NL2026403B1 - Next generation sequencing method of zoobenthos cytochrome oxidase subunit i gene and use thereof - Google Patents
Next generation sequencing method of zoobenthos cytochrome oxidase subunit i gene and use thereof Download PDFInfo
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Abstract
The present invention relates to the technical field of zoobenthos identification, and in particular to a next generation sequencing (NGS) method of a zoobenthos cytochrome oxidase subunit T (COT) gene and use thereof. The method 5 includes the following steps: Sl, amplifying the COT gene by polymerase chain reaction (PCR) with a DNA sample of a mixed zoobenthos sample as a template; SZ, amplifying a PCR.product obtained in step Sl as a template by PCR, to obtain a PCR product as a DNA library, where an amplified primer pair includes a 10 pyroseguencing—NGS adapter and each sample corresponds to one pyroseguencing—NGS adapter; and S3, conducting guality detectioncm1aresultinglflwxlibraryy andselectingeagualified DNA_library to conduct emPCR.amplification.and.NGS. The method obtains a high—throughput full—length (~658 bp) COT gene 15 sequence with read length covering large benthic communities by all—at—once sequencing, providing a large taxonomic resolutionandavoidingfalsepositivesdueixnexcessivelyhigh throughput.
Description
TECHNICAL FIELD The present invention relates to the technical field of zoobenthos identification, and in particular to a next generation sequencing (NGS) method of a zoobenthos cytochrome oxidase subunit I (COI) gene and use thereof.
BACKGROUND Benthic invertebrates usually refer to invertebrate communities that live on the bottom of a water body in the whole or most of life history, principally including molluscs, aquatic insects, annelida, and crustaceans. In water ecological monitoring, benthic invertebrates are essential for evaluating ecological conditions of the freshwater ecosystem. Morphological characteristics play a very important role in species identification but are easily limited by biological sex and specific developmental stages and influenced by phenotypic plasticity and genetic variability; moreover, ubiquitous cryptic species and high demands for morphological identification lead to misidentification easily. Scientists anticipate that there are a total of more than 10-15 million biological species globally. If these species are identified by using conventional morphological methods, all taxonomists worldwide will spend thousands of years working together to achieve complete identification. Therefore, species identification urgently needs a new technical method. DNA barcoding technology developed in recent years just fulfills this need. Cytochrome oxidase subunit I (COI) is ubiquitous in mitochondria. The gene is functionally conserved but has certain evolution rate; with a substantial amount of information, the gene is widely used in research on DNA barcoding. DNA barcoding is weak in mixed samples and environmental samples, but NGS solves the problem that DNA barcoding can merely identify a single individual. However, Illumina platforms in NGS are limited by read length, most of which are sequenced based on ~313 bp COI fragments; even if a maximum read length of 300 PE is used, 658 bp COI barcode still has a gap of 100 bp, such that a full length of barcode region cannot be obtained by all-at-once sequencing; the presence of gap substantially influences data integrity, so as not to meet the requirement for species identification. Moreover, Illumina sequencing, due to excessively high throughput, detects non-target DNA amplicons with an extremely low copy number very easily. With longer read length, third generation sequencing, such as Oxford nancpore and PacBio SMAT, can reduce splicing costs; however, because of high error rate of single read length, there is a need for error correction by repeated sequencing, leading to an increase in sequencing cost, less abundant bioinformatics software, and low data accumulation.
SUMMARY Therefore, the technical problem to be solved in the present invention is to provide an NGS method of a zoobenthos COI gene and use thereof. With high throughput of the sequencing method, data for ~658 bp COI standard barcode gene is acquired by all-at-once sequencing, with high accuracy, providing a larger taxonomic resolution.
For this purpose, the technical solutions of the present invention are as follows: An NGS method of a zoobenthos COI gene includes the following steps: 51, amplifying the COI gene by polymerase chain reaction (PCR) with a DNA sample of a mixed zoobenthos sample as a template; S2, amplifying a PCR product obtained in step Sl as a template by PCR, to obtain a PCR product as a DNA library, wherein an amplified primer pair comprises a forward primer and a reverse primer, the primer pair comprises a pyrosequencing-NGS adapter, and each sample corresponds to one pyrosequencing-NGS adapter; and 53, conducting quality detection on a resulting DNA library obtained in step S2, and selecting a qualified DNA library to conduct emulsion PCR (emPCR) amplification and NGS.
