high-throughput sequencer calibration.

[0056] This invention, through the technical path of "simulation design - absolute quantification - morphological simulation - precise mixing", not only overcomes the core problems in the background technology one by one, but also brings a series of breakthrough and verifiable beneficial effects: for the first time, it realizes the essential leap of standard substances from "sequence control" to "behavioral simulator", and solves the root cause of "simulation failure".

[0057] The DNA template length in this standard material is highly consistent with that of real genomic DNA, and its physical morphology simulates the binding state with complex matrices through the carrier material. This allows it to accurately reflect the recovery rate differences in DNA extraction steps and reproduce PCR amplification kinetics indistinguishable from real samples. It can detect quality control data obtained by users, thus possessing unprecedented "ecovalidity" and can be directly used to optimize and calibrate pretreatment wet laboratory procedures.

[0058] Simultaneously, this invention constructs a quantitative diagnostic tool capable of performing a "panoramic CT scan" of the entire sequencing process, completely changing the current situation of single-dimensional quality control assessment. Using it for a single experiment, it can simultaneously output multi-dimensional, quantitative performance indicator reports, including: quantitative performance: evaluating the dynamic range and quantitative linearity (R²) of the detection through abundance gradient. 2 Resolution and sensitivity: Through abundance design, the detection limit and accuracy of the process are determined; Algorithm performance: Through homologous sequences, the true positive rate and false negative rate of bioinformatics classification and filtering algorithms are objectively evaluated. In this way, a revolutionary upgrade of quality control has been achieved from "result verification" to "process diagnosis and optimization guidance".

[0059] This invention establishes an industry-shared, metrologically-grade "performance benchmark" by combining absolute quantification based on digital PCR (dPCR) with a precise calibration standard, laying the physical foundation for data comparability and methodological standardization. The value of each component in this standard reference material is traceable to dPCR, a primary measurement standard, making it a stable and reproducible metrological "ruler." Tests of this standard reference material by different laboratories, at different times, and using different platforms will all return to the same set of absolute "truth." This provides an objective and reliable technical basis for cross-research and cross-platform data comparison and integration for the first time, powerfully propelling research into a new stage of "reproducibility and interoperability."

[0060] This invention, through a total synthesis strategy and an engineered, modular preparation process, pioneers a new paradigm for standard substances that combines "high-end performance" with "universal availability," breaking through the bottleneck of unattainable high-fidelity standard preparation. This invention completely eliminates reliance on in vivo biological culture and complex biological processes, transforming the preparation of standard substances into a highly controllable, scalable, and biosafety-free in vitro biochemical and physical engineering process. This significantly reduces the cost of producing standard substances with complex diagnostic functions, fundamentally ensuring batch-to-batch consistency, thus transforming them from expensive "research luxuries" into "routine diagnostic consumables" that can be widely deployed in quality control systems, possessing enormous market transformation potential and social value. Attached Figure Description

[0061] Figure 1 Standard material DNA design process.

[0062] Figure 2 Add connector and index location map.

[0063] Figure 3 Database construction.

[0064] Figure 4 Standard material gene preparation process.

[0065] Figure 5 Image showing plasmid identification results.

[0066] Figure 6 PCR amplification procedure diagram for the target fragment.

[0067] Figure 7 Figure showing the results of PCR product purification and identification.

[0068] Figure 8 Electrophoresis results of sequence 33-49 on the chip.

[0069] Figure 9 Sequence 34 chip electrophoresis results.

[0070] Figure 10 The sequencing alignment results of gene sequence 1 are shown in the figure. Detailed Implementation

[0071] The present invention will be further described below through specific embodiments in order to better understand the present invention, but this does not constitute a limitation on the present invention.

[0072] Example 1

[0073] Step 1: Standard Material DNA Simulation Design

[0074] This study aims to construct a comprehensive system for the calibration, quality control, and internal control of high-throughput sequencing platforms. This system includes physical DNA labeling materials and their dedicated digital sequence reference database. The physical standard materials are named "DNA Standard Materials for High-Throughput Gene Sequencing Instrument Calibration," and their corresponding digital sequence information sets are constructed as the "Sequencing Standard Material Reference Database."

