A method for analyzing food residues in the gastrointestinal tract of Antarctic krill using molecular sequencing technology

Abstract

The present invention relates to the field of marine biology technology, and specifically to a method for analyzing food residues in the gastrointestinal tract of Antarctic krill using molecular sequencing technology. The method comprises the following steps: dissecting Antarctic krill in a low-temperature environment and obtaining total DNA and total RNA using a nucleic acid extraction kit, and amplifying target genes in combination with specific primer design; subsequently, barcode labeling and high-throughput sequencing library construction are performed, paired-end sequencing is performed using an Illumina NovaSeq platform, and data quality control and redundancy removal are completed to obtain high-quality sequence data, accurate species identification is performed based on multi-level sequence alignment and a lowest common ancestor algorithm, phylogenetic tree and network analysis methods are used to analyze the diversity and trophic level distribution of Antarctic krill food residues, and food sources are further traced through environmental DNA comparison. The present invention can significantly improve the accuracy and scope of species identification and is of great significance for understanding the energy flow and trophic level structure of the Antarctic ecosystem.

Description

Technical Field

[0001] The present invention relates to the field of marine biological technology, and in particular to a method for analyzing food residues in the gastrointestinal tract of Antarctic krill using molecular sequencing technology. Background Art

[0002] Antarctic krill plays a fundamental role in the Antarctic marine ecosystem. They are not only a key food source for top predators such as penguins, seals, and whales, but also play a central role in energy flow and nutrient cycling. As global climate change increasingly impacts environmental factors in Antarctic waters, the number and distribution of Antarctic krill populations are experiencing some fluctuations, which have profound implications for the stability of the Antarctic food web and the sustainability of the ecosystem.

[0003] Existing methods for studying Antarctic krill food residues primarily rely on microscopic morphological observations, stable isotope analysis, or fatty acid labeling. These methods often have limitations in identifying the soft tissues of target organisms, distinguishing micropredators, determining food links, and addressing community-level dynamics. First, microscopic observations require significant time and expertise, and lack the accuracy to identify species in partially digested soft tissues. Second, stable isotope and fatty acid methods can provide information on trophic levels or energy transfer pathways, but struggle to annotate specific species at the genus or species level. Third, faced with a wide variety of food sources, traditional methods struggle to track residues from different sources and compare them across databases, hindering a systematic and comprehensive understanding of the Antarctic krill diet. Summary of the Invention

[0004] Based on the above objectives, the present invention provides a method for analyzing food residues in the gastrointestinal tract of Antarctic krill using molecular sequencing technology.

[0005] A method for analyzing food residues in the gastrointestinal tract of Antarctic krill using molecular sequencing technology, comprising the following steps:

[0006] S1, sample collection and preservation: Antarctic krill individuals were captured from the target sea area, rapidly dissected in a low-temperature environment (below -20°C), gastrointestinal contents were collected, and fixed with a preservation reagent that was free of nucleic acid contamination;

[0007] S2, nucleic acid extraction and quality testing: extracting total DNA and total RNA from the gastrointestinal contents using a nucleic acid extraction kit, and detecting the purity and integrity of the total DNA and total RNA using fluorescence quantification and gel electrophoresis;

[0008] S3, PCR amplification and sequencing library construction: Based on the known taxonomic information of the food source, specific primers are designed to perform PCR amplification on the total DNA and total RNA to obtain amplified sequences, which include target regions of nuclear genes and mitochondrial genes;

[0009] Adding barcode tags to the amplified sequences to distinguish different samples, and performing adaptor ligation and fragmentation on the amplified sequences using sequencing library construction reagents to construct a high-throughput sequencing library, and ensuring the uniformity of the high-throughput sequencing library by fragment quality assessment;

[0010] S4, high-throughput sequencing and data quality control: using the Illumina NovaSeq high-throughput sequencing platform to perform paired-end sequencing on the high-throughput sequencing library to obtain high-quality sequencing data covering the target gene region;

[0011] Performing quality control on the sequencing data, including removing low-quality sequences and adaptor-contaminated sequences, and performing redundancy removal on the quality-controlled sequencing data to obtain valid sequencing data;

[0012] S5, sequence alignment and species annotation: use alignment tools and databases to identify species of the effective sequencing data, and construct species composition data of gastrointestinal food residues based on classification tree analysis;

[0013] Based on the species composition data and combined with the Antarctic ecosystem food web model, network analysis methods were used to analyze the trophic level distribution and food source diversity of gastrointestinal food residues;

[0014] S6, Food source traceability: Verify the source of the species composition data of the gastrointestinal food residues through environmental DNA (eDNA) comparison analysis and evaluate the ecological contribution of specific food populations.

[0015] Optionally, the S1 includes:

[0016] S11, Live Capture and Pre-processing: Live Antarctic krill samples are captured using a plankton net vertical trawling system in the target Antarctic waters, immediately transferred to a pre-cooled insulated box, and transported to the deck laboratory within 15 minutes;

[0017] S12, establishment of a low-temperature dissection environment: a sterile ice bed was laid in an ISO 5 clean bench, the temperature controller was set to -25±2°C, and liquid nitrogen vapor was continuously introduced to maintain a low-temperature environment in the operating area;

[0018] S13, aseptic dissection procedure: Using titanium dissection tools treated with DNase / RNase inactivation, the carapace of the Antarctic krill was dissected along the dorsal midline, and the gastrointestinal tissue was completely removed and placed in a pre-cooled sterile culture dish;

[0019] S14, preservation reagent permeation treatment: immersing the gastrointestinal tissue in a 4° C. pre-cooled preservation solution, and shaking the solution to fully release the gastrointestinal contents, thereby obtaining a gastrointestinal mixed suspension;

[0020] S15, solid-liquid separation and subpackaging: Filter the obtained gastrointestinal mixed suspension through a 50 μm nylon filter membrane to obtain a filtrate, and subpack the filtrate into 2 mL cryovials, with the filling volume of each tube not exceeding 1.5 mL to prevent cryovial bursting, and obtain subpacked filtrate samples;

[0021] S16, gradient freezing storage: the aliquoted filtrate samples were sequentially placed in a -20°C environment for 2 hours, then transferred to a -80°C environment for 12 hours, and finally placed in liquid nitrogen vapor phase for long-term storage;

[0022] S17, metadata association record: Use QR code labels to mark the cryopreservation tubes for gradient freezing preservation, and write the sample collection location (latitude and longitude), seawater temperature (-1.8℃ to 2.5℃) and krill body length (30-60 mm) information into the blockchain evidence system.