For the method, in step Sl, a system of the PCRamplification is as follows: 10x Buffer, containing 20 mM Mg?** plus, 5 ul; dNTP, 2.5 mM, 4 ul; forward primer COIF, 5 uM, 2 ul; reverse primer COIR, 5 uM, 2 ul; Ex Tag DNA Polymerase, 5 U/ul, 0.25 ul; DNA template, 5 ng; and ultrapure water, 35.75 ul.
For the method, in step Sl, a reaction program of the PCR amplification is as follows: initial denaturation at 95°C for 3 min; 30-35 cycles of denaturation at 95°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 45 s; 72°C for 7 min, and cryopreservation at 4°C.
For the method, in step S2, a system of the PCR amplification is as follows: 10x Buffer, containing 20 mM Mg?" plus, 5 ul; dNTP, 2.5 mM, 4 pl; 454AL+MID +COIF, 5 uM, 2 ul; 454B+ COIR, 5 uM, 2 ul; Ex Tag DNA Polymerase, 5 U/ul, 0.25 ul; DNA template, 1 ng; and ultrapure water, 35.75 ul.
For the method, in step S2, a reaction program of the PCR amplification is as follows: initial denaturation at 925°C for 3 min; 18 cycles of denaturation at 95°C for 30 s, annealing at 68°C for 30 s, and extension at 72°C for 45 s; 72°C for 7 min, and cryopreservation at 4°C.
For the method, in step S3, the quality detection is conducted on the library by quantitative competitive polymerase chain reaction (QC PCR), and a system of the QC PCR is as follows: 10x Buffer, containing 20 mM Mg?" plus, 5 ul;
dNTP, 2.5 mM, 8 nl; forward primer QC-F, 100 uM, 1 ul; reverse primer QC-R, 100 pM, 1 ul; Ex Taq DNA Polymerase, 5 U/ul, 1 ul; DNA library, 1 x 10% molecules/ul, 2 ul; and ultrapure water, 32 pl. For the method, in step S83, the quality detection is conducted on the library by QC PCR, and a program of the QC PCR is as follows: initial denaturation at 94°C for 11 min; 30 cycles of denaturation at 94°C for 1 min, annealing at 60°C for 1 min, and extension at 72°C for 1 min; 72°C for 10 min, and cryopreservation at 4°C. For the method, in step S3, a reaction system after QC PCR is incubated with exonuclease, reaction systems before and after restriction enzyme digestion are electrophoresed, and DNA libraries with a sequencing library length of ~750 bp are selected as qualified DNA libraries. For the method, in step 83, during the emPCR amplification, the DNA libraries are subjected to emPCR lv amplification in a ratio of the number of DNA molecules to the DNA capture beads count, 1.e., 3. For the method, step S3 further includes a step of data analysis of sequencing results. An NGS kit for a zoobenthos COI gene is provided, including a primer pair used for amplifying the zoobenthos COI gene, the system of the PCR amplification, the system of the PCR amplification, and/or the system of the QC PCR. Use of the NGS kit for a zoobenthos COI gene in obtaining a zoobenthos COI gene barcode is provided; preferably, the use includes use thereof in water ecological monitoring. The technical solutions of the present invention have the following advantages:
1. The NGS method of a zoobenthos COI gene provided by the present invention includes the following steps: Sl, amplifying the COI gene by PCR with a DNA sample of a mixed zoobenthos sample as a template; S2, amplifying a PCR product obtained in step S1 as a template by PCR, to obtain a PCR product as a DNA library, where an amplified primer pair includes a forward primer and a reverse primer, the primer pair includes a pyrosequencing-NGS adapter, and each sample corresponds to one pyrosequencing-NGS adapter; and S3, conducting quality 5 detection on a resulting DNA library obtained in step S2, and selecting a qualified DNA library to conduct emPCR amplification and NGS. The NGS method of a zoobenthos COI gene obtains a high-throughput full-length (~658 bp) COI gene sequence with read length covering large benthic communities by all-at-once sequencing, providing a large taxonomic resolution and avoiding false positives due to excessively high throughput.