[0075] Its design follows a progressive process from basic sequence selection and core sequence optimization to full-featured library simulation, ultimately forming a multi-level system that combines fragment-level accuracy with long-fragment structure. For the core design roadmap and outputs at each stage, please refer to [link to relevant documentation]. Figure 1 As shown:

[0076] 1. Reference sequence selection and basic database construction:

[0077] This invention uses the human reference genome GRCh38 (version GRCh38.p14) as the design basis. This genome integrates the core reliability of Sanger sequencing with the supplementary completeness of second-generation sequencing data, possessing authoritative sequence structure and rich genomic diversity. Based on the GRCh38 reference genome, 97 fragments (SEQ ID No: 1 to SEQ ID No: 97) of 320 bp in length were randomly selected from different chromosomes using bioinformatics methods to construct the "GRCh38 Basic Database". The database aims to broadly cover multiple regions of the genome, avoid technical bias, ensure its representativeness in various sequencing technologies (such as bridging amplification, rolling circle amplification, etc.), and serve as the source for subsequent sequence optimization and design.

[0078] 2. Core sequence (i.e., the selected 97 fragment sequences) optimization and generation of sequencing standard reference database:

[0079] To avoid cross-contamination between samples and achieve precise localization in complex biological contexts, the sequences were systematically optimized using bioinformatics methods based on the GRCh38 database. These optimizations included chiral-based reverse engineering, GC content homogenization, and modular sequence integration. Chiral-based reverse engineering: The chiral principle was used for reverse engineering sequence design, introducing sequence features significantly different from the natural genome to ensure high specificity and detectability as internal controls. GC content homogenization: The GC content of all fragments was strictly controlled within the range of 25%–65% to mitigate the GC bias of sequencing technology. Modular sequence integration: The optimized 97 fragments were further assembled into two comprehensive long reference sequences based on principles such as base balance, sequence complexity, and physical distribution uniformity. These were named Integrated Reference Sequence 1 and Integrated Reference Sequence 2 (RIS1 / RIS2).

[0080] 3. Full-function connector integration:

[0081] In high-throughput sequencing experiments, the sample to be tested needs to undergo a series of preprocessing steps (including DNA fragmentation, end repair, A-tailing, adapter ligation, fragment selection, and PCR amplification). The core purpose is to ligate sequencing adapters to both ends of the target DNA fragment. For the locations of adapters and indexes, please refer to [link to relevant documentation]. Figure 2 As shown, a sequencing library capable of being recognized by the instrument is constructed. The library adapter includes a P5 / P7 adapter, an index (barcode) sequence, and sequencing primer binding regions (R1 SP / R2 SP). Based on the above principles, a universal adapter sequence containing all the above elements was designed and added to both ends of all unit sequences in the optimized sequencing reference database, thereby constructing the sequencing reference library database SMLD and completing the final design scheme for the standard reference sequence used for high-throughput sequencer calibration.

[0082] 4. The sequencing standard reference database has been completed:

[0083] Through reference sequence design and balance optimization, a sequencing reference database of standard materials was designed, consisting of multiple fragments with completely known sequences. (This system fully includes 97 baseline unit sequences (RUS) and 2 assembled sequences, etc.) The reference sequence information table is shown in Table 1. Baseline unit sequences refer to known sequences used as alignment and assembly standards, typically validated, high-quality genomic sequences. Assembled sequences refer to continuous long sequences obtained by splicing and integrating a large number of short sequencing reads using sequence assembly software. Simultaneously, DNA standard materials were developed based on the design scheme of the sequencing reference material library database. A schematic diagram of the entire data construction is shown in [reference needed]. Figure 3 As shown. In subsequent calibration experiments, the data generated from sequencing this standard material will be compared with the "Sequencing Standard Material Reference Database" to achieve an accurate evaluation of the sequencing system's performance.

[0084] Step 2: Controlled Synthesis and Absolute Quantification

[0085] The purpose of this step is to accurately and traceably transform the digital design from step one into a physical entity.

[0086] 1. Preparation of Standard Reference Materials

[0087] The core preparation strategy is as follows: First, high-precision DNA samples containing 97 reference unit sequences (RUS) are obtained and validated using chemical synthesis and molecular cloning techniques. Then, the purified fragments are mixed in equal proportions to prepare the final standard substance. For the complete standard substance gene preparation process, please refer to [link to documentation]. Figure 4As shown in the diagram, the DNA samples were obtained by synthesizing 97 baseline unit sequences using sequence fragment synthesis. These sequences were then used to construct strains containing the fragments via plasmids and other vectors, followed by Sanger sequencing. Strains with correct sequencing results were streaked twice to form single clones, which were then preserved. Simultaneously, the single clones were enriched by shaking for plasmid extraction. The target fragments were then amplified using the plasmids as templates. The products were subjected to agarose gel electrophoresis (1.5%), and the target fragments were excised and purified using gel extraction methods. Finally, the purified products were identified using Sanger sequencing. Ultimately, 97 DNA samples with accurate sequence information were obtained.