[0023] Optionally, the S2 includes:

[0024] S21, sample pretreatment and lysis: transfer the aliquoted filtrate samples to -20°C for equilibration for 2 hours, and then thaw them in a 4°C refrigerator for 1 hour;

[0025] The cryovials were opened in an ultra-clean workbench (ISO class 5), and lysis buffer (pH 7.5, containing 20 mM Tris-HCl, 2% CTAB, and 0.8 M NaCl) containing 1% β-mercaptoethanol was added. 3 mm diameter zirconium oxide beads were placed in a grinder pre-cooled with liquid nitrogen and ground at 1800 rpm for 3 min to obtain a homogenized lysate.

[0026] S22, complex nucleic acid adsorption purification: transfer the homogenized lysate to a DNA / RNA co-extraction centrifuge column, add equal volumes of pre-chilled chloroform-isoamyl alcohol (24:1) in sequence, shake and mix, centrifuge at 12,000 × g for 15 minutes (4°C), take the supernatant and combine it with silica-based magnetic beads for 10 minutes, then apply a magnetic field for separation and discard the waste liquid.

[0027] S23, stepwise elution and concentration: Wash the magnetic beads twice with 80% ethanol containing 10 mM EDTA, then elute the total DNA with nuclease-free water preheated at 35°C (elution volume 50 μL). Then, enrich the total RNA by 70% ethanol precipitation and dissolve it in 20 μL of DEPC water to obtain purified total DNA / total RNA.

[0028] S24, double-strand integrity verification: The total DNA concentration (dsDNA HS channel) and total RNA concentration (RNA HS channel) were measured using a Qubit 4.0 fluorometer, and the integrity of the total DNA and total RNA was assessed by electrophoresis analysis using an Agilent 2100 bioanalyzer.

[0029] S25, antifreeze protein removal: add 0.1 U / μL Antarctic krill antifreeze protease (AFP-III) to the total DNA solution, incubate at 37°C for 30 minutes, and then purify the enzymatic products using AMPure XP magnetic beads.

[0030] S26, aliquot and freeze: The verified qualified nucleic acid samples are aliquoted into pre-cooled low-adsorption EP tubes, marked with batch codes, and stored in a -80°C ultra-low temperature freezer. At the same time, the electrophoresis pattern and concentration data are uploaded to the laboratory information management system and defined as qualified nucleic acid samples.

[0031] Optionally, the S3 includes:

[0032] S31, Food source classification information summary: Collect and organize references and database information related to Antarctic krill feeding targets, identify common or high-proportion potential food source species, determine the conserved regions of their nuclear and mitochondrial gene sequences, and construct an Antarctic target gene library to provide key references for primer design;

[0033] S32, Primer Design and Validation: Based on the conserved regions, use bioinformatics software to predict the specificity and annealing temperature of candidate primer sequences, select primer pairs that meet the amplification efficiency and specificity requirements, validate the primers in vitro, and perform small-scale PCR tests using target templates and negative controls;

[0034] S33, PCR reaction system configuration: Weigh the qualified nucleic acid sample packaged in S26, add specific primers, DNA polymerase, dNTP mixture, buffer, and the required magnesium ion solution, and adjust the concentration and volume of each component to ensure that the PCR reaction system is under optimal amplification conditions;

[0035] S34, Amplification Cycles and Preliminary Testing: Set the temperature and time for the initial denaturation, annealing, and extension steps, and set the number of cycles to effectively amplify the target fragments of nuclear and mitochondrial genes. Detect the size and specificity of the PCR reaction products by agarose gel electrophoresis to confirm that a clear band is generated in the expected segment, and define this product as the "amplified sequence."

[0036] S35, amplified sequence purification: Use column purification or magnetic bead purification methods to remove enzymes, primer residues and other impurities to obtain high-purity amplified sequences. Perform fluorescence quantitative measurement on the purified amplified sequences to obtain accurate concentration and quality information.

[0037] Optionally, the S3 further includes:

[0038] S36, dual-index barcoding: The purified amplicon sequences were aliquoted into separate reaction tubes, and a unique i5 / i7 dual-index combination was added to each sample using the Nextera XT Index Kit.

[0039] S37, adapter cryo-ligation: ligating Illumina TruSeq adapters to the dual-indexed labeled products of S36;

[0040] S38, antifreeze fragmentation treatment: the ligation product of S37 was fragmented using a Covaris S220 sonicator;

[0041] S39, Antarctic library purification: Fragmentation products were purified using AMPure XP magnetic beads (bead:sample volume ratio 0.8:1) and screened for 300-500 bp target fragments using the BluePippin system;

[0042] S40, library normalization and quality control: The purified library was quantified using Qubit 4.0, and the fragment distribution was detected using Agilent 4200 TapeStation.

[0043] Optionally, the S4 includes:

[0044] S41, library dilution and denaturation: Dilute the high-throughput sequencing library that has passed the quality inspection to 1.8 pM with 0.1 N NaOH solution, add 5% PhiX Control v3 library as an internal reference, denature at 95°C for 5 minutes, and immediately cool in an ice bath.

[0045] S42, sequencing chip loading: load the denatured library into the designated lane of the Illumina NovaSeq 6000 S4 flow cell.

[0046] S43, paired-end sequencing run: Start the NovaSeq 6000 system and run a 2×150 bp paired-end sequencing program.

[0047] S44, raw data generation and preliminary quality control: After sequencing is completed, the raw data in FASTQ format are exported through the Illumina BaseSpace platform and basic filtering is performed.

[0048] Optionally, the S4 further includes:

[0049] S45, adapter contamination filtering: Use Cutadapt (v3.4) to remove Illumina adapter residual sequences from the raw data and output decontaminated sequencing data.

[0050] S46, low-quality sequence trimming: Trimmomatic (v0.39) was used to perform quality trimming on the decontaminated sequencing data and output quality-controlled trimmed data.

[0051] S47, Antarctic contamination sequence removal: The quality control trimmed data were aligned with the Antarctic eDNA contamination database (AntEco v2.1), and exogenous contamination sequences were removed using Bowtie2 (v2.4.4). Unaligned reads were retained and defined as Antarctic-specific sequencing data.

[0052] S48, de-redundancy processing: VSEARCH (v2.18.0) was used to perform de-redundancy clustering on the Antarctic-specific sequencing data and output valid sequencing data.

[0053] Optionally, the S5 includes:

[0054] S51, multi-level sequence alignment: perform BLAST alignment of the output valid sequencing data with the Antarctic target gene library (e value ≤ 1e-5), and simultaneously align to the NCBI nt library to supplement unannotated sequences;

[0055] S52, LCA taxonomic assignment: Species taxonomy was assigned using the lowest common ancestor (LCA) algorithm of MEGAN6 (v6.21);

[0056] S53, classification tree construction: Based on the alignment results, a maximum likelihood phylogenetic tree was constructed using RAxML (v8.2.12);

[0057] S54, species composition data generation: Integrate classification results with phylogenetic tree topology, count read abundance by taxonomic level, and generate the species composition matrix of Antarctic krill food residues.

