Methods and compositions for nucleic acid analysis

Abstract

The present disclosure relates to compositions and methods for the detection, identification, sequence analysis and quantification of organisms in a single amplification reaction. The disclosed methods use next generation sequencing (NGS) to sequence the amplification products. The disclosure also relates to kits containing primers specific for microorganisms and viral organisms, cancer, genetic diseases, and forensic medicine.

Description

[0001] Related Applications

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 369,946, filed on July 30, 2022, entitled “Multiplexed Barcoding Amplification and Multi-locus Sequence Typing,” the entire contents of which are incorporated herein. Background of the Invention

[0004] The multiplex PCR (polymerase chain reaction) method allows for the simultaneous amplification of multiple target DNA sequences in a single reaction. It involves the use of multiple primer pairs, each specific for a different target sequence, along with a DNA polymerase and nucleotides. By incorporating multiple primer sets, each corresponding to a specific target region, into a single PCR reaction, multiple DNA fragments can be amplified simultaneously.

[0005] Multiplex PCR has a wide range of applications in many fields, including medical diagnosis, genetics, forensics, microbial and viral detection, and research. Some applications of multiplex PCR include: (1) genetic disease screening; (2) pathogen identification; (3) forensic DNA analysis; (4) human leukocyte antigen (HLA) typing; and (5) cancer mutation analysis.

[0006] For genetic disease screening, multiplex PCR can be used to screen for the presence of mutations or gene variations associated with specific genetic diseases, hereditary diseases, or cancer. By simultaneously amplifying multiple target genes or regions, it is possible to detect multiple disease-related variants in a single test. For pathogen identification, multiplex PCR can be used to quickly detect and identify pathogens such as bacteria, viruses, parasites, or fungi in clinical, food, or environmental samples. By targeting specific genomic regions unique to different pathogens, multiplex PCR can provide rapid and accurate diagnosis. For forensic DNA analysis, multiplex PCR is widely used in forensic science for DNA feature analysis and identification, enabling the amplification of multiple genetic markers, such as short tandem repeats (STRs), thereby allowing simultaneous analysis of multiple loci and improving the accuracy of DNA feature analysis. For HLA typing, which is critical in organ transplantation and immunogenetics, multiplex PCR-based methods can simultaneously amplify and identify specific HLA gene alleles, enabling rapid and comprehensive HLA typing for compatibility assessment. For cancer mutation analysis, multiplex PCR can be used to detect specific mutations or genetic changes associated with cancer. Multiplex PCR allows for efficient screening and characterization of tumor samples by amplifying target genes known to carry cancer-associated mutations.

[0007] Targeted DNA sequencing allows users to selectively analyze specific regions of the genome. Only specific target regions are sequenced instead of sequencing the entire genome. This approach is more targeted and more efficient because it allows those skilled in the art to collect information about specific genes or genomic regions without sequencing the entire genome. Targeted DNA sequencing typically involves the use of capture probes or primers designed to specifically bind to and capture the target region. These primers or probes are typically complementary to the targeted DNA sequence, enabling them to be selectively amplified and sequenced. This is particularly useful when studying specific genes or genomic regions known to be associated with certain pathogenicity, diseases, or traits. By targeting these regions, researchers can analyze variants, mutations, or structural changes that may be associated with specific conditions or traits. Compared with whole genome sequencing (WGS), targeted DNA sequencing is faster, less expensive, and requires fewer computing resources. It has become a valuable tool in various research fields, including cancer genomics, genetic disease diagnosis, forensics, and personalized medicine.

[0008] Detection and accurate identification of pathogens at the subtype level involve a variety of methods and technologies. The ability to accurately distinguish between different strains and substrains within bacterial / fungal / parasitic / viral species is an important requirement for accurate genotyping and epidemiological surveillance, especially in the agricultural and food industries and clinical settings. Globalization has increased the complexity of the food supply chain more than ever before; companies are relying on suppliers of ingredients and raw materials from more parts of the world. This in turn leads to a potential increase in food-related risks, as raw materials may not be produced to the same hygienic standards as domestically produced raw materials. This has led to changes in food safety regulations for domestic and foreign food suppliers, requiring them to identify all potential hazards associated with food and implement measures to prevent these risks from becoming a threat to public health. Despite these measures, food safety recalls due to product contamination and outbreaks (especially those related to pathogenic microorganisms) continue to occur, resulting in huge costs to the public and food producers. In the preventive measures of identification, better technologies are needed that allow various participants in the food supply chain to proactively detect, identify and remove contaminated food.

[0009] Although selective housekeeping genes are used for strain differentiation, the slow accumulation of variation within the housekeeping loci will lead to a lack of differentiation between extremely closely related strains. Therefore, more diverse loci with informative variation can provide high-resolution data, which can lead to genotyping at the substrain level. Currently, the food industry needs a direct, effective, widely applicable and cost-effective method to not only detect multiple strains in food and the environment, but also to determine their serotypes and potential sources. For example, the Salmonella genus includes more than 2,700 serotypes that cannot be distinguished using low-resolution methods. Although whole genome sequencing (WGS) can generate a complete genomic map of food pathogens, it is a laborious, time-consuming and expensive method that requires pure isolates. Therefore, WGS is not suitable for samples with complex microbial backgrounds, limiting its widespread application in the food industry.

[0010] Subtyping of bacteria, fungi, parasites, or viruses determines the similarities between separate isolates of the same species, which is an important measure for preventing foodborne illness outbreaks because it allows public health officials to quickly identify the source of contamination and take appropriate measures to prevent further spread. Several methods for subtyping microorganisms and viruses are currently in use. Serotyping is a phenotyping method that involves identifying the type of bacterial cell surface antigens. This method can be used to distinguish different strains of a species, but its ability to distinguish is lower than that of molecular methods. Pulsed field gel electrophoresis (PFGE) is a molecular typing method that uses restriction enzymes to digest bacterial DNA into large fragments, which are then separated by size using gel electrophoresis. The resulting banding patterns can be used to compare bacterial strains and identify relatedness.

[0011] Multilocus sequence typing (MLST) is a molecular typing method that involves sequencing several housekeeping genes within the genomes of microorganisms and viruses. The resulting sequences can be compared with sequences of other related strains / types to identify their relatedness and infer their evolutionary relationships. Traditional MLST classifies and compares bacterial strains based on the nucleotide sequences of several loci. The method involves PCR amplification and sequencing of internal fragments of 6-8 housekeeping genes, which are genes that encode essential metabolic functions and are conserved between bacterial species. Although traditional MLST can distinguish bacterial strains based on sequence variation in the targeted loci, it may not be able to distinguish closely related strains. Although traditional MLST has several advantages, it also has some limitations, such as: (1) limited genomic coverage, given that MLST only targets a few genes, typically six to nine, which may not provide a comprehensive understanding of the genetic diversity of bacteria / fungi / parasites / viruses; (2) insufficient differentiation ability, given that MLST may not always be able to distinguish closely related strains, which may be problematic in epidemiological studies or outbreak investigations; and 3) MLST requires pure colonies and cannot analyze samples with complex backgrounds.

[0012] WGS is a powerful technique for studying the genetic composition of organisms, including microbial and viral genomes. The method provides the highest level of resolution and can be used to identify SNPs and other genetic variations within pathogens. WGS can provide the highest level of specificity and sensitivity in identifying microbial and viral subtypes and tracking the spread of epidemics. However, the technology also has some limitations. First, the cost of WGS can be a limiting factor, especially for large-scale studies or routine clinical applications. However, sequencing costs have been rapidly declining in recent years, making it more accessible to researchers and clinicians, but it is still expensive for routine settings. Second, the amount of data generated by WGS can be very large, which poses challenges for storage, processing, and analysis. Processing and analyzing such large data sets requires specialized computing resources and expertise. Third, the quality of sequencing reads can affect the accuracy of the results, and there may be technical problems or errors in the sequencing process that need to be resolved. Fourth, interpreting the results of WGS can be challenging, especially for non-experts and routine laboratories, as it requires a detailed understanding of genomics, bioinformatics, and statistical analysis. There is also a risk of misinterpretation or drawing wrong conclusions due to the complexity of the data. Fifth, WGS is not always able to resolve fine variations within microbial populations, such as single nucleotide variants or small insertions and deletions, which can lead to difficulties in accurately tracking transmission or understanding the evolution of bacterial populations. Sixth, for microbial and viral genome sequencing, WGS requires pure colonies. Samples with complex backgrounds cannot be analyzed by WGS. In short, while WGS is a powerful and increasingly accessible tool for microbial, fungal, parasitic, and viral genomics, it requires careful consideration of its limitations and potential challenges in data management, analysis, and interpretation, which in turn requires skilled data analysis expertise and labor.

[0013] The solution is to order-check only a large group of informative variable / hypervariable regions in microorganism and virus genomes rather than order-checking the housekeeping genes of low discrimination power by traditional MLST to obtain significantly higher resolution of differentiation. The number of these polymorphic loci varies due to the bacteria, fungi, parasites and virus strains analyzed. Usually, up to 100 loci can be enough to distinguish the substrain level, but the invention proposed herein is not limited to the number of loci or regions. The number of loci can be 20, 50, 100, 200 or more. The sequence data from these variable data can provide the virulence potential of relevant species, strains, substrains, human infections, drug resistance monitoring and tracking the differential information of the source of infection from humans, animals, food and environmental storage hosts, without the need for WGS.

[0014] Timely and accurate screening, detection, typing, and subtyping minimize the health and economic burden of food, animal, and environmental pathogens. Efficient and cost-effective high-resolution methods are still needed. Furthermore, such methods should involve simple workflows and be easily automated to circumvent the complex workflows and barriers of WGS. SUMMARY OF THE INVENTION

[0016] In some embodiments, the present disclosure describes a method for screening and analyzing at least one sample for food, animal, human, and environmental pathogens, the method comprising the following steps: for each sample, hybridizing multiple target-specific primers to nucleic acids from the sample in the presence of a barcoded universal primer to form a test reaction in a single reaction vessel, wherein at least one target-specific primer is configured to bind to at least one target nucleic acid sequence; subjecting each test reaction to amplification conditions to generate amplicons; combining the amplicons from each sample; bead cleaning at least a portion of the combined amplicons to form enriched amplicons; and sequencing the combined and enriched amplicons formed from each sample by next-generation sequencing.

[0017] In some embodiments, each barcoded universal primer comprises: a universal priming portion at the 3' end; a barcode portion in the middle; and a universal priming portion at the 5' end. In some embodiments, each target-specific primer comprises a specific sequence portion for a target nucleic acid sequence and a universal priming portion. In some embodiments, each sample is obtained from a subject, a food, one or more plants, or an environmental source. In some embodiments, the target-specific primers comprise primers configured to amplify at least 20 variable regions of at least bacteria, fungi, parasites, or viruses for identification, genotyping, subtyping, and detection of the coexistence of multiple isolates.

[0018] In some embodiments, the methods described herein further include mapping and counting multiple pathogen genomes for microbial and viral typing, subtyping, and monitoring, including the following additional steps: (1) determining the score of locus A of the first genome as the ratio between the number of unique reads mapped to locus A of the first genome and the total number of unique reads mapped to locus A of the first genome and at least one other genome, wherein if no read is mapped to locus A of the first genome, the score is preset to a minimum number; repeating the determination step of part (1) for at least one other locus; and (3) determining the overall score of the first genome by multiplying the scores of all test loci in steps (1) and (2). In some embodiments, the method further includes the following steps: (a) determining the genome with the highest overall score for any remaining reads; (b) terminating the evaluation if the number of non-empty loci of the highest-scoring genome is less than a preset cutoff; (c) outputting the genome with the highest score; (d) removing reads mapped to all genomes with the highest score at that time; and (e) repeating steps (a)-(d) until the evaluation ends according to step (b). In some embodiments, the preset cutoff value is at least 10.