[0088] The following is the specific process for preparing standard material genes.

[0089] 1. Synthesis of gene fragments

[0090] We commissioned Shenzhen BGI Genomics Co., Ltd. to chemically synthesize all target DNA fragments based on the sequence information of 97 reference unit sequences (RUS) in the SMLD. The synthesis service covered the entire process from sequence design, oligonucleotide synthesis, splicing to complete fragments.

[0091] 2. Cloning and identification of fragments

[0092] To obtain a template that can be stably amplified and has a precise sequence, the synthesized fragment was cloned and identified:

[0093] 1) Cloning vector construction: Using homologous recombination technology, each target fragment was ligated into a cloning vector and transformed into competent E. coli cells;

[0094] 2) Screening of positive clones: The transformed bacterial culture was plated on LB agar plates containing the corresponding antibiotics and incubated at 37°C for 12-16 hours. Positive clones were then screened by colony PCR.

[0095] 3) Sequence Validation and Plasmid Preparation: Positive clones from the initial screening were validated using Sanger sequencing. Clones with 100% sequence identity with the designed sequence were amplified and high-purity plasmids were extracted using a plasmid extraction kit. The extracted plasmids were identified by agarose gel electrophoresis (1%) (band size equals vector plus sequence). See the results figure below. Figure 5 As shown. The correct strains, verified by sequencing, were purified by streak purification twice to form single clones, which were then prepared into a glycerol strain library and stored at -80℃ for long-term storage.

[0096] 3. Amplification and purification of the target fragment

[0097] This step aims to prepare high-purity, high-accuracy target DNA fragments in large quantities from validated, correct plasmid templates, providing high-quality raw materials for the subsequent construction of reference materials. The entire process undergoes rigorous optimization and multiple quality controls to ensure that the final 97 reference unit sequences (RUS) meet the requirements for reference materials in terms of sequence accuracy and physicochemical properties.

[0098] 1) PCR Amplification System Optimization and High-Fidelity Enzyme Selection: During the PCR amplification stage, the focus was on screening and optimizing high-fidelity DNA polymerases. This study compared two commonly used high-fidelity enzymes: NEB Q5 High-Fidelity DNA Polymerase and BGI's self-produced high-fidelity PFU enzyme. Through systematic comparison, the most suitable amplification system for this project was determined.

[0099] High-fidelity enzyme comparison experiment design: The same plasmid template and primer combination were used. A uniform amplification procedure was employed; see [link to relevant documentation]. Figure 6 As shown: 98℃ pre-denaturation for 30 seconds; 35 cycles (98℃ 10 seconds, 60℃ 30 seconds, 72℃ 30 seconds); final extension at 72℃ for 5 minutes. A comprehensive evaluation of the yield, accuracy, and fragment integrity of the amplified products was performed.

[0100] Technical parameter comparison and analysis: NEB Q5 High-Fidelity DNA Polymerase exhibits superior overall performance, with a fidelity approximately 200 times that of ordinary Taq enzymes and an error rate of 2.8 × 10⁻⁶. -7 It is particularly suitable for amplifying fragments of 400-500 bp in length. In comparison, while Pfu enzymes offer comparable fidelity, their amplification efficiency is slightly lower than that of Q5 enzymes. Sanger sequencing of the amplified products confirmed that the sequence amplified using Q5 enzymes was 100% identical to the reference sequence, with no base errors detected.

[0101] Final optimized reaction system: Based on the above comparison results, a 25 μL standard reaction system was selected, and its specific composition is shown in Table 2.

[0102] Table 2. Composition of the reaction system

[0103]

[0104] The system was validated by three independent replicate experiments, showing good reproducibility. The yield of amplified products remained stable between 850-950 ng, meeting the requirements of subsequent experiments.