[0058] Optionally, the S5 further includes:

[0059] S55, food web model loading: calling the Antarctic ecosystem food web model from the SCAR-EcoFoodWeb database and integrating the species composition matrix;

[0060] Energy transfer efficiency: 10% per trophic level;

[0061] S56, network topology construction: Cytoscape (v3.9.1) was used to construct the feeding relationship network;

[0062] S57, trophic level index calculation: Calculate the trophic level position of each food source based on network topology;

[0063] S58, Diversity quantification and visualization: The Shannon-Wiener index and Simpson index were used to quantify the diversity of food sources, and a network heat map was generated using Gephi (v0.9.7).

[0064] Optionally, the S6 includes:

[0065] S61, environmental DNA sequence matching: cross-matching the obtained valid sequencing data with the Antarctic environmental DNA pollution database (AntEco v2.1) and the generated species composition matrix;

[0066] S62, Geographical traceability analysis: Based on the collection location information stored in the S17 blockchain, environmental DNA reference data for the corresponding sea area is extracted from the SCAR-MarBIN geographic database to construct an Antarctic regional environmental DNA traceability database;

[0067] S63, Quantification of ecological contribution: Use linear mixed models to assess the relative contribution of target food groups.

[0068] Beneficial effects of the present invention:

[0069] This study incorporates a comprehensive pipeline encompassing the collection of Antarctic krill gastrointestinal contents, cryoanatomy, nucleic acid extraction, library construction and sequencing, data quality control, species annotation, and network analysis. This streamlines sample collection and cryoprotection while balancing nucleic acid integrity and efficient extraction, ensuring high precision and coverage from sample to sequencing library. Multi-level sequence alignment and LCA classification algorithms enable in-depth and precise food source identification at the species annotation level, significantly enhancing the accuracy and comprehensiveness of studies on Antarctic krill's diet composition.

[0070] This method incorporates stringent quality control and redundancy removal mechanisms during high-throughput sequencing and subsequent data processing, including filtering for adapter contamination, trimming low-quality sequences, and eliminating contaminants from the Antarctic eDNA database. This ensures high sequencing data quality and specificity. Non-redundant ASV / OTU tables generated through VSEARCH clustering minimize duplication noise. After species annotation, suspicious or conflicting sequences are verified for geographic and biological distribution using authoritative databases such as SCAR-EcoFoodWeb and AntEco v2.1, forming a multivariate cross-validation system to ensure the reliability and traceability of the final conclusions.

[0071] This paper not only uses the maximum likelihood phylogenetic tree to perform phylogenetic hierarchical analysis of the species composition information of Antarctic krill food residues, but also integrates it into the SCAR-EcoFoodWeb ecological model. Through network topology construction, trophic level index calculation and diversity quantification, it achieves an in-depth analysis of the feeding structure and nutrient transfer efficiency of Antarctic krill. Further combined with environmental DNA tracing and geographic information tracing, it can accurately locate the ecological contribution rate of key food sources, assist in evaluating the impact of marine environmental changes on the food source of Antarctic krill and the balance of the Antarctic ecosystem, and has important scientific research and conservation management value. BRIEF DESCRIPTION OF THE DRAWINGS

[0072] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only for the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0073] Figure 1 Schematic diagram of a method flow in an embodiment of the present invention;

[0074] Figure 2 Schematic diagram of the S1 process of an embodiment of the present invention. DETAILED DESCRIPTION

[0075] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments. It is also noted that, to provide a more detailed description, the following embodiments are best and preferred embodiments, and those skilled in the art may employ alternative methods for implementing certain known technologies. Furthermore, the accompanying drawings are intended only to provide a more detailed description of the embodiments and are not intended to limit the present invention.

[0076] like Figure 1-Figure 2 As shown, a method for analyzing food residues in the gastrointestinal tract of Antarctic krill using molecular sequencing technology comprises the following steps:

[0077] S1. Sample collection and preservation: Antarctic krill individuals are captured from the target sea area, rapidly dissected in a low-temperature environment (below -20°C), and gastrointestinal contents are collected. These samples are then fixed using a nucleic acid-free preservation reagent to ensure the integrity of the DNA and RNA in the gastrointestinal contents.

[0078] S2, Nucleic Acid Extraction and Quality Testing: Total DNA and RNA were extracted from the gastrointestinal contents using a nucleic acid extraction kit. The purity and integrity of the total DNA and RNA were tested using fluorescence quantification and gel electrophoresis to ensure suitability for subsequent library construction and sequencing.

[0079] S3, PCR amplification and sequencing library construction: Based on the known taxonomic information of the food source, specific primers are designed to perform PCR amplification on total DNA and total RNA to obtain amplified sequences, including the target regions of nuclear and mitochondrial genes;

[0080] Barcode tags are added to the amplified sequences to distinguish different samples. Sequencing library construction reagents are used to perform adapter ligation and fragmentation on the amplified sequences to construct high-throughput sequencing libraries. Fragment quality assessment is performed to ensure the uniformity of the high-throughput sequencing libraries.

[0081] S4, high-throughput sequencing and data quality control: Paired-end sequencing of the high-throughput sequencing library was performed using the Illumina NovaSeq high-throughput sequencing platform to obtain high-quality sequencing data covering the target gene region;

[0082] Perform quality control on sequencing data, including removing low-quality sequences and adaptor-contaminated sequences, and performing redundancy processing on the sequencing data after quality control to obtain valid sequencing data;

[0083] S5, Sequence alignment and species annotation: Species identification was performed on valid sequencing data using alignment tools and databases, and species composition data of gastrointestinal food residues were constructed based on classification tree analysis;

[0084] Based on species composition data and combined with the Antarctic ecosystem food web model, network analysis methods were used to analyze the trophic level distribution and food source diversity of gastrointestinal food residues;

[0085] S6, Food source tracing: Verify the source of species composition data of gastrointestinal food residues through environmental DNA (eDNA) comparison analysis and evaluate the ecological contribution of specific food populations.

