High-throughput Sequencing Approach Research Articles

Identification and Characterization of Citrus Concave Gum-Associated Virus Infecting Citrus and Apple Trees by Serological, Molecular and High-Throughput Sequencing Approaches.

Citrus concave gum-associated virus (CCGaV) is a negative-stranded RNA virus, first reported a few years ago in citrus trees from Italy. It has been reported in apple trees in the USA and in Brazil, suggesting a wider host range and geographic distribution. Here, an anti-CCGaV polyclonal antiserum to specifically detect the virus has been developed and used in a standard double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) that has been validated as a sensitive and reliable method to detect this virus both in citrus and apple trees. In contrast, when the same antiserum was used in direct tissue-blot immunoassay, CCGaV was efficiently detected in citrus but not in apple. Using this antiserum, the first apple trees infected by CCGaV were identified in Italy and the presence of CCGaV in several apple cultivars in southern Italy was confirmed by field surveys. High-throughput sequencing (HTS) allowed for the assembling of the complete genome of one CCGaV Italian apple isolate (CE-c3). Phylogenetic analysis of Italian CCGaV isolates from apple and citrus and those available in the database showed close relationships between the isolates from the same genus (Citrus or Malus), regardless their geographical origin. This finding was further confirmed by the identification of amino acid signatures specific of isolates infecting citrus or apple hosts. Analysis of HTS reads also revealed that the CE-c3 Italian apple tree, besides CCGaV, was simultaneously infected by several viruses and one viroid, including apple rubbery wood virus 2 which is reported for the first time in Italy. The complete or almost complete genomic sequences of the coinfecting agents were determined.

Plankton biodiversity and species co-occurrence based on environmental DNA – a multiple marker study

Metabarcoding in combination with high-throughput sequencing (HTS) allows simultaneous detection of multiple taxa by targeting single or several taxonomically informative gene regions from environmental DNA samples. In this study, a multiple-marker HTS approach was applied to investigate the plankton diversity and seasonal succession in the Baltic Sea from winter to autumn. Four different markers targeting the 16S, 18S, and 28S ribosomal RNA genes were employed, including a marker for more efficient dinoflagellate detection. Typical seasonal changes were observed in phyto- and bacterioplankton communities. In phytoplankton, the appearance patterns of selected common, dominant, or harmful species followed the patterns also confirmed based on 20 years of phytoplankton monitoring data. In the case of zooplankton, both macro- and microzooplankton species were detected. However, no seasonal patterns were detected in their appearance. In total, 15 and 2 new zoo- and phytoplankton species were detected from the Baltic Sea. HTS approach was especially useful for detecting microzooplankton species as well as for investigating the co-occurrence and potential interactions of different taxa. The results of this study further exemplify the efficiency of metabarcoding for biodiversity monitoring and the advantage of employing multiple markers through the detection of species not identifiable based on a single marker survey and/or by traditional morphology-based methods.

Paramyxoviruses from neotropical bats suggest a novel genus and nephrotropism.

Paramyxoviruses have a broad host range and geographic distribution, including human pathogens transmitted by bats, such as Nipah and Hendra viruses. In this study, we combined high-throughput sequencing and molecular approaches to investigate the presence of paramyxoviruses in neotropical bats (Microchiroptera suborder) in Brazil. We discovered and characterized three novel paramyxoviruses in the kidney tissues of apparently healthy common vampire bats (D. rotundus) and Seba's short-tailed bats (C. perspicillata), which we tentatively named Kanhgág virus (KANV), Boe virus (BOEV), and Guató virus (GUATV). In this study, we classified these viruses as putative species into the Macrojêvirus genus, a newly proposed genus of the Orthoparamyxovirinae subfamily. Using RT-PCR, we detected these viruses in 20.9% (9 out of 43) of bats tested, and viral RNA was detected exclusively in kidney tissues. Attempts to isolate infectious virus were successful for KANV and GUATV. Our results expand the viral diversity, host range, and geographical distribution of the paramyxoviruses.

Early assessment of circulating tumor DNA after curative-intent resection predicts tumor recurrence in early-stage and locally advanced non-small-cell lung cancer.