[0105] 2) Product Purification and Identification: The PCR products were separated by agarose gel electrophoresis (1.5%). A target band of approximately 400-500 bp (corresponding to the size of the target fragment) was excised and purified using an agarose gel DNA recovery kit. A small amount of the purified product was verified by agarose gel electrophoresis (1.5%). The results confirmed the correct fragment size and good purity. See the results figure below. Figure 7 As shown.

[0106] 3) Final quality control: The purified final DNA product is subjected to Sanger sequencing again to ensure that no mutations were introduced during the amplification and purification process.

[0107] Through the aforementioned rigorously optimized processes and quality control measures, 97 RUS DNA samples with completely accurate sequences and meeting purity requirements were successfully prepared. These samples met the stringent requirements for standard substances in terms of concentration, purity, and sequence accuracy.

[0108] To ensure the accuracy and purity of the reference material sequences, a systematic quality control process was performed on the 97 prepared reference unit sequence DNA samples. The testing included two key components: fragment purity analysis and Sanger sequencing verification. Multiple quality controls were implemented to ensure that each DNA sample met the stringent requirements for reference material preparation.

[0109] 1. Purity detection of DNA fragments

[0110] 1) Sample pretreatment

[0111] The 97 purified PCR products were precisely diluted to a uniform concentration of 10 ng / μL using TE buffer (10 mM Tris-HCl, 1 mM EDTA) at pH 8.0. Each sample was vortexed for 30 seconds, briefly centrifuged, and 100 μL of standardized DNA suspension was prepared for use.

[0112] 2) Chip testing process

[0113] Chip preparation: Using an Agilent high-sensitivity DNA chip, add 9 μL of gel-dye mixture to each well. Sample loading: Accurately transfer 1 μL from each standardized sample into the designated sample well. Instrument analysis: Microfluidic electrophoresis analysis was performed using an Agilent 2100 Bioanalyzer.

[0114] 3) Quality control standards and results

[0115] Purity determination: A qualified sample must exhibit a single main peak at the expected size (approximately 456 bp) in the electrophoresis pattern, without primer dimers (<100 bp) or other non-specific amplification products or other contaminating peaks. Integrity confirmation: The size of the fragment corresponding to the main peak must be consistent with the designed sample length, with fluctuations controlled within ±5%. For example, see the results for sequence 33-49. Figure 8 and Figure 9 As shown.

[0116] The test results showed that all 97 samples met the above quality control standards, proving that the DNA samples had high purity and good integrity, and met the requirements for subsequent preparation of standard substances.

[0117] 2. Detection of fragment DNA Sanger sequencing sequence information

[0118] To ensure sequence accuracy to the greatest extent possible, this study employs a multiple validation strategy:

[0119] 1) Tripartite verification: Each sample was sent to three authoritative sequencing institutions, namely BGI Genomics, Sangon Biotech, and Kangwei Century, for independent verification;

[0120] 2) Bidirectional sequencing: Each sample undergoes both forward and reverse bidirectional sequencing to ensure full-length sequence coverage;

[0121] 3) Repeated testing: Two independently prepared samples were provided for sequencing for each institution.

[0122] Sequence analysis quality control:

[0123] 1) Sequence alignment: Use BioEdit software to align the sequencing peak diagram with the reference sequence;

[0124] 2) Quality assessment: High clarity of sequencing peaks, low background noise, and Q values ​​greater than 30 are required;

[0125] 3) Consistency standard: The sequencing results of the three institutions are required to be 100% consistent with the reference sequence, and the forward and reverse sequencing results must be completely consistent.

[0126] All 97 samples underwent rigorous sequencing verification. The sequence verification results are shown in Table 3. For example, the alignment results for sequence 1 are shown in Table 3. Figure 10 As shown.

[0127] Table 3. Comparison of Sequencing Results with Reference Sequence

[0128]

[0129] Through purity testing and multiple sequencing verification using the above system, it was ensured that the 97 reference unit sequences met the stringent requirements for standard substances in terms of concentration, purity, and sequence accuracy, laying a solid foundation for the construction of high-quality standard substances.

[0130] 2. High-fidelity synthesis:

[0131] Using gene synthesis technology, all the DNA fragments designed in step one are prepared. Sequencing is required to ensure sequence accuracy, and purity verification is necessary to ensure the purity of the synthesized sample.