[0019] In some embodiments, the target-specific primers include primers configured to amplify multiple variable regions of at least bacteria, fungi, parasites, or viruses for genotyping, subtyping, detection, and identification of multiple genotypes, serotypes, or subtypes of the same species or different species in the same sample. In some embodiments, the target-specific primers include primers configured to amplify and detect target sequences of at least one species, type, or subtype of bacteria, fungi, parasites, or viruses in the same sample.

[0020] In some embodiments, the sample comprises at least one microbial and / or viral species, strain, or substrain.

[0021] In some embodiments, the target-specific primers comprise primers configured to amplify and analyze target sequences relevant to forensic testing.

[0022] In some embodiments, the method further comprises the step of combining the enriched amplicons from each sample prior to sequencing. In some embodiments, the method further comprises the step of quantifying each type and species in each sample after sequencing the enriched amplicons.

[0023] In some embodiments, the test reaction contains a polymorphic gene with a unique sequence for an internal control.

[0024] In some embodiments, the present disclosure describes a method for screening at least one sample for food, animal, human, plant and environmental pathogens, the method comprising the following steps: for each sample, in the presence of a barcoded universal primer, hybridizing multiple target-specific primers to nucleic acids from the sample to form a test reaction in a single reaction vessel, wherein at least one target-specific primer is configured to bind to at least one target sequence selected from the group consisting of: bacteria, fungi, parasites or viruses, wherein each barcoded universal primer comprises: a universal priming portion at the 3' end; a barcode portion in the middle; and a universal priming portion at the 5' end; and wherein each target-specific primer comprises a specific sequence portion for a target nucleic acid sequence and a universal priming portion; subjecting each test reaction to amplification conditions to generate amplicons; combining the amplicons from each sample; bead cleaning at least a portion of the combined amplicons generated from each sample to form enriched amplicons; and sequencing the combined and enriched amplicons formed from each sample by next-generation sequencing.

[0025] In some embodiments, the present disclosure describes a method for screening at least one sample for forensic DNA analysis, cancer, or a genetic disease, the method comprising the following steps: for each sample, hybridizing multiple target-specific primers to nucleic acids from the sample in the presence of a barcoded universal primer to form a test reaction in a single reaction vessel, wherein at least one target-specific primer is configured to bind to at least one target sequence, wherein each barcoded universal primer comprises: a universal priming portion at the 3' end; a barcode portion in the middle; and a universal priming portion at the 5' end; and wherein each target-specific primer comprises a specific sequence portion for a target nucleic acid sequence and a universal priming portion; subjecting each test reaction to amplification conditions to generate amplicons; combining the amplicons from each sample; bead cleaning at least a portion of the combined amplicons generated from each sample to form enriched amplicons; and sequencing the combined and enriched amplicons formed from each sample by next-generation sequencing.

[0026] In some embodiments, the present disclosure describes kits comprising a plurality of target-specific primers configured to bind to target sequences specific to biological samples associated with cancer, genetic diseases, forensic testing, and microbial and viral species.

[0027] In some embodiments, the present disclosure describes methods and compositions for amplifying selective target regions in nucleic acid samples. In some embodiments, the methods include the following steps: (1) contacting the nucleic acid sample with a target-specific primer in a PCR reaction in the presence of a barcoded universal primer; and (2) allowing the primers to extend to generate target amplification products (amplicons) of varying sizes. In some embodiments, the methods include a step of determining the presence or absence of a target amplification product. In some embodiments, the methods include a step of establishing the sequence of the target amplification product. In some embodiments, less than 50%, 40%, 30%, 20%, 10%, 5%, 0.5%, or 0.1% of the amplification products are primer dimers or artifacts.

[0028] In some embodiments, the concentration of each target-specific primer can be about 500nM, 250nM, 100nM, 80nM, 70nM, 50nM, 30nM, 10nM, 2nM or 1nM. In some embodiments, the GC content of the target-specific primer can be different, and for example, it can be 40% to 70%, or 30% to 60% or 50% to 80%. In some embodiments, the melting temperature (Tm) of the target-specific primer can be 55°C to 65°C, or 40°C to 70°C, or 55°C to 68°C. In some embodiments, the length of the target-specific primer can be 20 to 90 bases, 40 to 70 bases, 20 to 40 bases or 25 to 50 bases. In some embodiments, the 5' region of the target-specific primer is a universal primer binding site, which is not complementary or specific to any nucleic acid region in the sample. In some embodiments, the target amplicon is 50 to 500 bases, 90 to 350 bases, or 200 to 450 bases in length.

[0029] In various embodiments of any aspect of the present disclosure, the method of primer extension is based on the polymerase chain reaction (PCR) of the prior art. In various embodiments, the annealing time can be greater than 0.5, 1, 2, 5, 8, 10 or 15 minutes. In various embodiments, the extension time can be greater than 0.5, 1, 2, 5, 8, 10 or 15 minutes.

[0030] In some embodiments, the methods disclosed herein quantify the number of copies of a target sequence present in a sample.

[0031] In various embodiments of the present disclosure, the compatibility and incompatibility scores of the selected primers are calculated based on the target amplicon GC content, the target amplicon melting temperature, the target amplicon heterozygosity rate, the complementation rate of the candidate primers to the target region; the size of the candidate primers, the size of the target amplicon, and the different factors of amplification efficiency and off-target rate. The selected target-specific primers can hybridize with the nucleic acid target and selectively amplify the target region. In various embodiments, the test sample is from a subject, individual, food, plant, animal, soil or environment suspected of having an infection or disease or having an increased risk of infection or disease; and one or more of the target nucleic acids contains a sequence at a target region associated with an infection or disease or an increased risk of infection or disease. In some embodiments, the test sample is from a subject, individual, animal, soil or environment that is not associated with any disease or infection. The characteristics of the target region can be used as an identity marker for a subject, individual, animal or other sample, similar to a fingerprint. In some embodiments, the information can be used for disease screening, detection, disease management, pathogen monitoring, food recalls, epidemics or epidemics.

[0032] In one embodiment, the method disclosed herein can be used for screening, detecting and identifying microorganisms and virus strains and types. In one embodiment, the method disclosed herein can be used for screening, detecting, genotyping, serotyping, subtyping and tracking infection sources (monitoring) using a wide range of MLST methods targeting variable / hypervariable regions. In some embodiments, candidate primers contact nucleic acid samples; wherein forward strand and reverse strand target-specific primers hybridize with target regions (if present in samples), wherein nucleic acid samples may have microorganisms and / or viral organisms or are suspected of having microorganisms and / or viral organisms, amplify multiple target nucleic acids; perform second generation sequencing on amplicons; and analyze sequence data by software analysis. In some embodiments, detected infections can be clinically acted upon. In some embodiments, detected infections can be associated with drug resistance. In some embodiments, detection, identification and quantification of microorganisms and viral species, strains and substrains can be associated with disease. In some embodiments, the infection source of a biological sample can be monitored or monitored.

[0033] In one aspect, the methods and compositions disclosed herein are designed for detecting, identifying, and quantifying target nucleic acids in samples that may contain microbial and viral organisms. In some embodiments, the disclosed methods include the following steps: (1) contacting a nucleic acid target in a sample with primers, wherein forward and reverse strand target-specific primers hybridize to different target regions in a test reaction; (2) amplifying the target nucleic acid under optimal amplification conditions; (3) sequencing the amplified products by NGS; and (4) analyzing and quantitatively measuring the generated sequence reads by mapping and counting methods.

[0034] In one embodiment, the methods disclosed herein can be used to screen and analyze genomic target regions for diseases (e.g., cancer or hereditary diseases). In one embodiment, the methods disclosed herein can be used to analyze genomes to perform forensic DNA analysis based on DNA features (e.g., short tandem repeat (STR) regions). In some embodiments, the methods disclosed herein can be used for pharmacogenetics or drug resistance to detect genetic variations that affect individual responses to drugs. In some embodiments, candidate primers contact a nucleic acid sample; wherein forward and reverse strand target-specific primers hybridize to the target region, amplifying multiple target nucleic acids; performing second-generation sequencing on the amplicons; and analyzing the sequence data by software analysis. In some embodiments, the detected nucleic acid variations can be clinically acted upon. In some embodiments, biological samples can be monitored for prognosis.

[0035] In some embodiments, the nucleic acid sample comprises genomic nucleic acid. In some embodiments, the sample comprises nucleic acid molecules obtained from food, vegetables, agricultural products, plants, soil, spoilage, water, environment or food production facilities. In some embodiments, the sample comprises nucleic acid molecules obtained from urine, tissue, saliva, biopsy, sputum, swab, surgical resection, cervical swab, tumor tissue, fine needle aspirate (FNA), scrapings, swab, mucus, semen, other non-limiting clinical or laboratory samples.

[0036] In another aspect, the disclosure relates to a kit comprising target-specific primers for amplifying a target region of interest in a sample.

[0037] In some embodiments, the disclosed method comprises the following steps: performing a one-step multiplex barcoded amplification and sequencing the resulting amplicons by NGS. In certain embodiments, the sample is obtained from a subject suffering from a single or multiple co-infections. In some embodiments, the analytical sensitivity of the method is 10 copies of each microbial and viral species in the sample; highly multiplex PCR amplifies 20, 50, 100, 200, 500, 1,000 or more targets with minimal primer-primer interactions. In some embodiments, the method includes the step of performing a single reaction, single-step barcoded multiplex PCR. In some embodiments, the method can analyze 5, 10, 20, 50, 100, 200, 500, 1,000, 2,000, 5,000, 10,000 or more samples by a single NGS sequencing run.

[0038] In some embodiments, the disclosed methods include using an internal control for each sample in a reaction assay, wherein the internal control is typically a housekeeping gene, a mitochondrial sequence, or a target sequence and monitors amplification and serves as a normalization, qualitative, or quantitative control.

[0039] In some embodiments, the disclosure relates to methods, compositions and kits for applying multiple target amplification and target enrichment before downstream analysis (e.g., next-generation sequencing). The method relies on the use of multiple target-specific primers and target enrichment amplification in DNA samples suspected of having diseases (cancer or genetic diseases), drug resistance, forensic information, or microbial and viral pathogens. Target-specific primers amplify target nucleic acids under optimal conditions in the presence of amplification reagents (e.g., polymerases and dNTPs) to amplify at least one or more nucleic acid targets of interest.

[0040] In some embodiments, the present disclosure relates to compositions comprising a plurality of target-specific primers that contact a target sequence in a sample and have sequences complementary to a target DNA associated with disease (cancer / genetic disease), drug resistance, forensics, or microbial and viral organisms (non-limiting).

[0041] In some embodiments of the disclosed methods, the primer design method selects candidate target-specific primers based on the following steps: (1) extracting the genomic sequence of a microbial or viral organism; (2) designing a set of primers with appropriate GC content, Tg, and GC content for variable, hypervariable, housekeeping, or other target sequences; m and target-specific forward and reverse strand target-specific primers with different distances from each targeted region; (3) for each primer, search for off-target matches of the target genomic sequence; filter the primers and retain those that pass the off-target threshold; (4) search the 3' end portion of each primer for complementary matches with the set of primer sequences; filter the primers step by step, wherein the primers with the most complementary matches at their 3' ends are first removed; and (5) synthesize the primers and run the entire wet lab experiment using next-generation sequencing; calibrate the performance of each primer and filter out primers with undesirable performance. In some embodiments, the primer selection procedure steps 2 to 4 and steps 2 to 5 are repeated until each target sequence is covered by at least one forward strand target-specific primer and one reverse strand target-specific primer in the primer set.

