Construction method of amplicon library for sequencing intestinal flora

Abstract

The invention discloses an amplicon library construction method for intestinal flora sequencing, and relates to the technical field of biological sequencing, and the amplicon library construction method specifically comprises the following steps: step (1): sample collection: collecting a fresh excrement sample, and carrying out sealed storage; (2) DNA data extraction: extracting the DNA data in the excrement sample; according to the amplicon library construction method for intestinal flora sequencing, a sample collection and preservation and DNA extraction method is optimized, and the integrity of a flora structure and the DNA quality in a sample are ensured; specific primers and optimized PCR amplification conditions are adopted, so that the amplification deviation is reduced, and the amplification efficiency is improved; through strict purification and quality control steps, the quality of the amplicon library is ensured, and the method is suitable for high-throughput sequencing; blank control and negative control are arranged, so that pollution is effectively controlled, and the accuracy and reliability of results are improved.

Description

Technical Field

[0001] This invention relates to the field of biological sequencing technology, specifically to a method for constructing an amplicon library for gut microbiota sequencing. Background Technology

[0002] The gut microbiota is closely related to human health and plays a crucial role in the development of various diseases. High-throughput sequencing technology provides a powerful tool for gut microbiota research, and amplicon library construction is a key prerequisite for gut microbiota sequencing. As an important component of the human micro-ecosystem, the gut microbiota contains trillions of microorganisms, and the balance of its community structure and function is closely related to human health. From metabolic diseases such as obesity and diabetes to complex conditions such as inflammatory bowel disease and autism, all have been shown to be associated with gut microbiota dysbiosis. Gut microbiota sequencing technology, by analyzing the gene sequences of microorganisms, reveals the composition and functional characteristics of the microbiota, becoming a core method for studying the gut microbiome. Amplicon library construction is a crucial step before sequencing. It involves PCR amplification of target gene fragments using specific primers and the introduction of sequencing adapters and sample tags to make the sample compatible with high-throughput sequencing platforms.

[0003] High-quality amplicon libraries directly impact the accuracy and reliability of sequencing data. On one hand, primer design must balance coverage and specificity, ensuring effective amplification of different microbial groups while avoiding errors introduced by non-specific amplification. On the other hand, factors such as the number of PCR cycles and optimization of the reaction system during library construction can lead to differences in amplification preferences among samples, thus affecting the true reflection of bacterial abundance.

[0004] For example, Chinese invention patent application number 202110359724.5 discloses a method for constructing a 16S rDNA amplicon library for detecting gut microbiota in gestational diabetes mellitus. By mixing equal amounts of amplification products and using optimized PCR-free adapter ligation, this method avoids the biases generated by PCR and chimeras in environmental samples, resulting in good uniformity of sequencing data among different samples. This makes subsequent cluster analysis and technical reproducibility more reasonable and accurate, and can more realistically reflect the species abundance distribution of gut microbiota in pregnant women with gestational diabetes mellitus, providing rapid detection results for patients. However, this method still has certain shortcomings. The lack of a control group during sequencing resulted in inadequate contamination control, affecting the accuracy of the results and the instability of the library quality, which directly impacted the precision and reliability of gut microbiota analysis.

[0005] Therefore, we propose an amplicon library construction method for gut microbiota sequencing to address the problems mentioned above. Summary of the Invention

[0006] The purpose of this invention is to provide an amplicon library construction method for gut microbiota sequencing, in order to solve the problem mentioned in the background art that the sequencing process in the current market does not include a control, resulting in poor contamination control affecting the accuracy of the results and unstable library quality, thereby directly affecting the accuracy and reliability of gut microbiota analysis.

[0007] To achieve the above objectives, the present invention provides the following technical solution: a method for constructing an amplicon library for gut microbiota sequencing, wherein the amplicon library construction method specifically includes the following steps: Step (1): Sample collection: Collect fresh fecal samples and seal them for preservation; Step (2): DNA data extraction: Extract DNA data from the fecal sample; Step (3): First round of PCR amplification: The DNA from step (2) is amplified by primers with sample-specific barcodes and sequencing adapters. The primers are targeted at the V3-V4, V4 or V4-V5 regions of the 16S rRNA gene. Step (IV): Purification and Quantification: The first-round PCR amplification products from step (III) are purified and quantified; Step (5): Second round of PCR amplification: The purified amplification products are ligated with adapters and subjected to a second round of PCR amplification; Step (6): Generating amplicon libraries: Purify, merge, and quality control the second round of PCR amplification products to obtain amplicon libraries.