2. The use of the NGS kit for a zoobenthos COI gene provided by the present invention in obtaining a zoobenthos COI gene barcode is provided; the use includes the use thereof in water ecological monitoring; environmental stress factors influencing macrobenthos in the Wei River Basin are identified efficiently as conductivity and total nitrogen by the NGS kit for a zoobenthos COI gene and/or the NGS method of a zoobenthos COI gene.
BRIEF DESCRIPTION OF THE DRAWINGS To describe the technical solutions in the examples of the present invention or in the prior art more clearly, the following briefly describes the accompanying drawings required for describing the examples or the prior art. Apparently, the accompanying drawings in the following description show some examples of the present invention, and a person of ordinary skill in the art may still derive other drawings from these accompanying drawings without creative efforts.
FIG. 1 illustrates a standard curve of precise and quantitative double-stranded deoxyribonucleic acid (dsDNA) based on fluorescence analysis in Example 1; FIG. 2 is a gel electrophoresis map of detection of COI gene libraries by Agilent 2100 Bioanalyzer in Example 1; FIG. 3 is an electrophoresis map of detection of COI gene libraries by Agilent 2100 Bioanalyzer in Example 1; FIG. 4 is a plot of dilution curves of different samples in Example 1 of the present invention.
DETAILED DESCRIPTION The following examples serve to provide further appreciation of the present invention, but are not limited to the preferred examples and do not limit the spirit and scope of the invention; any product that is the same as or similar to the inventionmade in light of the invention or by combination of the present invention with other features of the prior art shall fall within the scope of the invention.
If specific experimental procedure or conditions are not indicated in the present invention, operations or conditions of conventional experimental procedures known in the art shall be used. If manufacturers of reagents and apparatus used are not indicated, conventional reagent products may be commercially available.
Example 1 The example provided an NGS method of a zoobenthos COI gene, including the following steps: First, samples related to the example were prepared as follows: Representative sampling points were set up according to the geographical and water quality characteristics of the water body. Zoobenthos samples were collected from different habitats using a Surber net (30 x 30 cm, tuck net aperture:
0.5 mm) or a Petersen grab (open area: 1/16 m*); three parallel samples were collected at each point, which were placed in 300 mL sample bottles after cleaning and rough selection and fixed in 95% alcohol for storage.
(1) Mixed biological samples prepared above were extracted by a tissue genomic DNA extraction kit; DNA concentrations were determined and uniformly diluted to a final concentration of 5 ng/ul. The COT gene was amplified by PCR using the same concentration of diluted DNA as a template. The forward primer COIF is shown as SEQ ID NO. 1, and the reverse primer COIR is shown as SEQ ID NO. 2. A system of the PCR amplification was as follows: 10x Buffer, containing 20 mM Mg** plus, 5 ul; dNTP, 2.5 mM, 4 ul; forward primer COIF, 5 uM, 2 ul; reverse primer COIR, 5 uM, 2 ul; Ex Tag DNA Polymerase, 5 U/ul, 0.25 ul; DNA template, 5 ng; and ultrapure water, 35.75 ul. A reaction program of the PCR amplification was as follows: initial denaturation at 95°C for 3 min; 30-35 cycles of denaturation at 95°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 45 5; 72°C for 7 min, and cryopreservation at 4°C. An amplified product was extracted and quantified by 1% agarose gel