[0086] S1 includes:

[0087] S11, Live Capture and Pre-processing: Live Antarctic krill samples are captured using a plankton net vertical trawling system in the target Antarctic waters. The samples are immediately transferred to a pre-cooled insulated box and transported to the deck laboratory within 15 minutes. The details include:

[0088] Trawling was carried out using a plankton net with a pore size of 500 μm and a trawling depth of 0–200 m;

[0089] The captured live krill samples were placed in a stainless steel insulated box (-80°C) pre-cooled with liquid nitrogen and filled with sterile ice packs to prevent temperature fluctuations;

[0090] S12, establishment of a cryogenic dissection environment: A sterile ice bed is laid in an ISO 5 clean bench, the temperature controller is set to -25±2°C, and liquid nitrogen vapor is continuously introduced to maintain a low-temperature environment in the operating area. Specifically, the following steps are required:

[0091] A liquid nitrogen vapor generator (flow rate 2 L / min) was used to maintain temperature stability within the workbench;

[0092] The operating table surface is covered with sterile aluminum foil and pre-cooled to below -20°C;

[0093] S13, sterile dissection procedure: Using DNase / RNase-inactivated titanium dissection tools, dissect the carapace along the dorsal midline of the Antarctic krill, completely remove the gastrointestinal tissue, and place it in a pre-chilled sterile culture dish. Specific procedures include:

[0094] After processing each sample, the dissection tools were immersed in 0.1% DEPC aqueous solution for 5 minutes to inactivate;

[0095] The weight of the excised gastrointestinal tissue was controlled within the range of 10-50 mg to avoid spillage of the contents;

[0096] S14, preservation reagent permeation treatment: immersing the gastrointestinal tissue in a 4°C pre-cooled preservation solution and shaking the gastrointestinal contents to fully release them, thereby obtaining a gastrointestinal mixed suspension, specifically comprising:

[0097] The preservation solution consisted of the following components: 2% guanidine thiocyanate, 25 mM disodium EDTA, 1% sodium lauryl sarcosinate, and 0.5 M Tris-HCl buffer (pH 8.0). The volume ratio of the preservation solution to the gastrointestinal tissue was 10:1 (1 mL of the preservation solution was used to treat 100 mg of tissue).

[0098] After shaking at 200 rpm for 30 minutes, the container was allowed to stand for 5 minutes to allow the undissolved shell fragments to settle to the bottom of the container.

[0099] S15, solid-liquid separation and packaging: Filter the obtained gastrointestinal mixed suspension through a 50 μm nylon filter membrane to obtain a filtrate, and dispense the filtrate into 2 mL cryovials. The filling volume of each tube should not exceed 1.5 mL to prevent the cryovial from bursting. Obtain the sample of the packaged filtrate, specifically including:

[0100] Before filtration, pre-cool the nylon filter membrane to 4°C. Immediately after filtration, quickly freeze the filtrate (i.e., the liquid portion obtained in this step) with liquid nitrogen.

[0101] Label each 2 mL cryovial with the batch number and collection time (accurate to the minute).

[0102] S16, Gradient Cryopreservation: The aliquoted filtrate samples were stored at -20°C for 2 hours, then transferred to -80°C for 12 hours, and finally placed in liquid nitrogen vapor phase for long-term storage, specifically including:

[0103] When storing in liquid nitrogen vapor phase, cryovials should be placed in stainless steel sleeves to avoid direct contact with liquid nitrogen;

[0104] The temperature transition rate is controlled to drop by 1°C per minute, forming a gradual cooling to protect the integrity of nucleic acids;

[0105] S17, metadata association record: Use QR code labels to mark the cryopreservation tubes after gradient freezing, and write the sample collection location (latitude and longitude), seawater temperature (-1.8°C to 2.5°C) and krill body length (30-60 mm) information into the blockchain evidence storage system, including:

[0106] The blockchain uses the Hyperledger Fabric framework, and the data hash value is synchronized to the Antarctic Research Data Center in real time;

[0107] The QR code label is made of low-temperature resistant polyimide and can withstand extreme environments of -196°C to 100°C.

[0108] S2 includes:

[0109] S21, sample pretreatment and lysis: transfer the aliquoted filtrate samples to -20°C for equilibration for 2 hours, and then thaw them in a 4°C refrigerator for 1 hour;

[0110] Open the cryovial in an ultra-clean workbench (ISO Class 5), add lysis buffer (pH 7.5, containing 20 mM Tris-HCl, 2% CTAB, 0.8 M NaCl) containing 1% β-mercaptoethanol, and place 3 mm diameter zirconium oxide beads in a liquid nitrogen pre-cooled grinder. Grind at 1800 rpm for 3 minutes to obtain a homogenized lysate. Specifically, the following steps are included:

[0111] Thawing was performed in a refrigerated environment at 4°C for 2 ± 0.5 h to reduce nucleic acid degradation.

[0112] The volume ratio of lysis buffer to sample was 5:1 (5 mL of buffer for 1 mL of filtrate) to ensure adequate lysis and cell disruption.

[0113] S22, complex nucleic acid adsorption purification: transfer the homogenized lysate to a DNA / RNA co-extraction spin column, add equal volumes of pre-chilled chloroform-isoamyl alcohol (24:1) and shake to mix thoroughly. Centrifuge at 12,000 × g for 15 minutes (4°C). Combine the supernatant with silica-based magnetic beads for 10 minutes, then apply a magnetic field for separation and discard the waste liquid. Specifically, the following steps are performed:

[0114] The centrifugal column should be pre-cooled to 4°C and the chloroform-isoamyl alcohol mixture should be prepared and used immediately to avoid component decomposition;

[0115] The particle size of the silica-based magnetic beads is 50 nm, and the binding time is accurate to 10±0.5 minutes to ensure the directional adsorption efficiency of nucleic acids.

[0116] S23, step-by-step elution and concentration: Wash the magnetic beads twice with 80% ethanol containing 10 mM EDTA, then elute the total DNA with nuclease-free water preheated to 35°C (elution volume 50 μL). Then, enrich the total RNA using 70% ethanol precipitation and dissolve it in 20 μL of DEPC water to obtain purified total DNA / total RNA. Specifically,

[0117] The pH value of the DNA elution buffer was 8.5, and the RNA elution buffer contained 1 mM DTT to protect the RNA structure;

[0118] After ethanol precipitation, centrifugation was performed at 12,000 × g, 4°C, for 15 min to ensure sufficient RNA precipitation;

[0119] S24, double-strand integrity verification: Use a Qubit 4.0 fluorometer to measure total DNA concentration (dsDNA HS channel) and total RNA concentration (RNA HS channel). Perform electrophoresis analysis on an Agilent 2100 bioanalyzer to assess the integrity of total DNA and total RNA. Select nucleic acid samples that meet the following criteria:

[0120] DNA integrity index (DIN) ≥7.0 and 28S / 18S rRNA ratio ≥1.8;

[0121] The RNA showed no degradation peaks and the RIN value was ≥8.0.

[0122] S25, antifreeze protein removal: Add 0.1 U / μL Antarctic krill antifreeze protease (AFP-III) to the total DNA solution. Incubate at 37°C for 30 minutes, and then use AMPure XP magnetic beads to remove the enzymatic products. Specifically,

[0123] The enzymatic reaction system contained 5 mM CaCl2 to activate protease activity and prevent residual antifreeze proteins from affecting sequencing;

[0124] After magnetic bead purification, the elution volume was 30 μL (10 mM Tris-HCl, pH 8.0) to ensure appropriate DNA concentration and purity.