[0132] 3. Absolute Quantification and Standardization:

[0133] For each synthesized fragment, absolute quantification was performed using a digital PCR-based (ddPCR) method to determine its copy number concentration. ddPCR is currently the "gold standard" for nucleic acid quantification, and its characteristic of not relying on a standard curve provides the most basic and traceable quantitative basis for this standard material.

[0134] Step 3: Corrective dilution and mixing based on the bias prediction model

[0135] This step is the core innovation of the present invention. Its purpose is to accurately convert the digitized "design ratio" (i.e., the predetermined ratio) in step one into a physical entity through a dilution and mixing process that includes a pre-correction mechanism, so as to offset the known systematic biases in the downstream analysis process and ensure that the final observation results truly reflect the design intent.

[0136] 1. Establishment and application of the calibration model

[0137] Based on historical data or preliminary experiments, a bias prediction model is established (i.e., the bias of other sequences is predicted by comparing the bias obtained from the data measured by one sequence with the predicted data). This model quantifies the signal intensity bias coefficient that different target DNA sequences may produce in a specific amplification or sequencing process due to differences in GC content, length, and corresponding primer and probe efficiencies (e.g., the observed signal of sequence A may be only 80% of the theoretical value, while that of sequence B may be 120%).

[0138] Before mixing, based on the "design ratio" (i.e. "target observation ratio") of each target preset in step one, the deviation prediction model is used to back-calculate and obtain the "actual input ratio" (i.e. "corrected mixing ratio") required by each component in absolute copy number during mixing to achieve this target observation ratio.

[0139] 2. Precision hybrid execution in a stabilized environment

[0140] Prepare a multi-component DNA stabilization dilution solution (e.g., containing Tris-HCl, EDTA, vector nucleic acid, and surfactant). The standard dilution solution and storage medium used are TE buffer with pH=8.0, which contains 10 mM Tris-HCl and 1 mM EDTA to provide a stable environment for mixing and prevent adsorption and degradation during the operation.

[0141] Using the DNA stock solutions that have been absolutely quantified in step two, precisely mix them with the above-mentioned stabilizing diluent according to the calculated "corrected mixing ratio" (in absolute copy number).

[0142] This process ensures that the initial (input) absolute copy number ratios of each DNA component in the mixture are pre-corrected.

[0143] 3. Formation of the final product

[0144] The homogenized solution is aliquoted and stored at low temperature to obtain the high-throughput sequencer calibration standard material of this invention (hereinafter referred to as this standard material). The characteristic of this material is that the proportion of its components in the physical mixing state is corrected, so that after undergoing the downstream analysis process, the final detected signal proportion can highly restore the "target observation proportion (design proportion)" preset in step one.

[0145] Step 4: Comprehensive Performance Verification

[0146] This step embodies the characteristic of this standard substance being "designed for application," and its verification process itself is a demonstration of its function.

[0147] 1. Multidimensional sequencing validation:

[0148] Use this standard material to perform repeat sequencing on at least two mainstream high-throughput sequencing platforms (such as Illumina MiSeq and NovaSeq).

[0149] 2. Generate a "Performance Evaluation Report":

[0150] Bioinformatics analysis of sequencing data not only verifies the detection rate but also systematically outputs key performance indicators.

[0151] Quantitative accuracy: the correlation coefficient (R²) between observed abundance and designed abundance. 2 ) and deviation.

[0152] Process challenge assessment: Evaluate the coverage uniformity and classification resolution of high GC sequences and homologous sequences.

[0153] Cross-platform comparability: Comparing the differences in analytical results of the same standard substance from different sequencing platforms.

[0154] This invention aims to provide a highly realistic and diagnostic standard substance for calibrating high-throughput sequencers. To achieve this objective, in addition to the core technical approach described above, other alternative solutions fall within the overall concept of this invention and should be considered within its scope of protection. Specific alternative solutions are as follows:

[0155] I. Alternative solutions for the "simulation design" stage:

[0156] 1. Alternatives to sequence sources:

[0157] DNA fragments on different chromosomes of the human reference genome can be "in vitro genomic fragments containing flanking sequences." Besides direct chemical synthesis, a "PCR amplification + in vitro splicing" approach can also be used. This involves first PCR amplifying the target region and its flanking sequences from the genomic DNA of a standard strain. Then, using techniques such as overlap extension PCR or Gibson assembly, these fragments are in vitro spliced ​​with synthesized "challenging sequence variants" (i.e., single sequences or variant sites with complex structures, high detection difficulty, and known truth values; these are the basic units constituting performance evaluation, such as high-GC blocks) to construct the required simulation template.