electrophoresis. (2) One nanogram of amplified product extracted in step (1) was amplified by PCR. A primer pair to be amplified included a forward primer and a reverse primer; the primer pair included a pyrosequencing-NGS adapter, and each sample corresponded to one pyrosequencing-NGS adapter, i.e., a forward pyrosequencing-NGS adapter was added at 5'-end of the primer COIF; a sequence of the forward pyrosequencing-NGS adapter (573') was composed of a 454AL sequence and an MID sequence successively. The 454AL sequence is shown as SEQ ID NO. 3. The MID sequence was composed of eight arbitrary nucleotides, and one MID sequence was designed for each sample (double~underlined sequences in Table 1) to further ensure that each sample corresponded to one pyroseguencing-NGS adapter. A reverse pyrosequencing-NGS adapter 454B sequence was added at 5'-end of the primer COIR. The 454B sequence is shown as SEQ ID NO. 4. Partial primer 454AL+MID (A1l-A12}) +COIF and primer 454B+ COIR are shown in Table 1. Table 1 Partial primer 454AL+MID{(A1-A12) +COIF and primer 454B+
454AL+A1+COIF ccatctcatccctgcgtgtctccgactcagacgagtgcggtcaacaaatcalaaagara SEQ ID NO:5 454AL+A2+COIF ccatctcatccctgcgtgtctccgactcagacgctcgaggtcaacaaatcalaaagara SEQ ID NO:6 454AL+A3+COIF ccatctcatccctgcgtgtctccgactcagagacgcacggtcaacaaatcalaaagara SEQ ID NO:7 454AL+A4+COIF ccatctcatccctgcgtgtctccgactcagagcactgtggtcaacaaatcalaaagara SEQ ID NO:3 454AL+A5+COIF ccatctcatccctgegtgtetccgactcagatcagacaggtcaacaaatrcataaagala SEQ ID NO:9 454ATL+A6+COIF ccatctcatccctgegtgtetccgactcagatetegcgggtcaacaaatrcataaagala SEQ ID NO:10
454AL+A7+COIF
Ccatctcatccctgcgtgtctccgactcagcgtgtctcggtcaacaaatcaltaaagara SEQ ID NO:11 454AL+A8+COIF ccatctcatccctgcgtgtctccgactcagctcgcgtgggtcaacaaatcalaaagara SEQ ID NO:12 454AL+A9+COIF ccatctcatccctgcgtgtctccgactcagtagtatcaggtcaacaaatcalaaagara SEQ ID NO:13 454AL+A10+COIF ccatctecatcecectgegtgtetecgactecagtctctatgggtcaacaaatcataaagata SEQ ID NO:14 454AL+A11+COIF ccatctcatccctgcgtgtctccgactcagtgatacgtggtcaacaaatcalaaagara SEQ ID NO:15 454AL+A12+COIF ccatctcatccctgcgtgtctccgactcagtactgagcggtcaacaaatcalaaagara SEQ ID NO:16
454B+COIR cctatcccectgtgtgcettggcagtctcagtaaacttcagggtgaccaaaaaatca
SEQ ID NO:17
A system of the PCR amplification was as follows: 10x Buffer, containing 20 mM Mg** plus, 5 ul; dNTP, 2.5 mM, 4 ul; 454AL+MID +COIF, 5 uM, 2 ul; 454B+ COIR, 5 uM, 2 ul; Ex Tag DNA Polymerase, 5 U/ul, 0.25 ul; DNA template, 1 ng; and ultrapure water, 35.75 ul.
A reaction program of the PCR amplification is as follows: initial denaturation at 95°C for 3 min; 18 cycles of denaturation at 95°C for 30 s, annealing at 68°C for 30 s, and extension at 72°C for 45 5; 72°C for 7 min, and cryopreservation at 4°C.
PCR amplification fragments were extracted by gel extraction.
Concentrations of extracted PCR amplification fragments were determined precisely by Promega Quant-iT dsDNA Assay Kit.
A standard curve was plotted as shown in FIG. 1, and the copy number of DNA molecules was calculated according to the following formula: copies/mL = (6,022x102: copies/mole) x concentration (ng/mL)/MW (g/mol). Each sample was diluted to 105 molecules/uL.
The above diluted samples were grouped; 10 ul each of each sample was pipetted and mixed well as a DNA library. (3) Quality detection was conducted on the DNA library by QC PCR.
Forward primer QC~-F was 5f GGTCAACAAATCATAAAGATATTGG-3 (SEQ ID NO: 18), and reverse primer QC-R was 5' -TAAACTTCAGGGTGACCAAAAAATCA-3’ (SEQ ID NO: 2). A system of the QC PCR was as follows: 10x Buffer, containing 20 mM Mg** plus, 5 ul; dNTP, 2.5 mM, 8 pul; forward primer QC-F, 100 pM, 1 ul; reverse primer QC-R, 100 pM, 1 ul; Ex Tag DNA Polymerase, 5 U/ul, 1 ul; DNA library, 1 x 10% molecules/ul, 2 ul; and ultrapure water, 32 ul.