[0125] S26, Aliquoting and freezing: Aliquot the qualified nucleic acid samples into pre-cooled low-adsorption EP tubes, label them with batch codes, and store them in a -80°C ultra-low temperature freezer. At the same time, upload the electrophoresis patterns and concentration data to the laboratory information management system. These samples are defined as qualified nucleic acid samples, including:

[0126] The EP pipe is made of polypropylene and pre-cooled to -20°C to reduce high and low temperature shock;

[0127] The LIMS system automatically generates a sample QC report (PDF format) containing DIN, RIN, and concentration data for subsequent query and traceability.

[0128] S3 includes:

[0129] S31, Food source classification information summary: Collect and organize references and database information related to Antarctic krill feeding targets, identify common or high-proportion potential food source species, determine the conserved regions of their nuclear and mitochondrial gene sequences, and construct an Antarctic target gene library to provide key references for primer design;

[0130] S32, Primer Design and Validation: Based on conserved regions, use bioinformatics software to predict the specificity and annealing temperature of candidate primer sequences. Select primer pairs that meet the amplification efficiency and specificity requirements. Perform in vitro primer validation and conduct small-scale PCR tests using target templates and negative controls to ensure that the primers have good amplification efficiency while excluding non-target amplification. Specific details include:

[0131] Use qualified nucleic acid samples packaged in S26 as templates;

[0132] Three redundant G bases were added to the 5′ end of the primer to compensate for the GC shift of the Antarctic diatom rbcL gene, and the annealing temperature was set at 58 ± 2°C;

[0133] S33, PCR reaction system configuration: Weigh the qualified nucleic acid sample packaged in S26, add specific primers, DNA polymerase, dNTP mixture, buffer, and the required magnesium ion solution, and adjust the concentration and volume of each component to ensure that the PCR reaction system is in optimal amplification conditions to avoid over-amplification or primer dimer formation. Specifically, include:

[0134] Prepare 10× antifreeze buffer (pH 8.3) containing 1.5 M betaine by mixing the following proportions:

[0135] Antifreeze buffer 5 μL;

[0136] dNTPs (2.5 mM each) 4 μL;

[0137] Antarctic-specific primer pair (10 μM), 1 μL each;

[0138] Hot start Taq enzyme (5 U / μL) 0.5 μL;

[0139] cDNA / DNA template (≥5 ng / μL) 2 μL;

[0140] MgCl2 (25 mM) 3 μL;

[0141] Make up to 50 μL with DEPC water;

[0142] S34, Amplification Cycles and Preliminary Testing: Set the temperature and time for the pre-denaturation, annealing, and extension steps, and set the number of cycles to effectively amplify the target fragments of nuclear and mitochondrial genes. Detect the size and specificity of the PCR reaction products by agarose gel electrophoresis to confirm that clear bands are generated in the expected segments. Define the product as the "amplified sequence." Specific details include:

[0143] Run the program: 95°C initial denaturation for 5 minutes → 35 cycles (95°C for 30 seconds → 58°C for 30 seconds → 72°C for 45 seconds) → final extension at 72°C for 10 minutes;

[0144] Use nucleic acid samples that have passed S24 verification as positive controls;

[0145] S35, amplified sequence purification: Use column purification or magnetic bead purification methods to remove enzyme, primer residues, and other impurities to obtain high-purity amplified sequences. Perform fluorescence quantitative measurement on the purified amplified sequences to obtain accurate concentration and quality information, providing a basis for subsequent barcode labeling and library construction. Specifically, it includes:

[0146] Purification was performed using S25 AMPure XP magnetic beads (bead:sample volume ratio 0.8:1);

[0147] The concentration (≥10 ng / μL) was determined using the Qubit 4.0 assay with an A260 / A280 ratio of ≥1.8.

[0148] S3 also includes:

[0149] S36, Dual-index barcoding: Aliquot the purified amplicon into separate reaction tubes and use the Nextera XT Index Kit to add a unique i5 / i7 dual-index combination (8 bp) to each sample, including:

[0150] Index Add Reaction System:

[0151] 5 μL of purified amplified sequence (≥10 ng / μL);

[0152] i5 index primer (10 μM) 1 μL;

[0153] i7 index primer (10 μM) 1 μL;

[0154] T4 DNA ligase (5 U / μL) 0.5 μL;

[0155] 10× ligation buffer (containing 0.5 M NaCl) 2 μL;

[0156] Make up to 20 μL with DEPC water;

[0157] Reaction conditions: incubation at 20°C for 15 minutes, followed by inactivation at 65°C for 10 minutes;

[0158] S37, Adapter Cold Ligation: Ligate Illumina TruSeq adapters to the dual-indexed labeled products from S36, including:

[0159] Ligation reaction system:

[0160] 15 μL of double-indexed labeled product;

[0161] TruSeq adaptor (15 μM) 2 μL;

[0162] Antarctic optimized ligase (3 U / μL) 1 μL;

[0163] 5× low-temperature ligation buffer (containing 1 M betaine) 4 μL;

[0164] Make up to 30 μL with DEPC water;

[0165] Reaction conditions: 16°C for 20 minutes, aptamer to labeled product molar ratio 1:1;

[0166] S38, antifreeze fragmentation treatment: The ligation product of S37 was fragmented using a Covaris S220 sonicator, specifically including:

[0167] Fragmentation parameters:

[0168] Peak energy: 5 W;

[0169] Action time: 45 seconds;

[0170] Number of cycles: 200 times;

[0171] Pre-cooling of reaction tubes: Pre-cooling with liquid nitrogen for 30 seconds to maintain an operating environment at 4°C;

[0172] Target fragment length: 350 ± 50 bp;

[0173] S39, Antarctic library purification: Fragmentation products were purified using AMPure XP magnetic beads (bead:sample volume ratio 0.8:1) and screened for 300-500 bp target fragments using the BluePippin system, including:

[0174] Purification conditions:

[0175] Washing solution: 80% ethanol (containing 10 mM EDTA, pH 8.0);

[0176] Eluent: 10 mM Tris-HCl (pH 8.5);

[0177] Elution volume: 30 μL;

[0178] Screening parameters: BluePippin selected 2% Agarose Cassette, cutoff range 300-500 bp;

[0179] S40, library normalization and quality control: The purified library was quantified using Qubit 4.0 and the fragment distribution was detected using Agilent 4200 TapeStation, including:

[0180] Homogenization standard:

[0181] Library concentration: 4 nM ± 5% (error range);

[0182] The main peak proportion is ≥90%, and the CV value is ≤10%;

[0183] The proportion of aptamers is <1%;

[0184] Quality inspection failure processing: If the quality inspection fails to meet the standards, repeat steps S38-S39 to adjust the fragmentation parameters.