[0008] Preferably, the fresh fecal sample in step (a) is a sample taken ≤2 hours after excretion. After collection, it is sealed in a sterile sampling tube and frozen at -80℃ or preserved with RNAlater protectant.

[0009] Preferably, in step (ii), DNA extraction is performed using CTAB / SDS, and bacterial cell walls are broken by bead milling. The extracted DNA concentration is ≥10 ng / μL, and the OD260 / 280 is between 1.8 and 2.0.

[0010] Preferably, the reaction system for the first round of PCR amplification in step (iii) is 20 μL, including 10 ng of DNA template, 10 μL of 2×PCR Mix, 0.5 μL each of upstream and downstream primers, and the primer concentration is 10 μM, which is then made up to 20 μL with dH2O.

[0011] Preferably, the conditions for the first round of PCR amplification are: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 30 s, 55-60℃ annealing for 30 s, 72℃ extension for 30 s, for a total of 25-30 cycles; and 72℃ final extension for 5 min.

[0012] Preferably, in step (iv), the purification of the first-round PCR amplification products is carried out by agarose gel electrophoresis followed by gel extraction or magnetic bead method to remove primer dimers and non-specific products, and the concentration of purified products is ≥5ng / μL.

[0013] Preferably, in step (v), the second round of PCR amplification cycles is 10-15 times, and the primer concentration is 5 μM.

[0014] Preferably, the quality control of the amplicon library includes using Agilent Bioanalyzer to detect the distribution of library fragments, with the target fragment accounting for ≥80% within ±50bp, and the Qubit quantitative concentration being ≥2nM.

[0015] Preferably, the amplicon library construction method further includes setting a blank control and a negative control during sample collection and library construction, wherein the blank control is template-free PCR and the negative control is a blank extraction reagent.

[0016] Preferably, in step (vi), the OD260 / 280 ratio is monitored by a UV-Vis spectrophotometer, and the fragment distribution is detected by electrophoresis using a microfluidic chip; the proportion of the target fragment ±50bp is ≥80%, and the concentration is ≥2nM by using a Qubit fluorometer to ensure quantification, thereby achieving comprehensive quality control of the library after purification and merging of the second round of PCR amplification products.

[0017] Compared with the prior art, the beneficial effects of the present invention are: the method for constructing amplicon libraries for gut microbiota sequencing: 1. Optimized sample collection, preservation, and DNA extraction methods to ensure the integrity of the bacterial community structure and DNA quality in the samples; employed specific primers and optimized PCR amplification conditions to reduce amplification bias and improve amplification efficiency; ensured the quality of the amplicon library through rigorous purification and quality control steps, making it suitable for high-throughput sequencing; and included blank and negative controls to effectively control contamination and improve the accuracy and reliability of the results.

[0018] 2. It enables precise analysis of the composition and diversity of gut microbiota, providing strong technical support for research on the relationship between gut microbiota and host health. Attached Figure Description

[0019] Figure 1 This is a schematic diagram of the amplicon library construction process of the present invention. Detailed Implementation

[0020] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0021] Please see Figure 1 This invention provides a technical solution: a method for constructing an amplicon library for gut microbiota sequencing, the method specifically including the following steps: Step (1): Sample collection: Collect fresh fecal samples and seal them for preservation; Step (2): DNA data extraction: Extract DNA data from the fecal sample; Step (3): First round of PCR amplification: The DNA from step (2) is amplified by primers with sample-specific barcodes and sequencing adapters. The primers are targeted at the V3-V4, V4 or V4-V5 regions of the 16S rRNA gene. Step (IV): Purification and Quantification: The first-round PCR amplification products from step (III) are purified and quantified; Step (5): Second round of PCR amplification: The purified amplification products are ligated with adapters and subjected to a second round of PCR amplification; Step (6): Generating amplicon libraries: Purify, merge, and quality control the second round of PCR amplification products to obtain amplicon libraries.