Circulating tumor DNA (ctDNA) has demonstrated great potential as a noninvasive biomarker to assess minimal residual disease (MRD) and profile tumor genotypes in patients with non‐small‐cell lung cancer (NSCLC). However, little is known about its dynamics during and after tumor resection, or its potential for predicting clinical outcomes. Here, we applied a targeted‐capture high‐throughput sequencing approach to profile ctDNA at various disease milestones and assessed its predictive value in patients with early‐stage and locally advanced NSCLC. We prospectively enrolled 33 consecutive patients with stage IA to IIIB NSCLC undergoing curative‐intent tumor resection (median follow‐up: 26.2 months). From 21 patients, we serially collected 96 plasma samples before surgery, during surgery, 1–2 weeks postsurgery, and during follow‐up. Deep next‐generation sequencing using unique molecular identifiers was performed to identify and quantify tumor‐specific mutations in ctDNA. Twelve patients (57%) had detectable mutations in ctDNA before tumor resection. Both ctDNA detection rates and ctDNA concentrations were significantly higher in plasma obtained during surgery compared with presurgical specimens (57% versus 19% ctDNA detection rate, and 12.47 versus 6.64 ng·mL−1, respectively). Four patients (19%) remained ctDNA‐positive at 1–2 weeks after surgery, with all of them (100%) experiencing disease progression at later time points. In contrast, only 4 out of 12 ctDNA‐negative patients (33%) after surgery experienced relapse during follow‐up. Positive ctDNA in early postoperative plasma samples was associated with shorter progression‐free survival (P = 0.013) and overall survival (P = 0.004). Our findings suggest that, in early‐stage and locally advanced NSCLC, intraoperative plasma sampling results in high ctDNA detection rates and that ctDNA positivity early after resection identifies patients at risk for relapse.

A Proposed Framework to Identify Dispensable and Essential Functions in Bifidobacteria: Case Study of Bifidobacterium breve UCC2003 as a Prototype of Its Genus.

Functional genomics of bacteria commonly aims at establishing genotype-phenotype links in microorganisms of industrial, technological and biomedical relevance. In this regard, random transposon mutagenesis coupled to high-throughput next-generation sequencing approaches, termed transposon-insertion sequencing (TIS), has emerged as a robust, genome-wide alternative to perform functional genome analysis. Though these approaches have been used in a large number of studies involving pathogenic and clinically relevant bacteria, they have received little attention in the fields of commensal and potentially beneficial bacteria, including probiotic microorganisms. In this chapter, we describe the implementation of the TIS method Transposon-Directed Insertion Sequencing to describe the set of essential genes in a representative strain of a genus encompassing several commensal and potentially probiotic bacteria and discuss considerations when applying similar methodological approaches to other Bifidobacterium species/strains of interest.

Editorial: Stressors Acting on Aquatic Ecosystems: High-Throughput Sequencing Approaches to Shed Light on Human-Nature Interactions

Editorial: Stressors Acting on Aquatic Ecosystems: High-Throughput Sequencing Approaches to Shed Light on Human-Nature Interactions

Automated genotyping of microsatellite loci from feces with high throughput sequences.

Ecological and conservation genetic studies often use noninvasive sampling, especially with elusive or endangered species. Because microsatellites are generally short in length, they can be amplified from low quality samples such as feces. Microsatellites are highly polymorphic so few markers are enough for reliable individual identification, kinship determination, or population characterization. However, the genotyping process from feces is expensive and time consuming. Given next-generation sequencing (NGS) and recent software developments, automated microsatellite genotyping from NGS data may now be possible. These software packages infer the genotypes directly from sequence reads, increasing throughput. Here we evaluate the performance of four software packages to genotype microsatellite loci from Iberian wolf (Canis lupus) feces using NGS. We initially combined 46 markers in a single multiplex reaction for the first time, of which 19 were included in the final analyses. Megasat was the software that provided genotypes with fewer errors. Coverage over 100X provided little additional information, but a relatively high number of PCR replicates were necessary to obtain a high quality genotype from highly unoptimized, multiplexed reactions (10 replicates for 18 of the 19 loci analyzed here). This could be reduced through optimization. The use of new bioinformatic tools and next-generation sequencing data to genotype these highly informative markers may increase throughput at a reasonable cost and with a smaller amount of laboratory work. Thus, high throughput sequencing approaches could facilitate the use of microsatellites with fecal DNA to address ecological and conservation questions.

Application of third-generation sequencing in cancer research.