[0042] In various embodiments of any aspect of the present disclosure, the methods and compositions are characterized by multiplex amplification and target enrichment of target nucleic acid regions. In some embodiments, the disclosed methods include the following steps: (1) contacting a target-specific primer with a target nucleic acid sequence in the presence of a barcoded universal primer and hybridizing with the target nucleic acid sequence in a sample; (2) amplifying the test reaction under optimal amplification conditions; (3) combining the amplification products from each individual sample; (4) performing a bead cleanup on a portion of the combined amplification products to remove unconsumed primers and primer dimers and create an enriched amplification product; (5) performing standard normalization and quantification on a portion of the enriched amplification product; and (6) sequencing the amplicons by next-generation sequencing.

[0043] In one embodiment, the barcoded universal primer comprises: a) a universal priming portion at the 3' end; b) a barcode portion in the middle; and c) a universal priming portion at the 5' end ( Figure 1 ). In one embodiment, each target-specific primer comprises a specific sequence portion for the target nucleic acid sequence and a universal priming portion.

[0044] In some embodiments, the composition comprises a plurality of target-specific primers, wherein at least one target-specific primer has at least 90% identity to any of the nucleic acid targets. In some embodiments, the composition comprises a plurality of target-specific primers, wherein the primers have at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the nucleic acid targets in the sample.

[0045] In some embodiments, the disclosure relates to compositions comprising a plurality of target-specific primers wherein the sequence complementary to a target nucleic acid of interest is about 15 to 40 bases in length.

[0046] In some embodiments, the disclosure relates to a composition of pre-calculated designed target primers that produce minimal cross-hybridization or primer-primer interactions with other target-specific primers in the composition. In some embodiments, the primers in the composition are designed to avoid non-specific priming that can lead to non-specific amplification. In some embodiments, amplification conditions (e.g., annealing temperature, annealing duration, and primer concentration) can be adjusted to minimize amplification artifacts (e.g., primer dimers).

[0047] In some embodiments, the disclosure relates to methods or compositions comprising a plurality of target-specific primers having minimal cross-hybridization with non-specific sequences present in a sample. In some embodiments, such cross-hybridization with non-specific targets can be monitored and evaluated by downstream analysis (e.g., next generation sequencing).

[0048] In some embodiments, the disclosure relates to methods or compositions comprising a plurality of target primers, the primers having a minimum self-complementary structure. In some embodiments, the composition comprises at least one target-specific primer that does not form a secondary structure (e.g., a hairpin or a loop). In some embodiments, the composition comprises a plurality of target-specific primers, wherein most or possibly all target-specific primers do not form a secondary structure, such as a hairpin and a loop.

[0049] In some embodiments, the target nucleic acid is obtained from a biological sample of food, fresh produce, water, soil, corrupt, environment or subject. In some embodiments, the sample comprises protein, cell, fluid, biological fluid, preservative and / or other substances. In certain embodiments, the sample is derived from urine, tissue, saliva, biopsy, sputum, swab, surgical resection, cervical swab, tumor tissue, fine needle aspiration (FNA), scrapings, swab, mucus, semen, other non-limiting clinical or laboratory samples.

[0050] In some embodiments, the target amplification product is sequenced by second-generation sequencing based on the current state-of-the-art second-generation sequencing technology or platform (e.g., Illumina platform (reversible dye terminator sequencing), 454 pyrophosphate sequencing, ion semiconductor sequencing (Ion Torrent), PacBioSMRT sequencing, Qiagen GeneReader sequencing technology, Oxford nanopore sequencing, and Element Biosciences sequencing technology). In some embodiments, the disclosed methods are not limited to these second-generation sequencing technology examples and can be applied to new sequencing innovations.

[0051] In certain embodiments, the foregoing methods may be performed at multiple time points. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] The present disclosure can be better understood with reference to the accompanying drawings. The elements in the accompanying drawings are not necessarily drawn to scale relative to each other, but rather emphasize the principle of clearly illustrating the present disclosure. In addition, in multiple views, the same reference numerals represent corresponding parts.

[0054] Figure 1 Schematic representation of target-specific primers and barcoded universal primers is depicted.

[0055] Figure 2 Depicted is a graphic representation comparing the expected resolution of PCR (one locus), conventional MLST (5-7 loci), multiplex barcoded amplification (50 to >100 loci), and WGS.

[0056] Figure 3 Depicted is a schematic representation of a method for multiplex barcoding PCR, a general method for a wide range of biological applications.

[0057] Figure 4 The workflow of multiplex barcoding PCR is shown.

[0058] Figure 5 Depicted is a graphic representation of high-resolution multiplex barcoded amplification of 47 variable and housekeeping targets of Salmonella enterica.

[0059] Figure 6 Graphic depiction of nine different Salmonella serovars amplified by multiplex barcoded amplification in the same PCR tube / reaction and differentiated by NGS.

[0060] Figure 7 Pairwise matrix distances between the tested Salmonella samples are listed.

[0061] Figure 8 Salmonella serovars called according to the best matching genome assembly are shown.

[0062] Fig. 9 Neighbor-joining trees of the tested Salmonella samples are shown.

[0063] Fig.10 Results are shown for 3, 6 and 9 mixtures of different Salmonella serotypes from the same sample.

[0064] Fig.11 Shown are the genotyping results of Cyclospora samples by analyzing 52 loci.

[0065] Fig.12 Shown are the genotyping results of Legionella samples by analyzing 52 loci.

[0066] Fig.13 Shown are the results of genotyping of monkeypox samples by analyzing 21 loci.

[0067] Fig.14 Shown are the genotyping results of Listeria samples by analyzing 69 loci.

[0068] Fig.15 The results of STEC serotyping by analyzing 61 loci are shown.

[0069] Fig.16 Sequence read counts for STEC serotype-specific wzx genes are shown.

[0070] Fig.17 Sequence read counts for other STEC gene markers are shown.

[0071] Fig.18 Shown are the results of genotyping of hepatitis A virus samples by analyzing 10 regions in the HAV genome.

[0072] Fig.19 Microbial identification results of two environmental samples are shown.

[0073] Fig. 20 Genotyping results for HBV, HCV, and HIV samples are shown.

[0074] Fig.21 Shown are mutations detected for breast cancer samples as well as a reference control BRCA somatic multiplex I (gDNA) control, with allele frequencies ranging from 7.5% to 60%.

[0075] Fig. 22 STR insertions and / or deletions in forensic testing by multiplex barcoded amplification and NGS are shown. The (-) sign indicates a deletion, and the (+) sign indicates an insertion. The number after the (+) or (-) indicates the copy number of the repeat unit detected in the assay. Two patterns are shown for each sample. All of these insertions and / or deletions are relative to the hgl9 genome reference.

[0076] Detailed Description

[0077] The present invention relates to a method for multiplex barcoded amplification, wherein amplification and barcoding occur simultaneously in the same PCR reaction (final product library), and then the barcoded amplicons are further analyzed by a system (e.g., next-generation sequencing). The present invention has a general method and can be applied to biological applications, such as: (1) screening, detection and identification of bacteria / viruses / parasites / fungi and microbiome organisms; (2) detection, genotyping / subtyping and monitoring of bacteria / viruses / parasites / fungi by analyzing a large number of variable / hypervariable sequences; (3) cancer / genetic disease nucleic acid sequence analysis; (4) pharmacogenetics and companion diagnostics for selecting the right treatment and prognosis; (5) drug resistance for applications such as antimicrobial therapy selection, monitoring and epidemiology and personalized medicine; (6) forensic DNA analysis for DNA signature analysis; or (7) any biological application using multiplex barcoded amplification.

[0078] The present invention can also measure DNA sequence variation in selective regions and characterize species by their unique allelic signatures. The present invention provides methods, compositions, kits, systems and instruments that allow for such target enrichment.

[0079] For microorganism and virus genotyping / subtyping analysis and monitoring, nucleic acid differences of multiple variable loci are analyzed to determine genotype / serotype / strain / substrain levels, including tracking the source of infection (monitoring). The present invention measures DNA sequence variations in selective loci and characterizes species by their unique allelic characteristics. The present invention provides methods, compositions, kits, systems and instruments that allow such target enrichment. The following examples, applications, descriptions and contents are exemplary and explanatory and are not intended to limit or restrict in any way.

[0080] Conventional MLST is a combination of PCR, sequencing, and data analysis for typing multiple loci, using variable regions of DNA sequences of the organism's genome to characterize isolates of microbial, viral, fungal, or parasitic species / subspecies. MLST has a wide range of applications and provides resources for the scientific, public health, and veterinary communities, as well as the food industry. The advent of next-generation sequencing technology has made it possible to obtain sequence information of entire microbial and viral genomes at relatively modest cost and effort. However, due to the sequence conservation of housekeeping genes, conventional MLST targeting housekeeping genes sometimes lacks the ability to distinguish microbial and viral species at the strain and substrain levels, which limits its application in epidemiological studies. Serological typing methods have been established earlier for distinguishing bacterial isolates, but immunological typing has disadvantages, such as dependence on a few antigenic loci and unpredictable reactivity of antibodies to different antigenic variants. Several molecular typing schemes have been proposed to determine the relatedness of pathogens, such as pulsed-field gel electrophoresis (PFGE), ribosome typing, and PCR-based fingerprinting. However, these DNA band-based subtyping methods do not provide meaningful evolutionary analysis. Although PFGE is considered the "gold standard" by many researchers, many strains cannot be typed by this technique due to DNA degradation during processing (gel smear).

[0081] Multiplexed barcoding amplification of large numbers of variable / hypervariable sequences of microorganisms in a single-tube, single-step reaction, combined with NGS-based MLST, significantly improves the resolution of microbial and viral species at the strain and substrain levels and will allow tracing of infection sources (surveillance).

[0082] The target enrichment method selectively captures genomic regions from DNA samples by several steps before sequencing reactions. The shortcoming of the existing target enrichment method is that there are multiple steps in its workflow, which increases time, cost and manpower. In the existing NGS workflow, amplification is carried out in one or two rounds, and the barcoding / indexing of each sample is carried out in a separate step, which makes automation more challenging, and may also increase the risk of cross contamination. In conventional MLST, about 5-7 housekeeping genes are generally used to balance between the acceptable identification ability, time and cost of strain typing. Although conventional MLST provides relatively acceptable differentiation ability, strain error typing is not uncommon, and substrain / subtype information cannot be obtained due to its low-resolution differentiation ability, which is essential for tracking and tracing the source of infection (microorganism and virus monitoring).

[0083] The present invention includes a one-step single-tube barcoded multiplex amplification step that is capable of amplifying a wide range of informative variable target sequences. In the PCR reaction, amplification and barcoding / indexing of nucleic acid targets occur simultaneously, followed by NGS and data analysis. For microbial and viral typing / subtyping analysis and monitoring, the analysis consists of: (1) data collection; (2) data analysis; and (3) multi-locus sequence analysis. In the data analysis step, all unique sequences are assigned allele numbers and combined into allele signatures and assigned sequence types, and the correlation of strains / substrains is determined by comparing allele signatures. Based on the organism, a large amount of data is generated during the sequencing process, and the generated sequence data is organized, managed, analyzed and merged by bioinformatics tools.

[0084] In data analysis, the generated sequences are designated as alleles. The alleles at the locus provide allelic characteristics. A series of characteristics can then be used as identification markers at the strain / substrain level. Even sequences that differ by a single nucleotide are designated as different alleles, and the number of nucleotide differences between alleles is not considered, because it is impossible to distinguish whether the differences in multiple nucleotide sites are the result of multiple point mutations or the result of a single recombination exchange.

[0085] In one aspect, the methods and compositions disclosed herein are designed for analyzing target nucleic acids in a sample that is analyzed for disease (cancer / genetic disease), drug resistance, genetic mapping, forensics, or microbial and viral organisms (bacteria, fungi, parasites, or viruses). In some embodiments, the disclosed methods include the following steps: (1) contacting a set of variable nucleic acid targets in a sample with primers, wherein forward and reverse strand target-specific primers hybridize to different target regions in a test reaction; (2) amplifying the target nucleic acid under optimal amplification conditions to determine the presence or absence of the target nucleic acid; (3) sequencing the amplified products by NGS; and (4) analyzing the generated sequence reads.