[0185] S4 includes:

[0186] S41, library dilution and denaturation: Dilute the high-throughput sequencing library that has passed quality inspection to 1.8 pM with 0.1 N NaOH solution, add 5% PhiX Control v3 library as an internal reference, denature at 95°C for 5 minutes, and immediately cool in an ice bath. Specifically, the following steps are performed:

[0187] Diluent: HT1 buffer containing 0.1% Tween-20 (Illumina);

[0188] Internal reference ratio: 5% PhiX is used to monitor sequencing error rate.

[0189] S42, sequencing chip loading: Load the denatured library into the designated lane of the Illumina NovaSeq 6000 S4 flow cell, including:

[0190] Loading volume: 600 μL per lane (concentration 1.8 pM);

[0191] Loading temperature: Maintain the flow cell temperature at 20 ± 0.5 °C.

[0192] S43, paired-end sequencing run: Start the NovaSeq 6000 system and run a 2 × 150 bp paired-end sequencing program, including:

[0193] Sequencing parameters:

[0194] Read1 length: 150 cycles;

[0195] Index1 length: 8 cycles (read i7 index);

[0196] Index2 length: 8 cycles (read i5 index);

[0197] Read2 length: 150 cycles;

[0198] Quality monitoring: Real-time tracking of cluster density (target range: 200-300 K / mm²).

[0199] S44, Raw Data Generation and Preliminary Quality Control: After sequencing is complete, the raw data is exported in FASTQ format through the Illumina BaseSpace platform and basic filtering is performed, including:

[0200] Filter by:

[0201] Sequences with N content > 5% in reads were removed;

[0202] Reads with a mean Phred quality score ≥ 30 were retained;

[0203] Output file: Paired-end sequencing raw data (file naming: SampleID_R1.fastq.gz / SampleID_R2.fastq.gz).

[0204] The S4 also includes:

[0205] S45, adapter contamination filtering: Use Cutadapt (v3.4) to remove Illumina adapter residual sequences from the raw data and output the decontaminated sequencing data, including:

[0206] Parameter settings:

[0207] Maximum error rate: 0.1;

[0208] Minimum match length: 5 bp;

[0209] Adaptamer sequence: TruSeq Universal / Indexed aptamer;

[0210] Output file: decontaminated sequencing data.

[0211] S46, low-quality sequence trimming: Trimmomatic (v0.39) is used to trim the quality of the decontaminated sequencing data and output quality-controlled trimmed data, including:

[0212] Pruning rules:

[0213] Sliding window: 4 bp window, average quality ≥ 20;

[0214] Remove bases with the first and last quality <Q20;

[0215] Minimum retention length: 50 bp;

[0216] Output file: QC trimmed data.

[0217] S47, Antarctic contamination sequence removal: The quality control trimmed data were aligned with the Antarctic eDNA contamination database (AntEco v2.1), and exogenous contamination sequences were removed using Bowtie2 (v2.4.4). Unaligned reads were retained and defined as Antarctic-specific sequencing data, including:

[0218] Comparison parameters:

[0219] Mode: --very-sensitive

[0220] Maximum mismatch: 3

[0221] Unaligned reads were retained and defined as Antarctic-specific sequencing data.

[0222] S48, redundancy removal: Use VSEARCH (v2.18.0) to perform redundancy clustering on the Antarctic-specific sequencing data and output valid sequencing data, including:

[0223] Parameter settings:

[0224] Similarity threshold: 97%;

[0225] Maximum allowed chimerism rate: 1%;

[0226] Reference database: Antarctic target gene library of S31;

[0227] Output file: valid sequencing data (non-redundant ASV / OTU table).

[0228] S5 includes:

[0229] S51, multi-level sequence alignment: Perform BLAST comparison (e-value ≤ 1e-5) on the output valid sequencing data with the Antarctic target gene library, and simultaneously align to the NCBI nt library to supplement the unannotated sequences, including:

[0230] Alignment tools: BLASTn (v2.12.0) for nuclear genes and BLASTx (v2.12.0) for mitochondrial genes;

[0231] Parameter settings:

[0232] Nuclear genes (18S / ITS2): maximum number of matches 100, coverage ≥ 90%;

[0233] Mitochondrial gene (COI / 16S): consistency ≥97%;

[0234] S52, LCA taxonomic assignment: Assign species taxonomy using the lowest common ancestor (LCA) algorithm of MEGAN6 (v6.21), specifically:

[0235] Classification threshold:

[0236] Phylum / class level: supporting read number ≥ 5 and relative abundance > 0.1%;

[0237] Genus / species level: supporting read number ≥ 10 and consistency ≥ 99%;

[0238] Eliminate species that do not match Antarctic biogeographic distributions (e.g. tropical plankton);

[0239] S53, classification tree construction: Based on the alignment results, RAxML (v8.2.12) was used to construct a maximum likelihood phylogenetic tree, including:

[0240] Model selection: GTR+GAMMA model (suitable for highly variable genes in Antarctica);

[0241] Bootstrap value: 1000 repetitions, node support rate ≥ 70%;

[0242] Output file: Antarctic food source phylogenetic tree (Newick format);

[0243] S54, species composition data generation: Integrate the classification results with the phylogenetic tree topology, count the read abundance by classification level, and generate the species composition matrix of Antarctic krill food residues, including:

[0244] Matrix dimensions: sample (row) × species (column), value is the normalized number of reads (CPM);

[0245] Data format: CSV file (including species classification information and confidence scores).

[0246] The S5 also includes:

[0247] S55, food web model loading: call the Antarctic ecosystem food web model from the SCAR-EcoFoodWeb database and integrate the species composition matrix, including:

[0248] Model parameters:

[0249] Basic trophic level data: phytoplankton (level 1), krill (level 2), penguins / cetaceans (level 3);

[0250] Energy transfer efficiency: 10% per trophic level;

[0251] S56, Network topology construction: Use Cytoscape (v3.9.1) to construct a feeding relationship network, including:

[0252] Node definition:

[0253] Food source node: species in the S54 matrix;

[0254] Krill node: central node connecting all ingested species;

[0255] Edge weight: log10-transformed value of normalized read abundance (CPM);

[0256] S57, trophic level index calculation: Calculate the trophic level position of each food source based on network topology, specifically including:

[0257] Calculation formula:

[0258] ;

[0259] in, is the trophic level of species i, is the feeding weight of krill on species j;

[0260] Iteration convergence condition: trophic level change < 0.01 or maximum number of iterations 50 times;

[0261] S58, Diversity Quantification and Visualization: The Shannon-Wiener index and Simpson index were used to quantify the diversity of food sources, and network heat maps were generated using Gephi (v0.9.7), including:

[0262] Index formula:

[0263] Shannon-Wiener: ;

[0264] Simpson: ;

[0265] in, is the relative abundance of species i;

[0266] Visual output: Antarctic krill feeding ecology map (PDF / PNG format).