[0158] 2. Alternatives to challenge types:

[0159] In addition to high-GC sequences, homologous sequences, and artificial chimeras, the "challenge matrix" (i.e., a complete evaluation system that systematically combines multiple challenging sequence variants according to dimensions such as type, abundance, and structure) can also introduce:

[0160] "Cross-boundary" simulation sequence:

[0161] Design a chimera that incorporates conserved regions of both bacteria and fungi to test the specificity of classification algorithms in extreme tests.

[0162] II. Alternative solutions for the "morphological simulation and carrier processing" stage:

[0163] 1. Substitution of carrier materials:

[0164] In addition to inert inorganic microspheres, carriers used to simulate sample matrices can also include:

[0165] Artificially simulated complex matrix:

[0166] Synthetic particles made from a specific mixture of proteins, polysaccharides, lipids, and humic acids.

[0167] The actual background material for inactivation:

[0168] If passed Standard soil, saliva, or fecal sample powders that have been completely inactivated and have had their background DNA removed by radiation are used as carriers.

[0169] Gel embedding system:

[0170] Synthetic DNA fragments are embedded in hydrogel microspheres (such as agarose or polyacrylamide) to mimic the spatial confinement effect of DNA in biological membranes or tissues.

[0171] 2. Alternative mechanisms of action:

[0172] In addition to the "binding-elution" method to simulate non-specific adsorption, the "covalent linkage-enzyme cleavage release" method can also be used. This involves first covalently coupling the DNA fragment to the vector via a cleavable linker arm (such as one containing restriction enzyme sites or chemical cleavage sites), and then releasing it by the user using a specific enzyme or chemical reagent before use, thus more rigorously simulating certain enzyme-based extraction methods.

[0173] III. Alternative solutions for the "absolute quantification and benchmark mixing" stage:

[0174] 1. Alternatives to quantitative methods:

[0175] Besides digital PCR (dPCR), the "gold standard" for absolute quantification can also be achieved under specific conditions using next-generation sequencing-based molecular counting (NGS-based counting) combined with external reference standards of known concentrations. This is acceptable as long as the accuracy and precision of the method are fully validated and can achieve traceable quantification.

[0176] 2. Replacement of mixed logic:

[0177] To achieve "corrective mixing," in addition to pre-correcting based on deviations calculated from GC content and length, "iterative feedback mixing" can also be used. This involves first performing preliminary mixing and a small-scale prediction sequence. Based on the deviation between the prediction sequence results and the design values, a correction coefficient is calculated, and the mixture ratio is then adjusted a second time. This process is iterated until the optimal match is achieved.

[0178] IV. Alternatives to the complete technology path:

[0179] A fundamental alternative approach is "RNA standard material based on in vitro transcription." This method first synthesizes a DNA template containing the target sequence, then generates a corresponding RNA mixture through in vitro transcription, and finally simulates the extraction and reverse transcription process of RNA samples (such as metatranscriptomics). This method aims to solve the quality control challenges of RNA-level sequencing, and its core concepts (simulation design, challenge matrix, morphological simulation, and absolute quantification) are consistent with the DNA method of this invention.

[0180] The preparation process of "simulated extracts" involves combining standard DNA with an inert carrier material and then eluting it, enabling the standard substance to evaluate the performance of the DNA extraction process, which goes beyond the scope of simple nucleic acid mixtures.

[0181] Multiplex PCR amplification strategy: While achieving multi-target amplification in a single reaction, the optimization of reaction conditions reduces primer competition, ensuring the effective detection and accurate proportion of low-abundance targets.

[0182] Practical evaluations have shown that the aforementioned high-throughput sequencer calibration standard material exhibits stable sequencing results, accurate fragment information, and good reproducibility across high-throughput gene sequencing platforms from different manufacturers, using different models, and with different chips. It can be used directly for NGS sequencer calibration or introduced as an additive in sample pretreatment to achieve end-to-end quality control from library construction to sequencing. It is also suitable for performance evaluation of various sequencing systems.

Claims

1. A method for preparing standard substances for high-throughput sequencer calibration, characterized in that, include: Step a) describes the design, synthesis, and absolute quantification of DNA simulation materials used for high-throughput sequencer calibration. Step b) Perform a corrective mixing to obtain a mixed standard substance, which is the standard substance for high-throughput sequencer calibration.