In the past several years, nanopore sequencing technology from Oxford Nanopore Technologies (ONT) and single-molecule real-time (SMRT) sequencing technology from Pacific BioSciences (PacBio) have become available to researchers and are currently being tested for cancer research. These methods offer many advantages over most widely used high-throughput short-read sequencing approaches and allow the comprehensive analysis of transcriptomes by identifying full-length splice isoforms and several other posttranscriptional events. In addition, these platforms enable structural variation characterization at a previously unparalleled resolution and direct detection of epigenetic marks in native DNA and RNA. Here, we present a comprehensive summary of important applications of these technologies in cancer research, including the identification of complex structure variants, alternatively spliced isoforms, fusion transcript events, and exogenous RNA. Furthermore, we discuss the impact of the newly developed nanopore direct RNA sequencing (RNA-Seq) approach in advancing epitranscriptome research in cancer. Although the unique challenges stillpresent for these new single-molecule long-read methods, they will unravel many aspects of cancer genome complexity in unprecedented ways and present an encouraging outlook for continued application in an increasing number of different cancer research settings.

Unraveling Protein Interactions between the Temperate Virus Bam35 and Its Bacillus Host Using an Integrative Yeast Two Hybrid–High Throughput Sequencing Approach

Bacillus virus Bam35 is the model Betatectivirus and member of the family Tectiviridae, which is composed of tailless, icosahedral, and membrane-containing bacteriophages. Interest in these viruses has greatly increased in recent years as they are thought to be an evolutionary link between diverse groups of prokaryotic and eukaryotic viruses. Additionally, betatectiviruses infect bacteria of the Bacillus cereus group, which are known for their applications in industry and notorious since it contains many pathogens. Here, we present the first protein–protein interactions (PPIs) network for a tectivirus–host system by studying the Bam35–Bacillus thuringiensis model using a novel approach that integrates the traditional yeast two-hybrid system and high-throughput sequencing (Y2H-HTS). We generated and thoroughly analyzed a genomic library of Bam35′s host B. thuringiensis HER1410 and screened interactions with all the viral proteins using different combinations of bait–prey couples. Initial analysis of the raw data enabled the identification of over 4000 candidate interactions, which were sequentially filtered to produce 182 high-confidence interactions that were defined as part of the core virus–host interactome. Overall, host metabolism proteins and peptidases were particularly enriched within the detected interactions, distinguishing this host–phage system from the other reported host–phage PPIs. Our approach also suggested biological roles for several Bam35 proteins of unknown function, including the membrane structural protein P25, which may be a viral hub with a role in host membrane modification during viral particle morphogenesis. This work resulted in a better understanding of the Bam35–B. thuringiensis interaction at the molecular level and holds great potential for the generalization of the Y2H-HTS approach for other virus–host models.

Identification and phylogenetic analysis of voltage-gated sodium channel haplotypes in the malaria vector Anopheles sinensis using a high-throughput amplicon sequencing approach

BackgroundAnopheles sinensis is a dominant vector for malaria transmission in Asian countries. Voltage-gated sodium channel (VGSC) mutation-mediated knock-down resistance (kdr) has developed in many A. sinensis populations because of intensive and long-term use of pyrethroids. Our previous study showed that multiple mutations at position 1014 of the VGSC were heterogeneously distributed in A. sinensis populations across Sichuan, China.MethodsTo understand resistance genotypes at the haplotype level and reconstruct the phylogenetic relationship of VGSC haplotypes, a cost-effective next-generation sequencing (NGS)-based amplicon sequencing approach was established to clarify haplotypes containing codon 1014 of the VGSC gene from a total of 446 adults collected in 12 locations of Sichuan, China.ResultsNineteen (19) haplotypes were identified, including 11 wild 1014L, 6 resistance 1014F, and 2 resistance 1014C haplotypes. We found that resistance haplotypes of A. sinensis VGSC were widely distributed at frequencies ranging from 3.67 to 92.61%. The frequencies of the 1014C haplotype in the southeast of Sichuan (Luzhou, Guangan, and Suining) were relatively higher than those in other sampling locations. Phylogenetic analyses support that kdr-type mutation at position 1014 is not singly originated and resistance 1014C haplotypes evolve from TTT-encoding 1014F.ConclusionsA cost-effective next-generation sequencing (NGS)-based amplicon sequencing approach has been established in this study. The data revealed the patchy distribution of VGSC resistance haplotypes with overall high frequencies in Sichuan, China. Phylogenetic analyses support multiple origins and sequential evolution (1014L → 1014F → 1014C) for kdr-type mutations in A. sinensis.Graphical abstract

Arbuscular mycorrhizal inoculum coupled with organic substrate induces synergistic effects for soil quality changes, and rhizosphere microbiome structure in long-term monocropped cucumber planted soil