[0086] In some embodiments, the present invention uses mapping and counting methods for microbial and viral typing, subtyping and monitoring. In some embodiments, the steps of the mapping and counting method are as follows: (1) The score of locus A of candidate genome X is defined as the ratio between the number of unique reads mapped to locus A of genome X and the total number of unique reads mapped to loci A of all analyzed genomes. If no reads are mapped to locus A of genome X, the score is preset to a minimum number, such as le -7; (2) The score of genome X is defined as the product of the scores of all test loci; (3) Step (a), find the genome with the highest score for the remaining reads (at the beginning of the program, the remaining reads mean all reads); if the number of non-empty loci of the genome with the highest score is less than a preset cutoff value, stop. In some embodiments, the preset cutoff value is at least 10. Then, step (b), output the genome with the highest score. Step (c), remove the reads mapped to all the genomes with the highest score so far; and then repeat steps (a) to (c). In some embodiments, the mapping and counting method can be performed using software.

[0087] Developing highly multiplexed amplification methods for nucleic acid samples with accuracy and high copy number sensitivity remains a challenge in the art. The present disclosure relates to NGS-based assays that combine balanced target-specific multiplexed amplification and sensitive copy number quantification, as well as balanced sequencing reads for each target, while barcoding of each target amplicon occurs simultaneously during the amplification step.

[0088] In another embodiment, for microbial and viral typing and subtyping, the present disclosure has the ability to detect and differentiate more than one serotype / genotype / strain / substrain in the same sample ( Figure 6 and 10 ). The graph depicts the detection of mixed strains of 3, 6, and 9 Salmonella serotypes in one sample.

[0089] All scientific terms used herein have the same meanings as commonly used and understood by one of ordinary skill in the art. The examples, materials, methods, figures and tables are illustrative only and are not intended to be limiting.

[0090] As used herein, "amplification conditions" means conditions suitable for amplification using polymerase chain reaction. The polymerase chain reaction can be multiplex PCR. Amplification conditions include, but are not limited to, the examples provided in Examples 1-6 disclosed herein.

[0091] As used herein, "barcoded universal primer" means a universal primer comprising a barcode sequence and at least one universal sequence. For example, see Figure 1 .

[0092] As used herein, "bead cleanup" means the use of bead-based purification, wherein the beads are configured to bind to one or more targets. As known to those skilled in the art, bead cleanup can use positive selection (i.e., the beads are configured to capture the target of interest) or negative selection (i.e., the beads are configured not to capture the target of interest). As known in the art, various beads can be used, such as streptavidin beads or magnetic beads.

[0093] As used herein, "compatibility score" means a score of a potential forward strand targeting primer or reverse strand targeting primer calculated based on different factors including target amplicon GC content, target amplicon melting temperature, target amplicon hybridization rate, complementarity rate of candidate primers to target region; candidate primer size, target amplicon size, primer-primer interaction, and amplification efficiency and off-target rate.

[0094] As used herein, "minimum number" means equal to or less than 1e -4 and preferably about 1e -7 The number of.

[0095] As used herein, "dsDNA" means double-stranded DNA.

[0096] As used herein, "environmental source" means any potential location in the natural and / or artificial environment where a sample can be collected. Environmental sources include, but are not limited to: water sources, such as oceans, lakes, ponds, rivers, and streams; soil sources, such as soil, sand, internal or external dust; gas sources, such as air.

[0097] As used herein, "FNA" means fine needle aspiration.

[0098] As used herein, "forward strand" means one strand of a dsDNA sample.

[0099] As used herein, "forward strand target-specific primer" means a primer configured to bind to a target sequence on the forward strand.

[0100] As used herein, "GC content" means guanine-cytosine content.

[0101] As used herein, "locus" means the specific physical location or position where a particular gene or genetic marker is located on the genome.

[0102] As used herein, "microbial and viral surveillance" means the systematic monitoring and tracking of bacterial and viral pathogens in a population or a specific geographic area. It involves the collection, analysis, and reporting of data related to the occurrence, distribution, and characterization of bacterial and viral infections.

[0103] As used herein, "multi-locus sequence typing (MLST)" means a technique for classifying microorganisms based on their gene sequences at several different conserved genes. By analyzing the DNA sequences of several conserved genes, the genetic relatedness of different strains can assign microorganisms to specific types. This information can be used to study the spread of microbial infections, track the evolution of microbial populations over time, and identify genetic factors that may lead to virulence or antibiotic resistance.

[0104] As used herein, "multiplex barcoded amplification" means multiplex target amplification, where amplification and indexing / barcoding of each target occurs simultaneously in a PCR reaction.

[0105] As used herein, "NGS" means next generation sequencing.

[0106] As used herein, "PCR" means polymerase chain reaction.

[0107] As used herein, "reverse strand" means the second strand in a dsDNA sample that is complementary to the forward strand.

[0108] As used herein, "reverse strand target-specific primer" means a primer configured to bind to a target sequence on the reverse strand.

[0109] As used herein, "sample" means a specimen or product in the field to which the present invention belongs. Samples can be obtained from various sources, such as subjects, foods, plants, and environmental sources. In the case of using the term in this specification, for example, with respect to a subject, "sample" means a "biological sample" or its equivalent. "Biological sample" means any product obtained from a biological material (e.g., an individual, a liquid, a body fluid, a cell line, a cultured tissue, or a tissue segment) as a source. Examples of "biological samples" include body fluids (e.g., blood, saliva, dental plaque, serum, plasma, urine, synovial fluid, and cerebrospinal fluid) and tissue sources.

[0110] As used herein, "species" means a group of microorganisms that share similar genetic and phenotypic characteristics.

[0111] As used herein, "species-specific primer" means a primer that is configured to bind to a target that is specific to a particular species.

[0112] As used herein, "strain" means a subgroup of a bacterial species having unique genetic and phenotypic characteristics that distinguish the subgroup of the bacterial species from other members of the same species. Strains may differ in their virulence, antibiotic resistance, or other traits that affect the behavior of the strain in the environment or the interaction of the strain with other organisms.

[0113] As used herein, "substrain" means a further subdivision of a bacterial strain that has additional genetic or phenotypic differences from other members of the strain. Substrains are typically identified by additional genetic markers or phenotypic traits.

[0114] As used herein, "subject" means an animal, preferably a mammal, most preferably a human.

[0115] As used herein, "subtype" means to further classify microorganisms and viruses within a particular type. In microbiology, bacterial subtypes can be identified based on specific genetic or phenotypic characteristics (e.g., antibiotic resistance or virulence factors). In virology, viral subtypes can be identified based on specific genetic mutations or changes in antigenic properties.

[0116] As used herein, "target-specific primer" means a primer configured to bind to a specific target. In some embodiments, the target-specific primer can be a type-specific primer. In some embodiments, the target-specific primer can be a species-specific primer. In some embodiments, the target-specific primer can be a specific sequence / region on a microorganism or viral genome.

[0117] As used herein, "type" in microbiology means a strain or specific type of microorganism. In virology, it means the classification of viruses based on their genetic and antigenic characteristics.

[0118] As used herein, "type-specific primers" means primers that are configured to bind to a target that is specific to a particular microorganism or viral genome.

[0119] As used herein, "universal sequence" means a sequence that is configured to be targeted by a universal sequence primer.

[0120] The present disclosure describes methods, compositions and kits for amplifying and enriching specific and known sequence nucleic acid targets to determine nucleotide sequence. The following examples, applications, descriptions and contents are exemplary and explanatory and are not limiting or restrictive in any way.

[0121] The present disclosure relates to selective amplification of a set of target sequences by multiplex barcoded amplification and further analysis by next generation sequencing. The present disclosure has general methods for a wide range of biological applications.

[0122] The disclosed methods offer a number of uniquely combined advantages, including, but not limited to: (1) single reaction PCR; (2) one round of highly multiplexed PCR covering a wide range of targets; (3) dual index barcoding (barcodes at both ends of each amplicon) to minimize cross-contamination; (4) target-specific amplification, allowing for uniform amplification; (5) quantification of target nucleic acids; (6) internal controls for each sample in the test reaction to monitor amplification, which can serve as a normalization factor for quantification; (7) amenability to high-throughput scaling; (8) the method is amenable to biological applications with multiplexed barcoded amplification; and (9) simple workflow and amenability to automation. See, for example, Figure 3-4 .

[0123] While WGS analyzes the entire genome, targeted target-specific panels continue to offer the following advantages: better coverage of targeted regions, more convenient detection of multiple variant types, significantly lower cost, higher throughput, simpler bioinformatics analysis, and targeted testing that avoids dealing with minor / incidental findings that inevitably occur in whole genome sequencing. In addition, targeted sequencing of specific regions of interest in a large number of samples is much more cost-effective in answering biological questions than sequencing the entire genome of a smaller number of organisms. Efficient and specific target enrichment methods enable more efficient targeted sequencing. The important parameters for target enrichment are: (i) sensitivity, (ii) specificity, (iii) uniformity, (iv) reproducibility, (v) cost, (vi) ease of use, and (vii) the amount of DNA required for each experiment (Nat Methods. 2010 Feb; 7(2): 111-8). Multiplexed barcoded amplification (in which amplification and barcoding are performed simultaneously in a PCR reaction) can be applied to a wide range of biological applications ( Figure 3 ).

[0124] Methods of multiplex amplification and target enrichment are described herein, which are analyzed by NGS. In various embodiments of any aspect of the present disclosure, methods and compositions are characterized by multiplex amplification and target enrichment of target nucleic acid regions of genomic materials (e.g., cancer, genetic diseases, drug resistance, forensics, and microbial and viral organisms).

[0125] In some embodiments, the present invention is applicable to detection, identification, genotyping and typing at the strain and substrain levels. In some embodiments, the disclosed method includes the following steps: (1) contacting a set of variable / hypervariable target nucleic acid sequences in the genome of a microorganism or virus with a target-specific primer in the presence of a barcoded universal primer and hybridizing with the target nucleic acid sequence in the sample; (2) amplifying the test reaction under optimal amplification conditions; (3) combining the amplification products from each individual or subject sample; (4) performing a bead cleanup on a portion of the combined amplification products to remove primers and primer dimers that may not have been consumed to create an enriched amplification product; (5) performing standard normalization and quantification on a portion of the enriched amplification product; and (6) sequencing the amplicons by next-generation sequencing. For example, see Figure 3-4 .

[0126] In some embodiments, the present invention is suitable for nucleic acid sequence analysis of target regions for cancer, genetic diseases, forensics, pharmacogenetics, and drug resistance. In some embodiments, the disclosed method includes the following steps: (1) contacting a target-specific primer with a target nucleic acid sequence in a sample in the presence of a barcoded universal primer and hybridizing with the target nucleic acid sequence in the sample; (2) amplifying the test reaction under optimal amplification conditions; (3) combining the amplification products from each individual or subject sample; (4) bead cleaning a portion of the combined amplification products to remove primers and primer dimers that may not have been consumed to create an enriched amplification product; (5) standardizing and quantifying a portion of the enriched amplification product; and (6) sequencing the amplicons by next-generation sequencing. For example, see Figure 3-4 .

[0127] In one embodiment, the barcoded universal primer comprises: (a) a universal priming portion at the 3' end; (b) a barcode portion in the middle; and (c) a universal priming portion at the 5' end ( Figure 1 ). In one embodiment, each target-specific primer comprises a specific sequence portion for the target nucleic acid sequence and a universal priming portion.

[0128] In some embodiments, the present disclosure provides methods, compositions and kits for detecting more than one genotype / serotype / strain / substrain / type / subtype in a single reaction vessel (e.g., a single tube) (e.g., see Figure 6 ). The number of genotypes / serotypes / strains / substrains / types / subtypes in the same sample can be 2, 3, 4, 8, 12, 16, 20, 30 or more.