[0267] S6 includes:

[0268] S61, Environmental DNA Sequence Matching: Cross-match the obtained valid sequencing data with the Antarctic Environmental DNA Contamination Database (AntEco v2.1) and the generated species composition matrix, including:

[0269] Comparison tool: VSEARCH (v2.18.0), parameter settings:

[0270] Similarity threshold: 97%;

[0271] Minimum match length: 100 bp;

[0272] Eliminate sequences that match non-Antarctic sources such as humans and ship equipment;

[0273] Output file: Antarctic local food source sequence collection;

[0274] S62, Geographical traceability analysis: Based on the collection location information stored in the S17 blockchain, the environmental DNA reference data of the corresponding sea area is extracted from the SCAR-MarBIN geographic database to build an Antarctic regional environmental DNA traceability database, specifically including:

[0275] Data Integration:

[0276] Matching latitude range: ±0.5° (centered on the collected longitude and latitude);

[0277] Matching depth range: ±50 meters (centered on the trawl depth);

[0278] Analytical methods:

[0279] The Bray-Curtis similarity index was calculated to quantify the consistency of species composition between krill food residues and local eDNA;

[0280] SIMPER analysis (similarity percentage decomposition) was used to identify key contributing species;

[0281] S63, Quantification of Ecological Contribution: Use linear mixed models to assess the relative contribution of target food groups, including:

[0282] Model parameters:

[0283] Fixed effects: species abundance (CPM), trophic level position (TL);

[0284] Random effects: collection area, season (from S17 metadata);

[0285] Output:

[0286] Key food source contribution rate report (CSV / PDF format), marking the ecological weight of species such as diatoms and copepods;

[0287] A heat map of Antarctic krill feeding traceability was generated (R ggplot2, PDF resolution ≥ 300 dpi).

[0288] The present invention encompasses any alternatives, modifications, equivalents, and solutions that fall within the spirit and scope of the present invention. To provide a thorough understanding of the present invention, specific details are described in detail in the following preferred embodiments of the present invention, but those skilled in the art will be able to fully understand the present invention without these detailed descriptions. Furthermore, to avoid unnecessary confusion regarding the essence of the present invention, well-known methods, processes, procedures, components, and circuits have not been described in detail.

[0289] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for analyzing food residues in the gastrointestinal tract of Antarctic krill using molecular sequencing technology, characterized in that: The following steps are involved: S1, sample collection and preservation: Antarctic krill individuals are captured from the target sea area, rapidly dissected in a low-temperature environment, gastrointestinal contents are collected, and the samples are fixed using a nucleic acid-free preservation reagent. S1 includes: S11, live capture and pre-processing: Live Antarctic krill samples were captured using a plankton net vertical trawling system in the target Antarctic waters, transferred to pre-cooled insulated boxes, and transported to the deck laboratory; S12, establishment of low-temperature dissection environment: a sterile ice bed is laid in an ISO 5 clean bench, and liquid nitrogen vapor is continuously introduced to maintain the low-temperature environment in the operating area; S13, aseptic dissection procedure: using inactivated titanium dissection tools, the thorax of the Antarctic krill was dissected along the dorsal midline, and the gastrointestinal tissue was completely removed and placed in a pre-cooled sterile culture dish; S14, preservation agent permeation treatment: immersing the gastrointestinal tissue in a pre-cooled preservation solution and shaking the solution to fully release the gastrointestinal contents, thereby obtaining a gastrointestinal mixed suspension; S15, solid-liquid separation and subpackaging: filtering the obtained gastrointestinal mixed suspension through a nylon filter membrane to obtain a filtrate, and subpackaging the filtrate into cryopreservation tubes to obtain subpacked filtrate samples; S16, gradient freezing storage: the aliquoted filtrate samples were sequentially placed in a -20°C environment for 2 hours, then transferred to a -80°C environment for 12 hours, and finally placed in liquid nitrogen vapor phase for long-term storage; S17, metadata association record: Use QR code labels to mark the cryovials that have been stored in gradient freezing, and write the sample collection location, seawater temperature, and krill body length information into the blockchain evidence storage system; S2, nucleic acid extraction and quality testing: extracting total DNA and total RNA from the gastrointestinal contents using a nucleic acid extraction kit, and detecting the purity and integrity of the total DNA and total RNA using fluorescence quantification and gel electrophoresis; S3, PCR amplification and sequencing library construction: Based on the known taxonomic information of the food source, specific primers are designed to perform PCR amplification on the total DNA and total RNA to obtain amplified sequences, which include target regions of nuclear genes and mitochondrial genes; Adding barcode tags to the amplified sequences to distinguish different samples, and performing adaptor ligation and fragmentation on the amplified sequences using sequencing library construction reagents to construct a high-throughput sequencing library, and ensuring the uniformity of the high-throughput sequencing library by fragment quality assessment; S4, high-throughput sequencing and data quality control: using the Illumina NovaSeq high-throughput sequencing platform to perform paired-end sequencing on the high-throughput sequencing library to obtain sequencing data covering the target gene region; Performing quality control on the sequencing data, including removing low-quality sequences and adaptor-contaminated sequences, and performing redundancy removal on the quality-controlled sequencing data to obtain valid sequencing data; S5, sequence alignment and species annotation: use alignment tools and databases to identify species of the effective sequencing data, and construct species composition data of gastrointestinal food residues based on classification tree analysis; The S5 includes: S51, multi-level sequence alignment: perform BLAST comparison on the output valid sequencing data and the Antarctic target gene library; S52, LCA taxonomic assignment: assign species taxonomy using the lowest common ancestor algorithm of MEGAN6; S53, classification tree construction: Based on the alignment results, RAxML was used to construct a maximum likelihood phylogenetic tree; S54, species composition data generation: integrating classification results with phylogenetic tree topology, counting read abundance by taxonomic level, and generating a species composition matrix of Antarctic krill food residues; Based on the species composition data and combined with the Antarctic ecosystem food web model, network analysis methods were used to analyze the trophic level distribution and food source diversity of gastrointestinal food residues; S6, Food Source Traceability: Verify the source of species composition data of gastrointestinal food residues through environmental DNA comparison analysis and assess the ecological contribution of specific food groups; The S6 includes: S61, Environmental DNA sequence matching: cross-matching the obtained valid sequencing data with the Antarctic environmental DNA pollution database and the generated species composition matrix; S62, Geographical traceability analysis: Based on the collection location information stored in the S17 blockchain, environmental DNA reference data for the corresponding sea area is extracted from the SCAR-MarBIN geographic database to construct an Antarctic regional environmental DNA traceability database; S63, Quantification of ecological contribution: Use linear mixed models to assess the relative contribution of target food groups.