The integrated soil biological amendments of mycorrhizal inoculation and organic substrate can be additive, synergistic, or antagonistic under plant-soil-microbe interaction. Underlying mechanisms of associated soil quality changes and microbiota structure are, however, less understood. To probe into this, consecutive greenhouse experiments (2016–2018) were conducted to study the effects of mycorrhizal inoculum (AMF: Diversispora epigaea L.) and soil organic substrate (GS: garlic stalk) were used as single and combined applications. In this study, a high-throughput sequencing approach was used to evaluate the rhizosphere microbiome structure of bacteria in the replanted substrate of 7-years of continuous cucumber cropping. From the two years of treatment, we found that co-amended GS + AMF significantly altered the soil quality changes through increasing soil organic matter, soil nutrient contents, and enzyme activities. Our 16 S rRNA amplicon revealed a distinct impact on alpha diversity and bacterial community. The observed species richness index (OTUs, Chao1, and ACE) and community diversity index (Shannon) were increased due to GS-amended soil and GS + AMF inoculation, respectively. Soil amendments also had apparent effects by altering bacterial community compositions at the phylum level, which were mostly associated with the higher abundance of Proteobacteria, Planctomycetes, Gemmatimonadetes, Actinobacteria and Bacteroidetes. Furthermore, correlation analyses revealed that the soil pH and enzyme activities emerged as the major determinants of the key microbial attributes. Our results suggest that GS + AMF treatment can directly regulate the soil environmental attributes or induce compositional shifts of microbes in the continuous cucumber cropping substrates. However, their co-inoculation impact was the shift of the key soil-microbiota consortium. Therefore, the study provides new insight into ways that can reduce the adverse effect of continuous cropping stress and eco-friendly contribute to the stable and healthy micro-ecological development in protected cultivation.

Niche differentiation of belowground microorganisms and their functional signatures in Assam type tea (Camellia sinensis var. assamica).

We employed an Illumina-based high-throughput metagenomics sequencing approach to unveil the rhizosphere and root endosphere microbial community associated with an organically grown Camellia population located at the Experimental Garden for Plantation Crops, Assam (India). The de novo assembled tea root endosphere metagenome contained 24,231 contigs (total 7,771,089 base pairs with an average length of 321 bps), while tea rhizosphere soil metagenome contained 261,965 sequences (total 230,537,174 base pairs, average length 846). The most prominent rhizobacteria belonged to the genera, viz., Bacillus (10.35%), Candidatus Solibacter (6.36%), Burkholderia (5.19%), Pseudomonas (3.9%), Streptomyces (3.52%), and Bradyrhizobium (2.77%), while the root endosphere was dominated by bacterial genera, viz., Serratia (46.64%), Methylobacterium (8.02%), Yersinia (5.97%), Burkholderia (2.05%), etc. The presence of few agronomically important bacterial genera, Bradyrhizobium, Rhizobium (each 0.93%), Sinorhizobium (0.34%), Azorhizobium, and Flavobacterium (0.17% each), was also detected in the root endosphere. KEGG pathway mapping indicated the presence of microbial metabolic pathway genes related to tyrosine metabolism, tryptophan metabolism, glyoxylate, and dicarboxylate metabolism which play important roles in endosphere activities, including survival, growth promotion, and host adaptation. The root endosphere microbiome also contained few important plant growth promoting traits related to phytohormone production, abiotic stress alleviation, mineral solubilization, and plant disease suppression.

De novo transcriptome assembly and EST-SSR markers development for Zelkova schneideriana Hand.-Mazz. (Ulmaceae).

The online version contains supplementary material available at 10.1007/s13205-021-02968-5.

A Standardized Procedure for Monitoring Harmful Algal Blooms in Chile by Metabarcoding Analysis.

Harmful algae blooms (HABs) monitoring has been implemented worldwide, and Chile, a country famous for its fisheries and aquaculture, has intensively used microscopic and toxin analyses for decades for this purpose. Molecular biological methods, such as high-throughput DNA sequencing and bacterial assemblage-based approaches, are just beginning to be introduced in Chilean HAB monitoring, and the procedures have not yet been standardized. Here, 16S rRNA and 18S rRNA metabarcoding analyses for monitoring Chilean HABs are introduced stepwise. According to a recent hypothesis, algal-bacterial mutualistic association plays a critical synergetic or antagonistic relationship accounting for bloom initiation, maintenance, and regression. Thus, monitoring HAB from algal-bacterial perspectives may provide a broader understanding of HAB mechanisms and the basis for early warning. Metabarcoding analysis is one of the best suited molecular-based tools for this purpose because it can detect massive algal-bacterial taxonomic information in a sample. The visual procedures of sampling to metabarcoding analysis herein provide specific instructions, aiming to reduce errors and collection of reliable data.