2. The method according to claim 1, characterized in that, The DNA simulation design for high-throughput sequencer calibration standard materials in step a) includes: (I) Reference sequence screening and basic database design: N fragments of length M are randomly selected from the biological genome (e.g., from different chromosomes of the human reference genome) using bioinformatics methods to construct the basic database of the design; preferably, the human reference genome is version GRCh38.p14; preferably, 50-200 fragments of length 200-400bp are selected, more preferably, 97 fragments of length 320bp are selected as the basic database of the design; (II) Optimization of the basic database and design of the sequencing reference database SMRD: Based on the basic database design, the sequences therein are optimized using bioinformatics methods to obtain N optimized reference unit sequences, and / or the optimized reference unit sequences are modularly integrated and further assembled into two integrated long reference sequences, named Integrated Reference Sequence 1 and Integrated Reference Sequence 2, abbreviated as RIS1 and RIS2. The designed reference database SMRD includes optimized reference unit sequences, and / or two integrated reference sequences RIS1 and RIS2 assembled from optimized reference unit sequences; preferably, the optimized reference unit sequences are obtained by using chiral principle reverse design, GC content homogenization, and structural stability control to eliminate potential secondary structure interference; preferably, the sequences are modularly integrated based on the principles of base balance, sequence complexity, and physical distribution uniformity. (III) Sequencing adapter design: The sequencing adapter includes a P5 / P7 adapter, an index or barcode sequence, and a sequencing primer binding region; preferably, the sequencing primer binding region includes R1 SP / R2 SP; preferably, the sequencing adapter is ligated using methods such as DNA fragmentation, end repair, A-tailing, adapter ligation, fragment screening, and PCR amplification. (IV) Sequencing Standard Material Library Database (SMLD) Design: Add a universal adapter sequence containing some or all of the elements in (III) to both ends of all the reference unit sequences of the sequencing standard material reference database optimized in step (II) to obtain the designed sequencing standard material library database (SMLD). (V) The design of the preparation of the standard material for the calibration of the high-throughput sequencer includes (1) designing a method for obtaining and verifying high-precision DNA samples of the N optimized design benchmark unit sequences respectively; (2) designing the signal ratio of the high-precision DNA samples of the N optimized design benchmark unit sequences after purification, i.e., the design ratio or "target observation ratio".

3. The method according to claim 2, characterized in that, The synthesis and absolute quantification of the high-throughput sequencer calibration standard material in step a) includes: (I) High-fidelity synthesis: Using gene synthesis technology, the complete DNA fragments of the N optimized design reference unit sequences are obtained and verified according to the method designed in step (V). Sequencing is performed to ensure sequence accuracy, and purity verification is performed to ensure the purity of the synthesized sample. Preferably, all DNA fragments can be chemically synthesized. Preferably, some DNA fragments can be generated using "PCR amplification + in vitro splicing," that is, the target region and its flanking sequences are first PCR amplified from the genomic DNA of the standard strain, and then these fragments are spliced ​​in vitro with the synthesized "challenge sequence variant" to construct the required simulation template. Preferably, the in vitro splicing can be achieved by overlap extension PCR or Gibson assembly technology. Preferably, the "challenge sequence variant" is a high-GC-content PCR variant. The sequence, homologous sequence, and artificial chimera are preferred. The "challenge sequence variant" is a chimera containing conserved regions of both bacteria and fungi. Preferably, the preparation via gene synthesis technology involves synthesizing the N optimized baseline unit sequences through sequence fragment synthesis, constructing a vector to obtain a strain containing the baseline unit sequence fragment, sequencing the strain, preserving the correctly sequenced monoclonal strains, enriching the correctly sequenced monoclonal strains by shaking, extracting plasmids, amplifying the target fragment using the extracted plasmid as a template, performing agarose gel electrophoresis on the product, excising and recovering the target fragment, purifying it using a gel recovery method, and finally sequencing the purified product to identify it, ultimately obtaining the N optimized DNA samples with accurate sequence information. (II) Absolute quantification: Absolute quantification is performed on each synthetic fragment to determine its copy number concentration. Preferably, absolute quantification is performed using a digital PCR-based method, i.e., ddPCR. More preferably, quantification is performed using "molecular counting based on next-generation sequencing" combined with an external reference standard of known concentration.