[0022] In step (1), the fresh fecal sample is the sample that was excreted ≤2 hours ago. After collection, it is sealed in a sterile sampling tube and stored at -80℃ or preserved with RNAlater protectant. By limiting the collection time and preservation method of fresh fecal samples, the originality of the microbial community structure can be effectively maintained and sequencing deviations caused by sample deterioration can be avoided.

[0023] In step (II), DNA extraction was performed using CTAB / SDS. Bacterial cell walls were disrupted via bead milling, resulting in an extracted DNA concentration ≥10 ng / μL and an OD260 / 280 between 1.8 and 2.0. The specific procedure was as follows: Fecal samples were mixed with bead milling media and disrupted in a bead mill at an appropriate speed and time. Then, DNA was purified using phenol-chloroform extraction or magnetic bead adsorption. The final extracted DNA concentration should be ≥10 ng / μL, with an OD260 / 280 between 1.8 and 2.0 to ensure DNA quality. Using CTAB / SDS combined with bead milling to disrupt cell walls improves DNA extraction efficiency, ensures the extracted DNA quality meets sequencing requirements, and reduces the risk of subsequent amplification failure.

[0024] In step (iii), the reaction system for the first round of PCR amplification is 20 μL, including 10 ng of DNA template and 2×PCR. Mix 10 μL of primers, 0.5 μL each of forward and reverse primers, with a primer concentration of 10 μM, and bring the volume to 20 μL with dH2O. Design primers targeting the V3-V4, V4, or V4-V5 regions of the 16S rRNA gene. Add sample-specific barcodes (6-10 bp in length) and sequencing adapters (such as Illumina's Nextera adapters) to the 5' ends of the primers. The PCR reaction system is 20 μL, including 10 ng of DNA template, 10 μL of 2×PCR Mix, 0.5 μL each of forward and reverse primers (10 μM), and bring the volume to 20 μL with ddH2O. Use a high-fidelity enzyme (such as Q5 HotStart High-Fidelity DNA Polymerase) for amplification to reduce the amplification error rate. Optimize primer concentrations and reaction components to ensure efficient and specific amplification of the target region of the 16S rRNA gene and reduce the generation of non-specific products.

[0025] The conditions for the first round of PCR amplification were: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 30 s, 55-60℃ annealing for 30 s, 72℃ extension for 30 s, for a total of 25-30 cycles; and 72℃ final extension for 5 min. By optimizing the denaturation, annealing, and extension parameters, the amplification efficiency was ensured while reducing base errors and biases caused by PCR cycles, thereby improving the accuracy of the amplification products.

[0026] In step (iv), the first round of PCR amplification products are purified by agarose gel electrophoresis followed by gel excision or magnetic bead method to remove primer dimers and non-specific products. The purified product concentration is ≥5ng / μL. The amplification products are purified by agarose gel electrophoresis or magnetic bead method to remove primer dimers and other impurities, ensuring the template quality for the subsequent second round of PCR and avoiding interference from impurities.

[0027] In step (5), the second round of PCR amplification should be performed 10-15 times with a primer concentration of 5 μM. Controlling the number of cycles and primer concentration can reduce deviations caused by over-amplification, ensure adapter ligation efficiency and library uniformity, and improve sequencing adaptability.

[0028] Quality control of amplicon libraries includes using Agilent Bioanalyzer to detect library fragment distribution, ensuring that the proportion of target fragments within ±50bp is ≥80%, and that the Qubit quantification concentration is ≥2nM. By detecting fragment distribution and concentration, we ensure that the library meets the requirements of high-throughput sequencing and avoid invalid or erroneous sequencing data due to insufficient library quality.

[0029] The amplicon library construction method also includes setting blank and negative controls during sample collection and library construction. The blank control is template-free PCR, and the negative control is extraction reagent blank. Sterile consumables are used throughout the sample collection and library construction process. Setting blank control (template-free PCR) and negative control (extraction reagent blank) eliminates environmental DNA contamination. Through blank and negative controls, the contamination situation during sample collection and construction is monitored in real time to ensure the authenticity and reliability of sequencing results.