Alteration of desert soil microbial community structure in response to agricultural reclamation and abandonment

Agricultural reclamation via the conversion of desert into cropland offers a strategy to utilize arid lands and combat desertification. However, limited irrigation water resources have forced the abandonment of reclaimed agricultural lands and succession towards unmanaged natural states, which may contribute greatly to erosion potential. Soil microorganisms can affect the degradation and wind erosion potential of soils in the desert region by mediating many ecological processes. However, little is known about how soil microbial community structures respond to desert agricultural reclamation with tillage-based farming and subsequential abandonment. Using quantitative PCR (qPCR) and 16S rRNA high-throughput sequencing approaches, we investigated the changes of bacterial, archaeal and fungal community biomasses, diversities and structures in a desert farmland soil (FS). We contrasted these to soils along a 10-year farming abandonment chronosequence (2-year, 5-year and 10-year) and with adjacent soil from a native desert (control) in the Badain Jaran desert, China. Soil bacterial and archaeal community biomasses significantly increased after reclamation compared with the control and generally decreased after farming abandonment. Species richness of communities decreased for archaea and increased for fungi in response to reclamation, whereas bacterial community species richness increased during the 5-year abandonment. Microbial community structures were divergent in reclaimed and abandoned soils and were closely related to soil moisture, total carbon (C), total nitrogen (N), available phosphorus (P) and the stoichiometry of C, N and P, which explained 24.4–89.0% of their variations. Our results indicate desert soil microbial biomass, diversity and compositions differently responded to agricultural reclamation and abandonment with some irreversible changes in compositions mainly driven by soil moisture and chemical properties changes. The insights are meaningful for sustainable development of agriculture and ethical land management in arid desert regions.

Air-dispersed aquatic microorganisms show establishment and growth preferences in different freshwater colonisation habitats.

We attempted to mimic aeolian ecosystems to examine how filters posed by regional characteristics can influence the establishment and growth of airborne microcolonisers of a common air source. Using a natural single source of aerosols we applied a combined microscopy and high-throughput sequencing approach to examine the diversity, settling and growth potential of air-dispersed microbes in water containers representing newly formed aquatic colonisation habitats of different trophic states and salinity. Heterotrophic microeukaryotes were favoured as initial settlers when nutrients were low, while autotrophs rapidly proliferated in the high-nutrient containers, possibly due to favourable germinating conditions for their preferred mode of dispersal with resting spores. Following settling of colonisers, we investigated two contrasting hypotheses: if the different water colonisation habitats harboured the same microbial communities after establishment and growth periods, this would point towards a selection of best-fit cosmopolitan colonisers, regardless of habitat-specific characteristics. Alternatively, community dissimilarities after the growth period would suggest a selection of settlers due to bottom-up controls combined with priority effects. Both analyses suggested that the structure of the microbial communities in the different colonisation habitats were driven by nutrient content and salinity, showing clustering to similar bottom-up forces and dissimilarities in significantly different colonisation habitats.

Genomic library preparation and hybridization capture of formalin-fixed tissues and allozyme supernatant for population genomics and considerations for combining capture- and RADseq-based single nucleotide polymorphism data sets.

Until recently many historical museum specimens were largely inaccessible to genomic inquiry, but high-throughput sequencing (HTS) approaches have allowed researchers to successfully sequence genomic DNA from dried and fluid-preserved museum specimens. In addition to preserved specimens, many museums contain large series of allozyme supernatant samples, but the amenability of these samples to HTS has not yet been assessed. Here, we compared the performance of a target-capture approach using alternative sources of genomic DNA from 10 specimens of spring salamanders (Plethodontidae: Gyrinophilus porphyriticus) collected between 1985 and 1990: allozyme supernatants, allozyme homogenate pellets and formalin-fixed tissues. We designed capture probes based on double-digest restriction-site associated sequencing (RADseq) derived loci from frozen blood samples available for seven of the specimens and assessed the success and consistency of capture and RADseq approaches. This study design enabled direct comparisons of data quality and potential biases among the different data sets for phylogenomic and population genomic analyses. We found that in phylogenetic analyses, all enrichment types for a given specimen clustered together. In principal component space all capture-based samples clustered together, but RADseq samples did not cluster with corresponding capture-based samples. Single nucleotide polymorphism calls were on average 18.3% different between enrichment types for a given individual, but these discrepancies were primarily due to differences in heterozygous/homozygous single nucleotide polymorphism calls. We demonstrate that both allozyme supernatant and formalin-fixed samples can be successfully used for population genomic analyses and we discuss ways to identify and reduce biases associated with combining capture and RADseq data.