A program of the QC PCR was as follows: initial denaturation at 94°C for 11 min; 30 cycles of denaturation at 94°C for 1 min, annealing at 60°C for 1 min, and extension at 72°C for
1 min; 72°C for 10 min, and cryopreservation at 4°C.
A reaction system after QC PCR was incubated with 1 ul of exonuclease for 1 hat 37°C, 1 ul each of reaction systems before and after restriction enzyme digestion was run on Agilent Bioanalyzer 2100 (DNA 7500 Chip). Sample libraries without small fragment amplification were qualified. Successfully constructed sequencing libraries had a length of ~750 bp, including a sequencing adapter and PCR primers.
The above qualified DNA libraries were subjected to emPCR amplification and high-throughput sequencing. The DNA libraries were subjected to emPCR lv amplification in a ratio of the number of DNA molecules to the DNA capture beads count, i.e., 3, and a given number of DNA capture bead sets were arranged in a sequencing plate for high-throughput sequencing.
Each sequencing region obtained 1,000,000 effective sequences, and each sample had a data quantity of approximately 1,000,000.
Example 2 Data analysis was conducted on sequencing results obtained in Example 1, including the following steps: 1) A list file of an MID sequence was input; Split-libraries.py in QIIME software was run to sort out MIDs of different samples, and noise sequences with degenerate bases, low-quality scores, chimeras, and a length of <150 bp were eliminated to acquire clean data.
2} The clean data acquired above were subjected to OTU clustering by using Usearch and CD-HIT software and OTU picking by using a plurality of thresholds (97%, 98%, etc.), to enhance the result reliability. Species annotation analysis was made by matching representative sequences of OTUs with 80% sequence similarity with sequences in the BOLD database, and a list of OTUs was generated.
3) Alpha-diversity indices of samples, such as Shannon index, Simpson index, and Chao index, were calculated based on relative abundances of OTUs. Sample cluster analysis was conducted by using the abundance data of sequences and the data for the presence or absence of OTUs; PCoA analysis was conducted on the dissimilarity of each sampling point using coefficients of the data of Jaccard (presence/absence) and Bray-Curtis (relative abundance). Example 3 Use in water ecological monitoring Environmental stress factors and main pollutants influencing macrobenthos communities in a river basin were determined based on the data acquired in Example 2: hydrochemical parameters were normalized by logarithmic transformation.
One-way ANOVA was conducted by analyzing the number of various environmental factors in operational taxonomic units among groups and differences in environmental factors, and then distance-based redundancy analysis (dbRDA) was conducted; meanwhile, ineffective environment variables were deleted gradually by previous picking (P>0.05); environmental stress factors and pollutants influencing the distribution of macrobenthos communities were selected by the Monte Carlo method, and an ordination plot was used to reflect the relationship between community distribution and pollutants and identify main stress factors and pollutants influencing macrobenthos communities in a river basin.
Example 4 The example provided an NGS kit for a zoobenthos COI gene, including a primer pair used for amplifying the zoobenthos COI gene, the system of the PCR amplification in step S1, the system of the PCR amplification in step S2, and/or the system of the QC PCR.
For the primer pair used for amplifying the zoobenthos COI gene, the forward primer COIF is shown as SEQ ID NO. 1, and the reverse primer COIR is shown as SEQ ID NO. 2. The system of the PCRamplification in step Sl was as follows: 10x Buffer, containing 20 mM Mg? plus, 5 ul; dNTP, 2.5 mM, 4 ul; forward primer COIF, 5 pM, 2 ul; reverse primer COIR, 5 uM, 2 ul; Ex Tag DNA Polymerase, 5 U/ul, 0.25 ul; DNA template, 5 ng; and ultrapure water, 35.75 nl.
The systemof the PCRamplification in step S2 was as follows:
10x Buffer, containing 20 mM Mg* plus, 5 ul; dNTP, 2.5 mM, 4 ul; 454AL+MID +COIF, 5 uM, 2 ul; 454B+ COIR, 5 uM, 2 ul; Ex Taq DNA Polymerase, 5 U/ul, 0.25 ul; DNA template, 1 ng; and ultrapure water, 35.75 ul.