[0030] In step (six), the OD260 / 280 ratio is monitored using a UV-Vis spectrophotometer, and the fragment distribution is detected by microfluidic chip electrophoresis. The proportion of the target fragment ±50bp is ≥80%, and the concentration is ≥2nM using a Qubit fluorometer. This achieves comprehensive quality control of the purified and merged library of the second round of PCR amplification products. By combining UV spectrophotometry, microfluidic electrophoresis, and fluorescence quantification, the library quality is controlled from multiple dimensions, including purity, fragment distribution, and concentration, to ensure the accuracy and reproducibility of sequencing data.

[0031] Construction and sequencing validation of amplicon libraries of gut microbiota from healthy adults: Experimental objective: To verify the reliability of the amplicon library construction method described in this invention in practical applications, and to evaluate the library quality and microbiome analysis capability through sequencing analysis of gut microbiota samples from healthy adults.

[0032] Experimental materials Sample source: Fresh fecal samples from 3 healthy adults (collected within 1.5 hours after excretion), sealed in sterile sampling tubes and frozen at -80°C; Main instruments: bead mill (MP Biomedicals), PCR instrument (Bio-Rad C1000), Agilent 2100 Bioanalyzer, Qubit 4.0 fluorometer; Experimental steps (1) DNA extraction Take 100mg of fecal sample and extract DNA using the CTAB / SDS combined bead milling method: The sample was mixed with 0.5 mm glass beads (volume ratio 1:2) and crushed in a bead mill at 3000 rpm for 2 min. The DNA was extracted twice with phenol-chloroform, precipitated with ethanol, and finally dissolved in 50 μL LTE buffer.

[0033] Test results: The DNA concentrations of the three samples were 12.5 ng / μL, 15.3 ng / μL, and 11.8 ng / μL, respectively, and the OD260 / 280 ratios were all between 1.85 and 1.92.

[0034] (2) First round of PCR amplification Primer design: Targeting the V4 region of the 16S rRNA gene, the primer sequences are as follows: 515F: 5'-GTGCCAGCMGCCGCGGTAA-3' (with added sample-specific barcode and Illumina adapter) 806R: 5'-GGACTACHVGGGTWTCTAAT-3'.

[0035] Reaction system (20 μL): 10 ng DNA template, 10 μL 2×Q5PCRMix, 0.5 μL each of forward and reverse primers (10 μM), and ddH2O to bring the total volume to 20 μL.

[0036] Amplification conditions: Pre-denaturation at 95℃ for 3 min; denaturation at 95℃ for 30 s, annealing at 55℃ for 30 s, extension at 72℃ for 30 s, 28 cycles; final extension at 72℃ for 5 min.

[0037] Product detection: Agarose gel electrophoresis showed a target band of approximately 290 bp, with no obvious non-specific amplification.

[0038] (3) Purification and quantification The first-round PCR products were purified using AMPureXP magnetic beads (1:1.8 beads / product volume), and the concentrations were 8.7 ng / μL, 10.2 ng / μL, and 9.1 ng / μL, respectively, as determined by Qubit quantification.

[0039] (4) Second round of PCR amplification Adapter ligation: Illumina P5 / P7 adapters were added to the first round of purified product in a reaction system of 25 μL. 10 μL of purified product, 12.5 μL of 2×PCRMix, 0.5 μL each of adapter primers (5 μM), and 1 μL of ddH2O.

[0040] (5) Library generation and quality control Purification and merging: The second-round product was purified by 0.8×AMPureXP magnetic beads and three samples were mixed at an equimolar concentration (10 nM).

[0041] Quality control and testing: Agilent Bioanalyzer showed that the target fragment (290bp±50bp) accounted for 88.5%; The Qubit quantification concentration is 3.2 nM, and OD260 / 280 = 1.91, which meets the requirements for sequencing.

[0042] Sequencing and Data Analysis Sequencing platform: Illumina MiSeq, 2×300bp paired-end sequencing.

[0043] Data processing: After removing connectors and low-quality sequences (Q≤20), an average of 4.2×10⁻⁶ sequences were obtained per sample. 4 Valid sequences; Based on 97% similarity, OTUs were clustered and annotated by comparison with the Greengenes database.

[0044] The results show: A total of 238 OTUs were detected in the three samples. The main bacterial groups were Bacteroidetes (42-48%), Firmicutes (35-41%), and Actinobacteria (5-8%). Alpha diversity analysis showed that the Shannon index was 4.5-5.2 and the Chao1 index was 210-235, indicating good library coverage. No valid sequences were detected in the blank control and negative control, confirming the absence of exogenous contamination.