[0129] In some embodiments, for each subject, the disclosed methods utilize one round of multiplex PCR in a single test reaction, which minimizes DNA cross-contamination and extra steps in the workflow. Typically, methods using more than one round of PCR are prone to DNA cross-contamination and can lead to inaccurate results.

[0130] In some embodiments, the disclosed methods are highly multiplexed microbial and viral identification panels that cover an unprecedented and unparalleled broad range of variable / hypervariable regions (including housekeeping genes) within microbial and viral genomes in a single reaction and one round of PCR.

[0131] In some embodiments, the disclosed methods include using housekeeping genes or control genes in microbial and viral genomes as internal controls for each subject in each test reaction, which can monitor amplification and serve as a normalization factor for quantifying the copy number of microbial and viral genomes for each sample in each test reaction. In some embodiments, the methods can quantify multiple strains / substrains in multiple co-infected samples in one test reaction.

[0132] In some embodiments, the disclosed methods include the use of dual barcode indexing, where amplicons are barcoded at both ends by universal barcoding primers, thereby minimizing cross-contamination and providing double confirmation of barcodes in case of amplification errors in the early stages of PCR.

[0133] In some embodiments, disclosed methods include using second generation sequencing for screening, detection, identification and quantitative microorganism or virus genome. In certain embodiments, the target nucleic acid sequence is amplified and sequenced to reveal the strain or substrain present in the sample, which can be used to track the source of infection (microorganism and virus monitoring). In some embodiments, disclosed methods include using second generation sequencing for analyzing the target nucleic acid region of the genomic material of samples related to cancer / hereditary diseases, drug resistance, forensics and other biological applications.

[0134] In some embodiments, the amplification conditions (e.g., number of cycles, annealing temperature, annealing duration, extension temperature, and extension duration) are adjusted to the optimal conditions for amplification. In some embodiments, based on the commercial DNA polymerase instructions, the number of cycles, amplification conditions (e.g., annealing temperature, annealing duration, extension temperature, and extension duration) are adjusted to the optimal conditions for amplification.

[0135] In some embodiments, nucleic acid sample comprises genomic DNA or RNA.In another embodiment, sample comprises nucleic acid molecules obtained from fresh agricultural products, food, imported food, food production facilities, farms, fresh agricultural products, animal farms, water, corrupt, soil and environment.In another embodiment, sample comprises nucleic acid molecules obtained from swabs or brushes.In some embodiments, sample comprises nucleic acid molecules obtained from saliva.In some embodiments, sample comprises nucleic acid molecules obtained from urine, tissue, saliva, biopsy, sputum, swab, formalin-fixed paraffin-embedded material (FFPE), surgical resection, cervical swab, tumor tissue, fine needle aspiration (FNA), scrapings, swab, mucus, urine, semen and other non-restrictive clinical or laboratory samples obtained nucleic acid molecules.

[0136] In some embodiments, the nucleic acid sample obtained can be from an animal, such as a human or mammalian subject. In another embodiment, the nucleic acid sample obtained can be from a non-mammalian subject, such as bacteria, parasites, viruses, fungi and plants.

[0137] In some embodiments, the present disclosure relates to target amplification of at least one target sequence from a biological sample of a normal or diseased subject. In some embodiments, the present disclosure relates to specific and selective target amplification of at least one target sequence and detection and identification of microbial and viral species / strains / substrains in nucleic acid samples.

[0138] In some embodiments, the target-specific primers include a plurality of primers designed to selectively amplify variable and hypervariable regions of microbial and viral target nucleic acid sequences; the amplification range varies depending on the size of the fragment and the position of the primer on the nucleic acid fragment, and the range of sizes can vary. In some embodiments, the target-specific primers include a plurality of primers designed to selectively amplify target nucleic acid sequences of genomic material of samples associated with cancer, genetic diseases, drug resistance, forensics, and other biological applications.

[0139] The amplification range varies due to the size of the fragment and the position of the primer on the nucleic acid fragment, and the range of sizes can vary. In some embodiments, the target-specific primers include a plurality of primers that are selectively designed to amplify a target nucleic acid sequence, wherein the lengths of the amplified target nucleic acid sequences differ from each other by no more than 90%, no more than 70%, no more than 50%, no more than 25%, or no more than 10%.

[0140] In some embodiments, the disclosed method involves target enrichment by multiplex target-specific PCR, which comprises the following steps: contacting the nucleic acid target with multiple target-specific primers in the presence of barcoded universal primers and PCR reagents (e.g., DNA polymerase, dNTPs, and reaction buffer); hybridizing and extending the primers to the complementary target nucleic acid sequence under optimal temperature and time conditions for given denaturation, annealing, and extension. In some embodiments, the amplification steps can be performed in any order. In some embodiments, when optimizing, amplification steps, purification steps, and clean-up steps can be added or removed to achieve optimal multiplex target amplification for downstream processing.

[0141] In some embodiments, described method uses PCR and as a kind of archaeal dna polymerase of component in reaction.In some embodiments, there is extensive archaeal dna polymerase to choose, and their feature is the different characteristics such as thermostability, fidelity, continuous synthesis ability and hot start.Depend on application, method can use archaeal dna polymerase with one or more of these characteristics.In some embodiments, the archaeal dna polymerase concentration of multiplex PCR can be higher than single PCR.

[0142] In some embodiments, the methods disclosed herein use multiple polymerase chain reactions to amplify target nucleic acid sequences, wherein more than one target sequence is amplified in a test reaction. In some embodiments, the amount of nucleic acid sample required for multiple amplification can be about 0.1 ng. In some embodiments, the amount of nucleic acid material can be about 1 ng, 5 ng, 10 ng, 50 ng, 100 ng or 200 ng.

[0143] In some embodiments, the method disclosed herein uses multiple polymerase chain reaction to amplify the target nucleic acid sequence, wherein more than one target sequence is amplified in the test reaction. The polymerase chain reaction of the prior art is carried out on a thermal cycler, and each cycle of PCR includes denaturation, annealing and extension. Each cycle of PCR includes at least a denaturation step, an annealing step and an extension step for extending nucleic acid. In some embodiments, annealing and extension can be merged. In some embodiments, the method disclosed herein includes 25 to 35 PCR cycles. Each cycle or cycle group can have different durations and temperatures, such as an annealing step can have a gradual increase and decrease of temperature and duration, or an extension step can have a gradual increase and decrease of temperature and duration. In some embodiments, the duration can have a reduction or increase with an increment of 5 seconds, 10 seconds, 30 seconds, 1 minute, 2 minutes, 4 minutes, 8 minutes or more. In some embodiments, the temperature can have a reduction or increase with an increment of 0.5, 1, 2, 4, 8 or 10 degrees Celsius.

[0144] In some embodiments of the present disclosure, the target-specific primer comprises a nucleotide modification at the 3' end or the 5' end or across the sequence. In some embodiments, the target-specific portion of the primer may be 15 to 40 bases in length. In some embodiments, the T of each target-specific primer may be 15 to 40 bases in length. m It may be from about 55°C to about 72°C.

[0145] In some embodiments, the present disclosure is characterized in that target enrichment and multiple amplification methods for target-specific nucleic acid amplification of microorganisms and viral species / strains / substrains are performed using target-specific primers. In some embodiments, the present disclosure is characterized in that target enrichment and multiple amplification methods for target-specific nucleic acid amplification of genomic materials related to cancer / hereditary diseases, drug resistance, forensics and other biological applications. In some embodiments, the selected target-specific primer contacts and hybridizes with a target nucleic acid sequence that may be associated with a disease. In one embodiment, the target-specific primer hybridizes with a nucleic acid sequence with different sizes in a test reaction. In some embodiments, the amplicon size selection can be used to sequence the amplified product of a specific length range. In some embodiments, the amplicon with a length range of 100 to 250 base pairs can be sequenced. In some embodiments, the amplicon of 150 to 300 base pairs, or the amplicon of 120 to 350 base pairs, or the amplicon of 200 to 500 base pairs or a larger length range can be sequenced.

[0146] In some embodiments, any procedure can be removed or repeated. In some embodiments, a purification step can be added to generate optimal results. These procedures are non-limiting, and those skilled in the art can easily add, remove or repeat these steps to obtain optimal results.

[0147] The ability to increase the number of target-specific primers in multiplex PCR allows for the simultaneous amplification of a large number of nucleic acid targets while reducing the amount of input DNA, labor, and time. This is particularly advantageous when the amount of starting input nucleic acid material is limited.

[0148] In some embodiments of the disclosed methods, the primer design method selects candidate target-specific primers based on the following step-by-step procedure: (1) extracting the genomic sequence surrounding each targeted variant position; (2) for each variant in the target sequence, designing a primer with appropriate GC content, T m and target-specific forward and reverse strand target-specific primers at different distances from each targeted variant; (3) for each primer, search for off-target matches of the target genomic sequence; filter primers and retain those that pass the off-target threshold; (4) search for complementary matches of the 3' end portion of each primer with the set of primer sequences; filter primers step by step, wherein the primers with the most complementary matches at the 3' end are first removed; (5) synthesize primers and run the entire wet lab experiment (including second-generation sequencing); calibrate the performance of each primer and filter out primers with undesirable performance. In some embodiments, the primer selection procedure steps 2 to 4 and steps 2 to 5 are repeated until each target variant is covered by at least one forward strand target-specific primer and one reverse strand target-specific primer in the primer set.

[0149] In some embodiments, the disclosure features a primer design method that eliminates low compatibility primers that form artifacts (e.g., primer dimers) in highly multiplexed PCR, thereby inhibiting effective amplification. This elimination system removes or significantly minimizes non-productive artifacts (e.g., primer dimers). In addition to downstream processing (e.g., high-throughput sequencing), removing low-compatibility and problematic primers significantly improves the overall performance and efficiency of highly multiplexed PCR. Artifacts and primer dimers lead to significant failures in obtaining optimal sequence results, and a large portion of sequencing reads may be non-specific and non-informative.

[0150] In some embodiments, the primer selection method is characterized by a primer compatibility score about primer-primer interaction and specific target nucleic acid hybridization (without non-specific initiation or hybridization with off-target region). The higher compatibility score of the candidate target-specific primer characterizes the specific hybridization with the target nucleic acid, and does not interact or has the minimum interaction with other primers in the primer set. Primers that do not meet the compatibility score (i.e., above the minimum threshold) will be removed. In various embodiments of the disclosed method, the compatibility score is calculated for at least 80%, 90%, 95%, 98%, 99% or 99.5% of the possible combinations of candidate primers in the group. The compatibility score in the primer selection is calculated based on multiple parameters (such as target amplicon GC content, target amplicon melting temperature, target amplicon heterozygosity, complementation rate of candidate primers to target region, candidate primer size, target amplicon size and amplification efficiency). Due to the fact that determining the compatibility score involves multiple aspects, the average score is calculated based on multiple parameters, and the average value may vary due to specific applications. The primer selection method will continuously eliminate low-compatibility primers and repeat the elimination process until it is equal to or below a minimum threshold until the best selected primer set is obtained, thereby generating a highly multiplexed target amplification PCR with no or minimized primer dimers.

[0151] In some embodiments, the primer selection method is characterized in that the primer compatibility score about primer-primer interaction and specific target nucleic acid hybridization (not having and off-target region hybridization).Primers with low compatibility score (i.e. higher than minimum threshold) will be excluded.However, if there is restriction in primer selection in some applications, the minimum threshold can be raised to a second threshold of a higher level, to promote the primer selection of primer sets.In some embodiments, repeat the selection process until selecting a candidate primer equal to or lower than the minimum threshold of the second level.