2. The method for analyzing food residues in the gastrointestinal tract of Antarctic krill using molecular sequencing technology according to claim 1, characterized in that: The S2 includes: S21, sample pretreatment and lysis: transfer the aliquoted filtrate samples to -20°C for equilibration for 2 hours, and then thaw them in a 4°C refrigerator for 1 hour; Open the cryotube in a clean bench, add lysis buffer containing 1% β-mercaptoethanol, place zirconium oxide beads in a grinder pre-cooled with liquid nitrogen, and grind for 3 minutes to obtain a homogenized lysate; S22, complex nucleic acid adsorption purification: transfer the homogenized lysate to a DNA / RNA co-extraction spin column, add equal volumes of pre-chilled chloroform-isoamyl alcohol, shake and mix thoroughly, centrifuge for 15 minutes, collect the supernatant and bind it to silica-based magnetic beads for 10 minutes, then apply a magnetic field for separation and discard the waste liquid; S23, stepwise elution and concentration: first wash the magnetic beads twice with ethanol, then elute the total DNA with preheated nuclease-free water, then enrich the total RNA by ethanol precipitation and dissolve it in DEPC water to obtain purified total DNA / total RNA; S24, double-strand integrity verification: the total DNA concentration and total RNA concentration were measured using a fluorescence quantitative instrument, and electrophoresis analysis was performed using a bioanalyzer to evaluate the integrity of the total DNA and total RNA; S25, antifreeze protein removal: Antarctic krill antifreeze protease was added to the total DNA solution and incubated for 30 minutes, and then the enzymatic products were purified using AMPureXP magnetic beads; S26, aliquot and freeze: The verified qualified nucleic acid samples are aliquoted into pre-cooled low-adsorption EP tubes, marked with batch codes, and stored in ultra-low temperature refrigerators. At the same time, the electrophoresis patterns and concentration data are uploaded to the laboratory information management system and defined as qualified nucleic acid samples.

3. The method for analyzing food residues in the gastrointestinal tract of Antarctic krill using molecular sequencing technology according to claim 2, characterized in that: The S3 includes: S31, Food source classification information summary: Collect and organize references and database information related to Antarctic krill feeding targets, identify common or high-proportion potential food source species, determine the conserved regions of their nuclear and mitochondrial gene sequences, and construct an Antarctic target gene library to provide key references for primer design; S32, Primer Design and Validation: Based on the conserved regions, use bioinformatics software to predict the specificity and annealing temperature of candidate primer sequences, select primer pairs that meet the amplification efficiency and specificity requirements, validate the primers in vitro, and perform small-scale PCR tests using target templates and negative controls; S33, PCR reaction system configuration: Weigh the qualified nucleic acid sample packaged in S26, add specific primers, DNA polymerase, dNTP mixture, buffer, and the required magnesium ion solution, and adjust the concentration and volume of each component to ensure that the PCR reaction system is under optimal amplification conditions; S34, Amplification Cycles and Preliminary Testing: Set the temperature and time for the initial denaturation, annealing, and extension steps, and set the number of cycles to effectively amplify the target nuclear and mitochondrial gene fragments. Detect the size and specificity of the PCR reaction products by agarose gel electrophoresis to confirm that a clear band is generated in the expected segment. This product is defined as the "amplified sequence." S35, amplified sequence purification: Use column purification or magnetic bead purification methods to remove enzymes, primer residues and other impurities to obtain high-purity amplified sequences. Perform fluorescence quantitative measurement on the purified amplified sequences to obtain accurate concentration and quality information.

4. The method for analyzing food residues in the gastrointestinal tract of Antarctic krill using molecular sequencing technology according to claim 3, characterized in that: Said S3 further comprises: S36, dual-index barcoding: The purified amplicon sequences were aliquoted into separate reaction tubes, and a unique i5 / i7 dual-index combination was added to each sample using the Nextera XT Index Kit. S37, adapter cryo-ligation: ligating Illumina TruSeq adapters to the dual-indexed labeled products of S36; S38, antifreeze fragmentation treatment: the ligation product of S37 was fragmented using a Covaris S220 sonicator; S39, Antarctic library purification: The fragmentation product was purified using AMPure XP magnetic beads and screened for 300-500 bp target fragments using the BluePippin system; S40, library normalization and quality control: The purified library was quantified using Qubit 4.0, and the fragment distribution was detected using Agilent 4200 TapeStation.

5. The method for analyzing food residues in the gastrointestinal tract of Antarctic krill using molecular sequencing technology according to claim 4, characterized in that: The S4 includes: S41, library dilution and denaturation: Dilute the high-throughput sequencing library that has passed the quality inspection to 1.8 pM with 0.1 N NaOH solution, add 5% PhiX Control v3 library as an internal reference, denature at 95°C for 5 minutes, and cool in an ice bath; S42, sequencing chip loading: load the denatured library into the designated lane of the Illumina NovaSeq 6000 S4 flow cell; S43, paired-end sequencing run: Start the NovaSeq 6000 system and run a 2 × 150 bp paired-end sequencing program; S44, raw data generation and preliminary quality control: After sequencing is completed, the raw data in FASTQ format are exported through the Illumina BaseSpace platform and basic filtering is performed.

6. The method for analyzing food residues in the gastrointestinal tract of Antarctic krill using molecular sequencing technology according to claim 5, characterized in that: Said S4 further comprises: S45, adapter contamination filtering: Use Cutadapt to remove Illumina adapter residual sequences from the raw data and output the decontaminated sequencing data; S46, low-quality sequence trimming: Trimmomatic is used to perform quality trimming on the decontaminated sequencing data and output quality-controlled trimmed data; S47, Antarctic contamination sequence removal: The quality control trimmed data were compared with the Antarctic eDNA contamination database, and exogenous contamination sequences were removed using Bowtie2. Unaligned reads were retained and defined as Antarctic-specific sequencing data; S48, de-redundancy processing: Use VSEARCH to perform de-redundancy clustering on the Antarctic-specific sequencing data and output valid sequencing data.

7. The method for analyzing food residues in the gastrointestinal tract of Antarctic krill using molecular sequencing technology according to claim 6, characterized in that: The S5 further includes: S55, food web model loading: calling the Antarctic ecosystem food web model from the SCAR-EcoFoodWeb database and integrating the species composition matrix; S56, network topology construction: construct feeding relationship network using Cytoscape; S57, trophic level index calculation: Calculate the trophic level position of each food source based on network topology; S58, Diversity Quantification and Visualization: The Shannon-Wiener index and Simpson index were used to quantify the diversity of food sources, and a network heat map was generated using Gephi.

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