Detection of bat-associated circoviruses in Korean bats.

In recent years, several novel circular single-stranded DNA viruses have been detected in various mammals, birds, insects, and environmental samples using metagenomic and high-throughput sequencing approaches. In this study, we tested for the presence of circoviruses in 243 bat fecal samples collected between 2018 and 2019 from 48 sampling sites across Korea. To detect circoviruses, nested PCR was performed with degenerate primers targeting a conserved replication-associated protein (rep) gene of circovirus/cyclovirus. Among 243 samples tested, a total of 37 fecal samples from 14 sampling sites were PCR-positive for circoviruses at a frequency rate of 15.23%. We obtained 36 partial rep gene sequences of circoviruses and one complete genome sequence of bat-associated circovirus 12, encompassing a genome size of 2097 nt containing two inversely arranged open reading frames and a conserved nonamer sequence in the apex of a stem-loop structure. In addition, we found four bat species that were harboring circoviruses in Korea based on species identification PCR of circovirus-positive bat fecal samples. Detailed sequence analysis indicated that the bat-associated circovirus sequences identified in this study were related to those of known bat and avian groups of circoviruses. Herein, we report evidence for the presence of bat-associated circoviruses in Korean bats.

Integrative Transcriptomics and Metabolomics Analysis of Body Color Formation in the Leopard Coral Grouper (Plectropomus leopardus)

Body color is an important economic and ecological trait in aquatic animals, which influence their economic values and determine the animal ability to survive in the marine environment. Red-colored Plectropomus leopardus is in high demand owing to its bright color and potential value for artificial breeding. High-throughput transcriptome sequencing and liquid chromatography tandem-mass spectrometry (LC-MS/MS) approaches were used to identify transcript and metabolic differences between black-colored and red-colored P. leopardus. Compared with the black-colored group, 218 and 181 differentially expressed genes were up- and down- regulated, respectively, in the red-colored group. 425 and 56 significantly different metabolites were identified in LC-MS positive and LC-MS negative ion models, respectively, between two colored groups. Based on the integrative analysis, the red-colored group exhibited greater carotenoid uptake, transport, and accumulation activity potential than those in the black-colored group and may consume more arachidonic acid for body color formation. The black-colored group showed greater melanin synthesis activity compared with the red-colored group. These results substantially improve the understanding of molecular and metabolic mechanisms underlying body color formation in P. leopardus and the potential of body color for the environment adaptation.

Impacts of biochar-based fertilization on soil arbuscular mycorrhizal fungal community structure in a karst mountainous area.

The application of biochar-based fertilizer can improve soil properties in part by stimulating microbial activity and growth. Karst ecosystems, which make up large areas of Southwest China, are prone to degradation. Understanding the response of arbuscular mycorrhizal fungal (AMF) community structure to biochar-based fertilizer application is of great significance to karst soil restoration. A field experiment was conducted in a typical karst soil (calcareous sandy loam) in Southwest China. A high-throughput sequencing approach was used to investigate the effect of biochar-based fertilization on AMF community structure in the karst soil. With the control (CK), compost with NPK fertilizer (MF), biochar (B), a lower amount of biochar with compost and NPK fertilizer (B1MF), biochar with compost and NPK fertilizer (BMF), and a higher amount of biochar with compost and NPK fertilizer (B4MF), the field trials were set up for 24 months. Soil amendments increased soil nutrient content and AMF diversity. The composition and structure of the AMF community varied among the treatments. AMF community composition was significantly impacted by soil chemical properties such as TC (total carbon), TN (total nitrogen), TP (total phosphorus), and AP (available phosphorus). Furthermore, network analysis showed that biochar-based fertilization increased the scale and complexity of the microbial co-occurrence network. Biochar-based fertilization enabled more keystone species (such as order Diversisporales and Glomerales) in the soil AMF network to participate in soil carbon resource management and soil nutrient cycling, indicating that biochar-based fertilizer is beneficial for the restoration of degraded karst soils.