The system of the QC PCR was as follows: 10x Buffer, containing 20 mM Mg* plus, 5 ul; dNTP, 2.5 mM, 8 ul; forward primer QC-F, 100 pM, 1 ul; reverse primer QC-R, 100 pM, 1 ul; Ex Taq DNA Polymerase, 5 U/ul, 1 ul; DNA library, 1 x 10% molecules/ul, 2 ul; and ultrapure water, 32 ul.
Example 5 The example provided use of the NGS method of a zoobenthos COI gene in Example 1 or the NGS kit for a zoobenthos COI gene in Example 3 in water ecological monitoring of the Wei River Basin, including the following steps: Sample collection and determination of physical and chemical parameters of water: Macrobenthos samples were collected from different habitats of river reaches of the Weil River Basin, fixed in 95% alcohol for storage, and taken back to the laboratory to complete DNA extraction. Water samples were collected in amber bottles, and national standard method was used to determine the total nitrogen (TN), total phosphorus (TP), ammonia-nitrogen (NH4'-N), biological oxygen demand (BODs)}), chemical oxygen demand (COD), and silicate content of the water body. A water quality meter (YSI) was used to determine the water temperature, pH, conductivity, salinity, dissolved oxygen, suspended matter, and total dissolved solids (TDS) .
COI standard barcoding and morphological identification: Macrobenthos samples were collected in duplicate from each sampling point: one was used for morphological identification, and the other was used for NGS based on COI barcoding. Herein,
morphological identification of zoobenthos refers to the Fauna Sinica and local faunas; the NGS method was the same as that in Example 1, the data analysis method was the same as that in Example 2, and the water ecological monitoring method was the same as that in Example 3.
Water ecological monitoring results of the Wei River Basin were as follows. Representative sequences of OTUs were annotated in the BOLD database. Creatures included aquatic insects (Ephemeroptera, Trichoptera, Plecoptera, Diptera, and Coleoptera), oligochaeta, and molluscs; 84.7% of sequences could be annotated at a genus level. Finally, conductivity and TN were identified as significant environmental stress factors influencing the benthic community distribution in the Wei River Basin.
Effect Example
1. Optimum ratio of the number of DNA molecules to the DNA capture beads count DNA libraries qualified in Example 1 were subjected to emPCR sv (SV EMPCR KIT (LIB-L)) assay to determine an optimum ratio of the number of DNA molecules to the DNA capture beads count. The number of DNA molecules with an enrichment ratio of 10%-20% was the optimum ratio. Table 2 shows the effects of the use of the DNA libraries in Example 1 in ratios of the number of DNA molecules to the DNA capture beads count (0.5, 1, and 4).
From the table, when CPB = 1, the enrichment ratio is 11%; when CPB = 4, the enrichment ratio is 25%. Thus, in order to ensure a high enrichment ratio and meet the optimum ratio, CPB = 3 is used for subsequent emPCR lv (LV emPCR Kit (Lib-1L)) amplification.
Table 2 emPCR sv data table Initial Ratio Library 1x107 mole/uL CPB = 4
2. QC PCR systems before and after exonuclease digestion in Example 1 were detected by Agilent Bicanalyzer 2100. A gel electrophoresis map is shown in FIG. 2, and an electrophoresis map is shown in FIG. 3. From these figures, DNA libraries prepared in Example 1 show no small fragment amplification after quality detection, indicating that the method of Example 1 is mature and reliable.
3. Dilution curves were plotted for different samples in Example 1. As shown in FIG. 4, the number of NGS sequences of the mixed sample at each sampling point reaches a plateau, and the number of sequences was reasonable, indicating that throughput conditions of the NGS method of a zoobenthos COI gene provided by the present invention fully satisfy the demands for subsequent research and use.
Obviously, the above examples are merely intended to describe the examples clearly, but not to limit the embodiment. Different forms of variations or alterations may also be made by those of ordinary skill in the art based on the above descriptions. All examples do not have to and cannot be presented herein. Obvious variations or alterations extended thereby still fall within the scope of the claimed invention.
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| CN112359119A (en) * | 2020-11-18 | 2021-02-12 | 中国科学院生态环境研究中心 | Environmental DNA macro-bar code method for large-scale benthonic animal community structure research |
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| CN104630244A (en) * | 2015-01-26 | 2015-05-20 | 中国科学院海洋研究所 | COI gene sequence of species of coral reef benthos and species identification method utilizing COI gene sequence |
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