4. The method according to claim 1, characterized in that, In step a), the high-throughput sequencer calibration standard material DNA simulation design, synthesis, and absolute quantification select DNA sequences according to gradient difference ratios.

5. The method according to claim 2, characterized in that, Step b) includes: (I) Establishment and application of the calibration model: Based on historical data or pre-experiments, a deviation prediction model is established. This model quantifies the signal intensity deviation coefficient that different DNA sequences in the sequencing standard reference database may produce in a specific amplification or sequencing process due to differences in GC content, length and corresponding primer and probe efficiency of the target DNA sequence. According to the design ratio of step (V), the deviation prediction model is used to back-calculate and obtain the "calibrated mixing ratio" required for each component to achieve this design ratio, i.e., the actual input ratio. (II) Precise mixing in a stabilizing environment: Prepare a multi-component DNA stabilizing diluent containing Tris-HCl, EDTA, vector nucleic acid, and a surfactant, preferably EDTA. Specifically, use a TE buffer at pH 8.0, i.e., 10 mM Tris-HCl and 1 mM EDTA. Using the DNA stock solutions that have been absolutely quantified in step a), precisely mix them with the above-mentioned stabilizing diluent according to the calculated "corrected mixing ratio". Preferably, the "corrected mixing ratio" is in absolute copy number. Preferably, the corrective mixing adopts "iterative feedback mixing", that is, a preliminary mixing is first carried out and a small-scale prediction sequence is performed. Based on the deviation between the prediction sequence result and the design value, the correction coefficient is calculated, and the proportion of the mixture is adjusted a second time. This process is repeated until the optimal match is achieved. Preferably, the DNA stock solutions, which have been quantitatively determined in step (II), are bound to an inert carrier material and then eluted; more preferably, the inert carrier material is selected from inert inorganic microspheres, artificially simulated complex matrices, inactivated real background substances, and gel embedding systems; preferably, the artificially simulated complex matrix is ​​selected from synthetic particles made of proteins, polysaccharides, lipids, and humic acids mixed in a specific ratio; preferably, the inactivated real background substance is selected from... Standard soil, saliva, or fecal sample powders that have been completely inactivated and have had their background DNA removed by radiation irradiation; the gel embedding system is used to embed synthetic DNA fragments in microspheres to simulate the spatial confinement effect of DNA in biological membranes or tissues; the hydrogel is preferably made of agarose or polyacrylamide. (III) Formation of the final product: The solution after uniform mixing is the standard material for high-throughput sequencer calibration, which may also be aliquoted and stored at low temperature.

6. The method according to any one of claims 1 to 5, characterized in that, The method further includes: Step c): Comprehensive performance verification, including: (I) Multidimensional sequencing verification: Using the high-throughput sequencer calibration standard material obtained in step (III), repeat sequencing is performed on at least two mainstream high-throughput sequencing platforms. Preferably, the mainstream high-throughput sequencing platforms are Illumina MiSeq and NovaSeq. (II) Generate a “performance evaluation report”: Perform bioinformatics analysis on sequencing data to not only verify the detection rate, but also systematically output key performance indicators; Preferably, the key performance indicators are: Quantitative accuracy: Correlation coefficient (R²) and bias between observed abundance and designed abundance; Process challenge assessment: Evaluate the coverage uniformity and classification resolution of high GC sequences and homologous sequences; Cross-platform comparability: Comparing the differences in analytical results of the same standard substance from different sequencing platforms.

7. The method according to any one of claims 1 to 6, characterized in that, Replace "standard material DNA simulation design" in step a) with "RNA standard material based on in vitro transcription". That is, first synthesize a DNA template containing the target sequence, then generate the corresponding RNA mixture through in vitro transcription, and then simulate the RNA sample.

8. A set of standard substances for calibrating a high-throughput sequencer, characterized in that, The set of standard materials for high-throughput sequencer calibration includes 97 nucleic acid sequences shown in SEQ ID NO: 1-SEQ ID NO:

97. Preferably, each of the sequences in SEQ ID NO: 1-SEQ ID NO: 97 is mixed in equal proportions.

9. An article comprising the set of standard substances for high-throughput sequencer calibration as described in claim 8.

10. The use of the set of standard substances for high-throughput sequencer calibration as described in claim 8 or the article described in claim 9 as standard substances for high-throughput sequencer calibration.

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