[0152] In embodiments, the method disclosed herein is characterized in that multiple amplification and target enrichment are performed using target-specific primers, and the target-specific primers contact the target nucleic acid sequence of the genomic material of the sample related to cancer / hereditary disease, drug resistance, forensics, microorganisms, fungi, parasites, viruses and other biological applications, wherein primer dimers can be reduced or minimized by adjusting different parameters (such as the duration of the annealing step, the increase or decrease of the temperature increment together with the number of cycles). In some embodiments, the primer concentration can be reduced, and the annealing temperature and duration can be increased to allow specific amplification (primers have more time intervals to hybridize with the target nucleic acid), and primer dimers can also be reduced or minimized. In some embodiments, the concentration of the primer can be 500nM, 250nM, 100nM, 80nM, 70nM, 50nM, 30nM, 10nM, 2nM, 1nM or less than 1nM. In some embodiments, the annealing temperature can be 1 minute, 3 minutes, 5 minutes, 8 minutes, 10 minutes or longer. In some embodiments, amplification with longer annealing times uses 1 cycle, 2 cycles, 3 cycles, 5 cycles, 8 cycles, 10 cycles, or more cycles followed by a standard annealing duration.

[0153] In one aspect, the disclosed method includes the step of amplifying a selective target nucleic acid sequence of a sample associated with cancer / genetic diseases, drug resistance, forensics, microorganisms, fungi, parasites, viruses, and other biological applications. In some embodiments, the method includes the step of contacting a nucleic acid sample with a target-specific primer in the presence of a barcoded universal primer in a test reaction. In some embodiments, the method includes the step of determining the presence or absence of a target amplification product. In some embodiments, the method includes the step of determining the sequence of the amplified target product. In some embodiments, the method identifies microorganisms at the strain or substrain level. In some embodiments, less than 50%, 40%, 30%, 20%, 10%, 5%, 0.5% or 0.1% of the amplified products are primer dimers or artifacts. In one embodiment, there may be more than one set of target-specific primers, for example, there may be two sets of target-specific primers for two test reactions, there may be 3 sets of target-specific primers for 3 test reactions, or there may be 5 sets of target-specific primers or more for 5 test reactions. In some embodiments, due to practical reasons (eg, limitations in primer design or selection), samples may also be split into multiple parallel multiplex testing reactions with multiple sets of target-specific primers.

[0154] In various embodiments, the concentration of each primer can be 500nM, 250nM, 100nM, 80nM, 70nM, 50nM, 30nM, 10nM, 2nM, 1nM or less than 1nM. In various embodiments, the primer concentration of each primer can be 1 μM to 1nM, 1nM to 80nM, 1nM to 100nM, 10nM to 50nM or 1nM to 60nM. In some embodiments, the GC content of the target-specific primer can be 40% to 70%, or 30% to 60% or 50% to 80% or 30% to 80%. In some embodiments, the primer GC content range can be less than 20%, 15%, 10% or 5%. In some embodiments, the melting temperature (T m ) can be 55°C to 65°C, or 40°C to 72°C, or 50°C to 68°C. In some embodiments, the melting temperature range of the primer can be less than 20°C, 15°C, 10°C, 5°C, 2°C, or 1°C. In some embodiments, the length of the target-specific primer can be 20 to 90 bases, 40 to 70 bases, 20 to 40 bases, or 25 to 50 bases. In some embodiments, the length range of the primer can be 60, 50, 40, 30, 20, 10, or 5 bases. In some embodiments, the 5' region of the target-specific primer is a universal priming site that is not complementary or specific to any target nucleic acid region.

[0155] In one aspect, the present disclosure relates to a kit comprising target-specific primers in the form of a set; the primers are designed and selected based on the described criteria to have minimal primer-primer interactions or non-specific priming. In another embodiment, the kit can be formulated for detection, screening, diagnosis, prognosis, and treatment of a disease. In another embodiment, the kit can be formulated for detection of drug resistance. In some embodiments, the kit can be used for screening, detection, identification, genotyping, subtyping, and monitoring of bacteria, fungi, parasites, and viruses. In some embodiments, the kit can be used to analyze samples associated with cancer / genetic diseases, forensics, drug resistance, pharmacogenetics, and other biological applications.

[0156] In some embodiments, the disclosed method comprises the following steps: (1) contacting a set of target-specific primers with a target nucleic acid sequence in the presence of a barcoded universal primer and hybridizing with the target nucleic acid sequence in each sample in the test reaction; (2) amplifying the test reaction under optimal amplification conditions; (3) combining the amplification products from each individual sample; (4) performing a bead cleanup on a portion of the combined amplification products to remove possible primer dimers, thereby creating an enriched amplification product; (5) performing standard normalization and quantification on a portion of the enriched amplification products; and (6) sequencing the amplicons by next-generation sequencing. The method may further include additional steps, such as purification. In one aspect, the disclosed method utilizes highly multiplexed PCR. In some embodiments, for PCR, 1 to 10 cycles of PCR may be performed; in some embodiments, 1 to 15 cycles, or 1 to 20 cycles, or 1 to 25 cycles, or 1 to 30 cycles, or 1 to 35 cycles or more cycles may be performed.

[0157] In another embodiment, when more than two targets are amplified, the disclosed methods can be used in a multiplexed fashion and are not limited to any number of multiplexes.

[0158] In some embodiments, the amplified product can be sequenced by a second generation sequencing platform. Second generation sequencing refers to a large-scale parallel DNA nucleic acid sequencing technology that is not based on Sanger (sanger), which can sequence millions to billions of DNA chains in parallel. The examples of the second generation sequencing technology and platform of the current prior art are Illumina platform (reversible dye terminator sequencing), 454 pyrophosphate sequencing, ion semiconductor sequencing (Ion Torrent), PacBio SMRT sequencing, QiagenGeneReader sequencing technology, Element Biosciences sequencing platform and Oxford nanopore sequencing. The present disclosure is not limited to these second generation sequencing technology examples.

[0159] Example 1

[0160] High-resolution Salmonella genotyping and subtyping

[0161] Materials and methods

[0162] Samples and DNA extraction: For this study, 12 ATCC Salmonella isolates and 3 other isolates were used: S. abaetetuba, S. anatum, S. bispebjerg, S. diarizoniae, S. infantis, S. Mbandaka, S. Montevideo, S. Poona, S. Seftenberg, S. Tennessee, S. Typhi, and S. Typhimurium. Stocks of bacterial strains stored in glycerol were recovered from -80°C stock storage. The isolates were streaked on tryptic soy agar plates with 5% sheep blood and incubated overnight at 35°C. One to three colonies were picked and their DNA was extracted using the QIAamp Mini DNA kit following the manufacturer's protocol. The Salmonella isolates were propagated and DNA extracted.

[0163] Assay Design: This assay was designed to examine 47 polymorphic loci in the Salmonella enterica genome for substrain level genotyping of Salmonella species. The 47 loci include seven loci of housekeeping genes used in traditional MLST assays (aroC, dnaN, hemD, hisD, purE, sucA, and ihrA). Although the assay was designed based on the Salmonella enterica genome, more than 20 loci could be amplified and sequenced for Salmonella bongori.

[0164] Amplification: One-step multiplex PCR was performed in a 96-well plate in a Veriti thermal cycler (ThermoFisher, CA, USA) in a final volume of 15 μl. The PCR reaction consisted of target-specific primers, barcoded universal primers, sample DNA, DNA polymerase, dNTPs, and PCR buffer.

[0165] Next-generation sequencing: All amplicons were combined into one tube. Then, a portion of the sample was purified using SPRIselect beads (Beckman Coulter, CA, USA) according to the manufacturer's instructions. The concentration of the purified sample was measured on Qubit 3 and normalized for sequencing. The library was sequenced using the Illumina Mid Output Sequencing Kit using the Illumina MiniSeq system.

[0166] Sequence data analysis software and interpretation: Decoding: Sequencing reads (FASTQ format) of forward and reverse reads and forward and reverse index reads are input into ChapterDx analysis software. In the absence of mismatches, the software assigns sequencing reads to samples based on the sequences of the forward and reverse index reads; Mapping: Sequencing reads are mapped to reference sequences using the Smith-Waterman algorithm, with options of nucleotide match reward of 1, nucleotide mismatch penalty of -3, cost of opening a gap of 5, and cost of extending a gap of 2. For each sequencing read, only the best matching alignment is retained if the alignment score exceeds 60. Subtype analysis: (1) Find the genome (X) with the most mapped loci; (2) Exit if the number of mapped loci is less than a preset cutoff; (3) Output genome (X) and remove all reads mapped to genome (X); and (4) Repeat steps 1-3. In some embodiments, the preset cutoff is at least 10. For each locus, generate a paired (one forward and one reverse) consensus sequence. Pairwise distances were calculated as the number of allelic differences between two strains. Then, a neighbor-joining tree was constructed based on the resulting distance matrix.

[0167] result

[0168] Distance between samples: Figure 7 The distances between each pair of test samples are listed. Pairwise distances are calculated as the number of allele differences between the two strains, which is the same as traditional MLST. In traditional MLST, the number of nucleotide differences between alleles is ignored, and different allele numbers are assigned regardless of whether the sequences differ at a single nucleotide site or at multiple sites. The basic principle is that a single genetic event that leads to a new allele can occur through a point mutation (changing only a single nucleotide site) or a recombination substitution (which will usually change multiple sites). Figure 7 Shown that the isolates of different serotypes have more than 30 loci differences, which indicates that the loci examined by multiple barcoding amplification NGS assays are heterogeneous. Samples 12 and 15 are both Salmonella typhimurium serotypes. However, these two samples belonging to the same serotype have differences in the alleles at four loci. None of these four loci overlap with the seven housekeeping genes (aroC, dnaN, hemD, hisD, purE, sucA and thrA) used in traditional MLST. Therefore, traditional MLST is unlikely to distinguish them.

[0169] Genotyping: Genotype calling methods infer serotypes based on the best matching genome assembly. Usually, the best matching genome assembly is annotated with serotype information. However, sometimes, the best matching genome assembly lacks serotype information. In order to fill the missing serotype information in the genome assembly, several different methods have been tested to estimate the evolutionary distance between Salmonella assemblies downloaded from the NCBI database. Ultimately, the program was chosen to calculate the distance between two Salmonella genome assemblies because the resulting distance matrix reflects the relationship between Salmonella genomes. Based on the calculated distance matrix, a number of Salmonella genome assemblies without serotype information were grouped together with Salmonella genome assemblies with serotype information using a single linkage clustering method. Figure 8 The NCBI accession number of the best matching genome assembly and its corresponding inferred serotype are listed. Sample 13 had no serotype information and was identified as S. Enteritidis by our assay. Sample 14 was originally labeled as S. Typhimurium, but our assay identified it as S. muenster with the best matching genome accession number CP019198.1. Neighbor-joining tree based on allelic differences ( Fig. 9 ) showed that sample 14 was separated from sample 12 (Salmonella typhimurium) and sample 15 (Salmonella typhimurium) with 45 loci differences. Figure 7 This indicates that sample 14 is unlikely to be Salmonella typhimurium.

[0170] Detection of the coexistence of multiple strains in the same sample: It has been reported that the same food or environmental sample may be contaminated with different Salmonella strains at the same time. Therefore, the frequency of multiple serovar Salmonella outbreaks may be higher than generally recognized. Identification of all Salmonella serovars involved in an outbreak may help indicate the source of the outbreak, clarify the scope of the outbreak, and determine the selection of appropriate control measures. To evaluate the ability of the assay to detect the coexistence of multiple strains in the same sample, DNA from multiple samples was mixed to simulate samples contaminated with multiple strains. Experiments were performed on mixtures of 3, 6, and 9 serovars, where (1) three tubes each contained a mixture of three serovars, (2) three tubes each contained a mixture of six serovars, and (3) two tubes each contained a mixture of nine serovars. The two mixed tubes with nine serovars were identical. The results show that the multiplexed barcoded amplification NGS assay was able to identify and distinguish all serovars from all mixtures using ChapterDx data analysis software, indicating that the assay has excellent ability to detect the coexistence of multiple strains in the same sample ( Fig.10 ).