[0045] Experimental conclusions The amplicon library construction method provided by this invention can be applied to gut microbiota sequencing efficiently and stably. The constructed library quality meets the requirements of high-throughput sequencing and can accurately analyze the composition and diversity of gut microbiota, providing reliable technical support for gut microbiota research.

[0046] The contents not described in detail in this specification are existing technologies known to those skilled in the art.

[0047] Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for constructing an amplicon library for gut microbiota sequencing, characterized in that: The amplicon library construction method specifically includes the following steps: Step (1): Sample collection: Collect fresh fecal samples and seal them for preservation; Step (2): DNA data extraction: Extract DNA data from the fecal sample; Step (3): First round of PCR amplification: The DNA from step (2) is amplified by primers with sample-specific barcodes and sequencing adapters. The primers are targeted at the V3-V4, V4 or V4-V5 regions of the 16S rRNA gene. Step (IV): Purification and Quantification: The first-round PCR amplification products from step (III) are purified and quantified; Step (5): Second round of PCR amplification: The purified amplification products are ligated with adapters and subjected to a second round of PCR amplification; Step (6): Generating the amplicon library: Purify, merge, and quality control the second round of PCR amplification products to obtain the amplicon library.

2. The method for constructing an amplicon library for gut microbiota sequencing according to claim 1, characterized in that: In step (a), the fresh fecal sample is the sample that was excreted ≤2 hours ago. After collection, it is sealed in a sterile sampling tube and frozen at -80℃ or preserved with RNAlater protectant.

3. The method for constructing an amplicon library for gut microbiota sequencing according to claim 1, characterized in that: In step (ii), DNA extraction was performed using CTAB / SDS, and bacterial cell walls were broken by bead milling. The extracted DNA concentration was ≥10 ng / μL, and the OD260 / 280 was between 1.8 and 2.

0.

4. The method for constructing an amplicon library for gut microbiota sequencing according to claim 1, characterized in that: The reaction system for the first round of PCR amplification in step (iii) is 20 μL, including 10 ng of DNA template, 10 μL of 2×PCR Mix, 0.5 μL each of upstream and downstream primers, and the primer concentration is 10 μM. The mixture is then made up to 20 μL with dH2O.

5. The method for constructing an amplicon library for gut microbiota sequencing according to claim 4, characterized in that: The conditions for the first round of PCR amplification were: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 30 s, 55-60℃ annealing for 30 s, 72℃ extension for 30 s, for a total of 25-30 cycles; and 72℃ final extension for 5 min.

6. The method for constructing an amplicon library for gut microbiota sequencing according to claim 1, characterized in that: In step (iv), the purification of the first-round PCR amplification products is carried out by agarose gel electrophoresis followed by gel excision and recovery or magnetic bead method to remove primer dimers and non-specific products. The concentration of purified products is ≥5ng / μL.

7. The method for constructing an amplicon library for gut microbiota sequencing according to claim 1, characterized in that: In step (v), the second round of PCR amplification cycles is 10-15, and the primer concentration is 5 μM.

8. The method for constructing an amplicon library for gut microbiota sequencing according to claim 1, characterized in that: The quality control of the amplicon library includes using Agilent Bioanalyzer to detect the distribution of library fragments, with the target fragment accounting for ≥80% within ±50bp, and the Qubit quantitative concentration being ≥2nM.

9. The method for constructing an amplicon library for gut microbiota sequencing according to claim 1, characterized in that: The method for constructing the amplicon library also includes setting a blank control and a negative control during sample collection and library construction. The blank control is template-free PCR, and the negative control is a blank extraction reagent.

10. The method for constructing an amplicon library for gut microbiota sequencing according to claim 1, characterized in that: In step (vi), the OD260 / 280 ratio is monitored by a UV-Vis spectrophotometer, and the fragment distribution is detected by electrophoresis using a microfluidic chip. The proportion of the target fragment ±50bp is ≥80%, and the concentration is ≥2nM by using a Qubit fluorometer to ensure quantification, thereby achieving comprehensive quality control of the library after purification and merging of the second round of PCR amplification products.

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