[0171] Example 2

[0172] High-resolution genotyping of Cyclospora cayetanensis

[0173] Materials and methods

[0174] Samples and DNA extraction: DNA was extracted from various fresh produce inoculated with purified C. cruzi oocyst preparations from patients, including salad mix (romaine and iceberg lettuce, carrots, and red cabbage), cilantro, and basil samples (25 g each). In addition, 50 liters of pond water samples were inoculated with 200 to 20,000 oocysts. Sample preparation and DNA extraction were performed according to previously described procedures (Durigan, M., Murphy, HR, and Da Silva, AJ (2020). Dead-End Ultrafiltration and DNA-Based Methods for Detection of C. cayetcmensis in Agricultural Water (Appl Environ Microbiol 86; Almeria, S., and Shipley, A. 2021). Cyclospora cruzi in packaged pre-cut salad mixes within and after the shelf life was detected by a method validated by the US Food and Drug Administration (Food Microbiol 98, 103802; Assurian, A., Murphy, H., Ewing, L., Cinar, HN, Da Silva, A., and Almeria, S. 2020). Evaluation of the US Food and Drug Administration validated molecular method for detection of Cyclospora cayetanensis oocysts on fresh and frozen berries. Food Microbiol 87,103397).

[0175] Assay design: Contigs of Cyclospora cruzi isolate NF1 (accession number MSEL00000000) were concatenated as a reference genome, from which fragments of 280 to 350 bases were obtained. The fragments were used as BLAST queries against 40 WGS assemblies of Cyclospora cruzi, and fragments with BLAST hits in all 40 WGS assemblies were retained. The entropy of each fragment was calculated, and fragments with entropy values ​​> 1.0 were considered for inclusion. Then, fragments that were evenly distributed along the artificial genome were selected as templates for primer design. 52 loci were selected for Cyclospora cruzi.

[0176] Multiplex barcoded amplification: One-step multiplex barcoded PCR was performed using the Cyclospora NGS assay. Briefly, for each sample, 5 μl of DNA was used in a single multiplex PCR containing target-specific primers for all amplicons in the presence of barcoded universal primers, DNA polymerase, dNTPs, and PCR buffer.

[0177] Next-generation sequencing: All amplicons were combined into one tube. Then, a portion of the sample was purified using SPRIselect beads (Beckman Coulter, CA, USA) according to the manufacturer's instructions. The concentration of the purified sample was measured on Qubit 3 and normalized for sequencing. The library was sequenced using the Illumina Mid Output Sequencing Kit using the Illumina MiniSeq system.

[0178] result

[0179] Fig.11 The genotyping results of the Cyclospora samples are shown. The table lists the genotyping results of the Cyclospora samples. Although most of the targeted loci were amplified and sequenced, the best scoring genome only matched about half of the loci. Nevertheless, this example shows the feasibility of this objective technology in genotyping Cyclospora.

[0180] Example 3

[0181] Legionella genotyping by multiplex barcoded amplification and extensively variable multilocus sequence typing

[0182] Materials and methods

[0183] Samples and DNA extraction: DNA was extracted from the enriched 12 Legionella samples.

[0184] Assay Design: To perform Legionella genotyping at the substrain level, the assay was designed to cover 55 polymorphic loci of the Legionella pneumophila genome. The assay also detects other Legionella species such as L. longveachae and L. bozemanii through conserved gene targets.

[0185] Amplification: Extracted DNA was subjected to one-step multiplex barcoded PCR with target-specific primers in the presence of barcoded universal primers, DNA polymerase, dNTPs and PCR buffer.

[0186] Next-generation sequencing: All amplicons were combined into one tube. Then, a portion of the sample was purified using SPRIselect beads (Beckman Coulter, CA, USA) according to the manufacturer's instructions. The concentration of the purified sample was measured on Qubit3 and normalized for sequencing. The library was sequenced using the Illumina Mid Output Sequencing Kit using the Illumina MiniSeq system.

[0187] result

[0188] Fig.12 Genotyping results for Legionella samples are listed. Most targeted loci were amplified and sequenced. However, the best scoring genomic matches were for about half of the loci. Nevertheless, most targeted loci were amplified and sequenced, demonstrating the feasibility of this technology for Legionella genotyping.

[0189] Example 4

[0190] High-resolution Monkeypox Genotyping and Surveillance NGS Assay

[0191] Materials and methods

[0192] Samples and DNA extraction: DNA was extracted from clinical samples diagnosed with monkeypox infection.

[0193] Assay Design: The assay is designed to amplify 21 highly polymorphic regions and distinguish monkeypox from poxviruses that infect other mammals. The assay distinguishes between different lineages of monkeypox as well as between monkeypox of the same lineage.

[0194] Amplification: Extracted DNA was subjected to one-step multiplex barcoded PCR with target-specific primers in the presence of barcoded universal primers, DNA polymerase, dNTPs and PCR buffer.

[0195] Next-generation sequencing: All amplicons were combined into one tube. Then, a portion of the sample was purified using SPRIselect beads (Beckman Coulter, CA, USA) according to the manufacturer's instructions. The concentration of the purified sample was measured on Qubit 3 and normalized for sequencing. The library was sequenced using the Illumina Mid Output Sequencing Kit using the Illumina MiniSeq system.

[0196] result

[0197] Fig.13The genotyping results of monkeypox virus are shown. The table lists the genotyping results of monkeypox virus samples, where all the best scoring genomes have accession number KJ642617.1, indicating that these samples may have similar original sources. The sample with barcode / index ID P5.02.06_P7.04.G only had 8 loci amplified and sequenced, which is mainly due to the low virus titer in the sample.

[0198] Example 5

[0199] High-resolution Listeria genotyping by multiplex barcoded PCR NGS assay

[0200] Materials and methods

[0201] Samples and DNA Extraction: DNA of eight Listeria samples was extracted from the enriched samples.

[0202] Assay Design: The Listeria assay was designed to target 69 polymorphic regions in the Listeria monocytogenes genome, 59 of which target polymorphic loci, 9 target traditional MLST loci (abcZ, bgl, cat, dap, dat, ldh, lhk, pgm, sod), and an internal control. The assay also detects other Listeria species: L. grayi, L. innocua, L. ivanovii, L. marthii, L. seeligeri, and L. welshimeri.

[0203] Amplification: Extracted DNA was subjected to one-step multiplex barcoded PCR with target-specific primers in the presence of barcoded universal primers, DNA polymerase, dNTPs and PCR buffer.

[0204] Next-generation sequencing: All amplicons were combined into one tube. Then, a portion of the sample was purified using SPRIselect beads (Beckman Coulter, CA, USA) according to the manufacturer's instructions. The concentration of the purified sample was measured on Qubit 3 and normalized for sequencing. The library was sequenced using the Illumina Mid Output Sequencing Kit using the Illumina MiniSeq system.

[0205] result

[0206] Fig.14Results of Listeria genotyping by barcoded multiplex amplification and variable width MLST are shown. Results for Listeria samples containing Listeria monocytogenes and Listeria innocua are listed in the table. For Listeria monocytogenes samples, 68 loci can be amplified and sequenced. For Listeria innocua samples, 36 loci can be amplified and sequenced.

[0207] Example 6

[0208] High-resolution Shiga Toxin-Producing E. Coli (STEC) genotyping by multiplex barcoded PCR NGS assay

[0209] Materials and methods

[0210] Samples and DNA extraction: Glycerol stocks of 15 strains were streaked on LB plates, single colonies were picked and grown overnight in LB medium, and then DNA was extracted using the Qiagen DNeasy kit according to the manufacturer's protocol. E. coli isolates were propagated and DNA extracted.

[0211] Assay design: The STEC E. coli assay examines 61 polymorphic loci, including nine loci of housekeeping genes used in conventional MLST assays (arcA, aroE, dnaE, espA, gapA, gnd, mdh, ompA, and pgm). In addition, the assay includes 7 serotype-specific loci (O26, O45, O103, O111, O121, O145, and O157) and 11 other loci (uidA, stxla, stx2a, eaeA, est, elt, aggR, ipaH, bfpA, GFP) of the wzx gene encoding the O-antigen flippase. Typical enteropathogenic E. coli (EPEC) strains produce bundled pili (BFP). BFP is a bundled polymer that is a pilus protein encoded by the bfpA gene found on large EPEC plasmids.

[0212] Amplification: Extracted DNA was subjected to one-step multiplex barcoded PCR with target-specific primers in the presence of barcoded universal primers, DNA polymerase, dNTPs and PCR buffer.

[0213] Next-generation sequencing: All amplicons were combined into one tube. Then, a portion of the sample was purified using SPRIselect beads (Beckman Coulter, CA, USA) according to the manufacturer's instructions. The concentration of the purified sample was measured on Qubit 3 and normalized for sequencing. The library was sequenced using the Illumina Mid Output Sequencing Kit using the Illumina MiniSeq system.

[0214] result

[0215] Fig.15 The number of loci amplified and sequenced for each sample is listed. Most non-serotype specific loci are amplified and sequenced. The O antigen responsible for the strain antigenic specificity determines the O-serogroup. For each of the E. coli O26, O45, O103, O111, O113, O121, O145 and O157 serogroups, the wzx (O-antigen flippase) gene was selected as the target in this assay.

[0216] Fig.16 The read counts of the serogroup-specific wzx genes for each sample are listed. The serogroup information of those STEC E. coli isolates was consistent with the read count prediction results. In addition to O-antigen, ten gene markers were selected as targets in this assay to examine the virulence gene profile. Fig.17 The read counts for each gene marker for each sample are listed. For those STEC E. coli isolates, read counts for stx1 and stx2 information were available, which was consistent with the read count prediction results. In addition, the assay showed that all those STEC E. coli isolates were positive for the uidA gene as expected, and also positive for the eaeA gene. The assay showed that all those STEC E. coli isolates were negative for the bfpA, LTa, STa, STb, aggR, and ipaH genes.

[0217] Example 7

[0218] High-resolution multiplex barcoded PCR NGS assay for hepatitis A (Hep A) genotyping

[0219] Materials and methods

[0220] Samples and DNA extraction: RNA from three HAV-contaminated samples was used in this experiment.

[0221] Assay Design: Primers used for the HAV assay were designed to cover 10 conserved regions in the HAV genome.

[0222] Amplification: First, reverse transcription (RT) reaction was performed with random RT primers. In the presence of barcoded universal primers, DNA polymerase, dNTPs and PCR buffer, one-step multiplex barcoded PCR was performed on cDNA by target-specific primers.

[0223] Next-generation sequencing: All amplicons were combined into one tube. Then, a portion of the sample was purified using SPRIselect beads (Beckman Coulter, CA, USA) according to the manufacturer's instructions. The concentration of the purified sample was measured on Qubit 3 and normalized for sequencing. The library was sequenced using the Illumina Mid Output Sequencing Kit using the Illumina MiniSeq system.

[0224] result

[0225] Fig.18 The genotyping results of HAV samples are shown. The table lists the results of HAV samples where 5 to 8 loci were amplified and sequenced. Since HAV is an RNA virus with a high mutation rate, the multi-locus sequencing approach increases the chances of detecting the HAV virus because even if the assay fails to amplify some loci due to primer mismatches, it can amplify other loci. In addition, sequence data from multiple loci provide information to track and trace the source of contamination by comparing the sequences of these sequenced loci.

[0226] Example 8

[0227] Microbial identification by multiplex barcoded PCR NGS assays

[0228] Materials and methods

[0229] Samples and DNA extraction: DNA extracted from environmental samples was used in this experiment.

[0230] Assay Design: Primers were designed to target the V3 and V4 regions of bacterial 16s rRNA. Primers were designed to target the internal transcribed region (ITS) between fungal 18s rRNA and 28S rRNA.

[0231] Amplification: Extracted DNA was subjected to one-step multiplex barcoded PCR with target-specific primers in the presence of barcoded universal primers, DNA polymerase, dNTPs and PCR buffer.

[0232] Next-generation sequencing: All amplicons were combined into one tube. Then, a portion of the sample was purified using SPRIselect beads (Beckman Coulter, CA, USA) according to the manufacturer's instructions. The concentration of the purified sample was measured on Qubit3 and normalized for sequencing. The library was sequenced using the Illumina Mid Output Sequencing Kit using the Illumina MiniSeq system.

[0233] result

[0234] Fig.19 Results for environmental samples are listed. Species with more than 100 reads are listed in the table. This assay detects a wide range of species. In addition, this assay detects bacteria and fungi in the same reaction.

[0235] Example 9

[0236] Single-tube detection and genotyping of HIV, HBV, and HCV by multiplex barcoded PCR NGS assay

[0237] Materials and methods

[0238] Samples and DNA extraction: RNA from clinical samples infected with HIV and HCV and DNA from clinical samples infected with HBV were used in this experiment.

[0239] Assay Design: Primers for 5 amplicons targeting conserved regions of the HBV genome, primers for 4 amplicons targeting conserved regions of the HCV genome, and primers for 5 amplicons targeting conserved regions of the HIV1 genome and primers for 4 amplicons targeting conserved regions of the HIV2 genome were designed.

[0240] Amplification: First, RNA was converted to cDNA by reverse transcription (RT) reaction with random RT primers. One-step multiplex barcoded PCR was performed on cDNA (HIV and HCV) by target-specific primers in the presence of barcoded universal primers, DNA polymerase, dNTPs and PCR buffer.

[0241] Next-generation sequencing: All amplicons were combined into one tube. Then, a portion of the sample was purified using SPRIselect beads (Beckman Coulter, CA, USA) according to the manufacturer's instructions. The concentration of the purified sample was measured on Qubit 3 and normalized for sequencing. The library was sequenced on the IlluminaMiniSeq system using the Illumina Mid Output Sequencing Kit.

[0242] result

[0243] Due to the high mutation rate, mismatches between primers and the targeted HBV, HCV, and HIV genomes are common. To minimize the risk of false-negative results, a multilocus amplification and sequencing strategy was used to detect and genotype HBV, HCV, and HIV1 / 2. Fig. 20 Genotyping results are presented, demonstrating the feasibility of this targeted technology for genotyping HBV, HCV, and HIV1 / 2 in a single tube via multiplex barcoded amplification.

[0244] Example 10

[0245] Comprehensive genetic profiling of BRCA1, BRCA2, and TP53 using multiplexed barcoded amplification NGS assays

[0246] Materials and methods

[0247] Samples and DNA extraction: Six human DNA samples were selected for this study, of which one sample was a healthy control and five samples had the following mutations: (1) NM_007294.3 (BRCA1): c.3700_3704delGTAAA, 2) (p.Val1234Glnfs); (2) NM_000059.4 (BRCA2): c.6468_6469del (p.Gln2157fs); (3) NM_007294.3 (BRCA1): c.5266dupC (p.Gln1756Profs); (4) NM_007294.3 (BRCA1): c.302-2A>C, and 5) NM_000059.3 (BRCA2): c.9097dupA (p.Thr3033Asnfs). As a reference control, the BRCA Somatic Multiplex I (gDNA) control from Horizon Discovery (Waterbeach, UK) was used, which includes 15 variants with allele frequencies ranging from 7.5% to 60%.

[0248] Assay Design: Primers were designed to cover all coding regions of BRCA1, BRCA2, and TP53 genes. Overlapping amplicons were designed to cover exons greater than 300 bp. Two PCR reactions were performed per sample to separate overlapping amplicons.

[0249] Amplification: Extracted DNA was subjected to one-step multiplex barcoded PCR with target-specific primers in the presence of barcoded universal primers, DNA polymerase, dNTPs and PCR buffer.

[0250] Next-generation sequencing: All amplicons were combined into one tube. Then, a portion of the sample was purified using SPRIselect beads (Beckman Coulter, CA, USA) according to the manufacturer's instructions. The concentration of the purified sample was measured on Qubit 3 and normalized for sequencing. The library was sequenced using the Illumina Mid Output Sequencing Kit using the Illumina MiniSeq system.

[0251] result

[0252] Fig.19 A depicts the results of a genomics analysis performed by the Integrative Genomics Viewer (IGV, https: / / software.broadinstitute.org / software / igv / ) shows a screenshot of the BRCA1 exon read alignment, and Fig.21 The mutations detected in the test samples are shown. The results show that all known mutations were detected in each sample as well as the BRCA Somatic Multiplex I (gDNA) control reference sample from Horizon Discovery, indicating that this assay is sensitive and specific for detecting somatic mutations in clinical samples.

[0253] Embodiment 11

[0254] STR Sequencing by Multiplexed Barcoded Amplification NGS Assay

[0255] Materials and methods

[0256] This assay uses genomic DNA from two human samples. Additional replicates of sample 2 were performed to test the reproducibility of this assay.

[0257] Assay Design: PCR primers were designed around D1S1656, D10S1248, D10S1435, D10S2325, D13S317, D13S325, D15S659, D16S539, D17S1301, D18S1364, D18S51, D19S433, D20S482, D21S11 STR loci.

[0258] Amplification: Extracted DNA was subjected to one-step multiplex barcoded PCR with target-specific primers in the presence of barcoded universal primers, DNA polymerase, dNTPs and PCR buffer.

[0259] Next-generation sequencing: All amplicons were combined into one tube. Then, a portion of the sample was purified using SPRIselect beads (Beckman Coulter, CA, USA) according to the manufacturer's instructions. The concentration of the purified sample was measured on Qubit 3 and normalized for sequencing. The library was sequenced using the Illumina Mid Output Sequencing Kit using the Illumina MiniSeq system.

[0260] result

[0261] Fig. 22 The insertions and / or deletions detected in both samples are listed. Note that the (-) sign indicates a deletion, and the (+) sign indicates an insertion. The number following the (+) or (-) indicates the copy number of the repeat unit detected in the assay. Each sample shows two patterns. All of these insertions and / or deletions are relative to the hgl9 genome reference.

[0262] The insertion and / or deletion patterns between the two replicate samples were consistent with each other. Most of the insertion and / or deletion patterns matched the STR patterns of the loci. However, there were a few exceptions, such as the deletion of T in the D1S1656 locus in sample 1 and the insertion of TA at the D21S11 locus. This assay shows the feasibility of the proposed technology for amplifying STR loci for forensic testing.

Claims

1. A method for screening and analyzing at least one sample for food, animal, human and environmental pathogens, the method comprising the steps of: for each sample, hybridizing a plurality of target-specific primers to nucleic acid from the sample in the presence of a barcoded universal primer to form a test reaction in a single reaction vessel, wherein at least one target-specific primer is configured to bind to at least one target nucleic acid sequence; subjecting each test reaction to amplification conditions to generate an amplicon; Amplicons from each sample were combined, performing a bead cleanup on at least a portion of the pooled amplicons to form enriched amplicons; and The pooled and enriched amplicons formed from each sample were sequenced by next-generation sequencing.

2. The method of claim 1, wherein each barcoded universal primer comprises: The universal priming moiety at the 3' end, The middle barcode portion; and Universal priming moiety at the 5' end.

3. The method of claim 1, wherein each target-specific primer comprises a specific sequence portion for the target nucleic acid sequence and a universal priming portion.

4. The method of claim 1, wherein each sample is obtained from a subject, a food, one or more plants, or an environmental source.

5. The method of claim 1, wherein the target-specific primers comprise primers configured to amplify at least 20 variable regions of at least bacteria, fungi, parasites or viruses for identification, genotyping, subtyping and detection of co-existence of multiple isolates.

6. The method of claim 1, further comprising mapping and counting multiple pathogen genomes for microbial and viral typing, subtyping and monitoring, comprising the following additional steps: (1) determining a score for locus A of a first genome as the ratio of the number of unique reads mapped to locus A of the first genome to the total number of unique reads mapped to locus A of the first genome and at least one other genome, wherein if no read is mapped to locus A of the first genome, the score is preset to a minimum number; repeating the determination step of part (1) for at least one other locus; and (3) determining an overall score for the first genome by multiplying the scores of all test loci in steps (1) and (2).

7. The method according to claim 6, further comprising the steps of: (a) determining the genome with the highest overall score for any remaining reads; (b) ending the evaluation if the number of non-empty loci in the highest-scoring genome is less than a preset cutoff; (c) outputting the highest-scoring genome; (d) removing reads mapped to all the highest-scoring genomes at that time; and (e) repeating steps (a)-(d) until the evaluation ends according to step (b). The method according to claim 7 , wherein the preset cut-off value is at least 10.

9. The method of claim 1, wherein the target-specific primers comprise primers configured to amplify multiple variable regions of at least bacteria, fungi, parasites or viruses for genotyping, subtyping, detection and identification of multiple genotypes or subtypes of the same species or different species in the same sample.

10. The method of claim 1, wherein the target-specific primers comprise primers configured to amplify and detect a target sequence of at least one species / type / subtype of bacteria, fungi, parasites, or viruses in the same sample.

11. The method of claim 1, wherein the sample comprises at least one microbial and viral species, strain / type, or substrain / subtype that is differentiated.

12. The method of claim 1, wherein the target-specific primers comprise primers configured to amplify and analyze target sequences relevant to forensic testing.

13. The method of claim 1, further comprising the step of pooling the enriched amplicons from each sample prior to sequencing.

14. The method of claim 1, further comprising the step of quantifying each type and species in each sample after sequencing the enriched amplicons.

15. The method of claim 1, wherein the test reaction comprises a polymorphic gene with a unique sequence for an internal control.

16. A method for screening at least one sample for food, human, animal and environmental pathogens, the method comprising the steps of: For each sample, a plurality of target-specific primers are hybridized to nucleic acid from the sample in the presence of a barcoded universal primer to form a test reaction in a single reaction vessel, wherein at least one target-specific primer is configured to bind to at least one target sequence selected from the group consisting of: bacteria, fungi, parasites, or viruses, wherein each barcoded universal primer comprises: a universal priming moiety at the 3' end; The middle barcode portion; and a universal priming portion at the 5' end; and wherein each target-specific primer comprises a sequence portion specific for the target nucleic acid sequence and a universal priming portion; subjecting each test reaction to amplification conditions to generate an amplicon; Amplicons from each sample were pooled; performing a bead cleanup on at least a portion of the pooled amplicons generated from each sample to form a pooled and enriched amplicon; as well as The pooled and enriched amplicons formed from each sample were sequenced by next-generation sequencing.

17. A method of screening at least one sample for forensic DNA analysis, cancer or genetic disease, the method comprising the steps of: For each sample, a plurality of target-specific primers are hybridized to nucleic acid from the sample in the presence of a barcoded universal primer to form a test reaction in a single reaction vessel, wherein at least one target-specific primer is configured to bind to at least one target sequence, wherein each barcoded universal primer comprises: a universal priming moiety at the 3' end; The middle barcode portion; and a universal priming portion at the 5' end; and wherein each target-specific primer comprises a sequence portion specific for the target nucleic acid sequence and a universal priming portion; subjecting each test reaction to amplification conditions to generate an amplicon; Amplicons from each sample were pooled; performing a bead cleanup on at least a portion of the pooled amplicons generated from each sample to form enriched amplicons; as well as The enriched amplicons formed from each sample were sequenced by next-generation sequencing.

18. A kit comprising a plurality of target-specific primers configured to bind to target sequences specific to biological samples associated with cancer, genetic diseases, forensic testing, and microbial and viral species.

Images