Amplicon Sequencing Method Research Articles

High-quality single amplicon sequencing method for illumina MiSeq platform using pool of ‘N’ (0–10) spacer-linked target specific primers without PhiX spike-in

BackgroundIllumina sequencing platform requires base diversity in the initial 11 cycles for efficient cluster identification and colour matrix estimation. This limitation yields low-quality data for amplicon libraries having homogeneous base composition. Spike-in of PhiX library ensures base diversity but reduces the overall number of sequencing reads for data analysis. To overcome such low diversity issues during amplicon sequencing on illumina platforms, we developed a high throughput single amplicon sequencing method by introducing ‘N’ (0–10) spacers in target gene amplification primers that are pooled for simple handling.ResultWe evaluated the efficiency of ‘N’ (0–10) spacer-linked primers by targeting bacterial 16S V3-V4 region, demonstrating heterogeneous base library construction. The addition of ‘N’ (0–10) spacers causes sequencing frameshift at every base that leads to base diversity and produces heterogeneous high quality reads within a single amplicon library. We have written a python based command-line software,“MetReTrim”, to trim the ‘N’ (0–10) spacers from the raw reads (https://github.com/Mohak91/MetReTrim). We further demonstrated the accuracy of this method by comparative mock community analysis with standard illumina V3-V4 primer method. The ZymoBIOMICS™ microbial community DNA standard was used as a control for this study. We performed data analysisusing the DADA2 pipeline where taxonomy was assigned using SILVA database as reference. We observed no difference between the communities represented by our method and standard illumina V3-V4 primer method.ConclusionThis method eliminates the need for PhiX spike-in for single amplicon sequencing on illumina MiSeq platform. This allows for sequencing of more number of samples in a run and a reduction in the overall cost. Given that Illumina sequencing works on SBS chemistry irrespective of the platform (such as HiSeq, MiSeq, NextSeq, NovaSeq, etc.) we propose that this strategy of using ‘N’ (0–10) spacer-linked primer design can be adopted for generating high-quality single locus amplicon sequencing in a high throughput manner across the illumina platform subject to further validation.

Identification and in vitro functional assessment of 10 CYP2C9 variants found in Chinese Han subjects.

Cytochrome P450 2C9 (CYP2C9) participates in about 15% of clinical drug metabolism, and its polymorphism is associated with individual drug metabolism differences, which may lead to the adverse drug reactions (ADRs). In this study, 1163 Chinese Han individuals were recruited to investigate their distribution pattern of CYP2C9 gene and find out the variants that may affect their drug metabolic activities. We successfully developed a multiplex PCR amplicon sequencing method and used it for the genetic screening of CYP2C9 in a large scale. Besides the wild type CYP2C9*1, totally 26 allelic variants of CYP2C9 were detected, which included 16 previously reported alleles and 10 new non-synonymous variants that had not been listed on the PharmVar website. The characteristics of these newly detected CYP2C9 variants were then evaluated after co-expressing them with CYPOR in S. cerevisiae microsomes. Immunoblot analysis revealed that except for Pro163Ser, Glu326Lys, Gly431Arg and Ile488Phe, most of newly detected variants showed comparable protein expression levels to wild type in yeast cells. Two typical CYP2C9 probe drugs, losartan and glimepiride, were then used for the evaluation of metabolic activities of variants. As a result, 3 variants Thr301Met, Glu326Lys, and Gly431Arg almost lost their catalytic activities and most of other variants exhibited significantly elevated activities for drug metabolism. Our data not only enriches the knowledge of naturally occurring CYP2C9 variants in the Chinese Han population, but also provides the fundamental evidence for its potential clinical usage for personalized medicine in the clinic.

N2O emission associated with shifts of bacterial communities in riparian wetland during the spring thawing periods.

Soil freeze-thaw processes lead to high nitrous oxide (N2O) emissions and exacerbate the greenhouse effect. The wetlands of the Inner Mongolia Plateau are in the pronounced seasonal freeze-thaw zone, but the effect of spring thaw on N2O emissions and related microbial mechanisms is still unclear. We investigated the effects of different periods (freeze, freeze-thaw, and thaw) on soil bacterial community diversity and composition and greenhouse gas emissions during the spring freeze-thaw in the XiLin River riparian wetlands in China by amplicon sequencing and static dark box methods. The results showed that the freeze-thaw periods predominantly impact on the diversity and composition of the bacterial communities. The phyla composition of the soil bacteria communities of the three periods is similar in level, with Proteobacteria, Chloroflexi, Actinobacteria, and Acidobacteria dominating the microbial communities. The alpha-diversity of bacterial communities in different periods varies that the freezing period is higher than that of the freeze-thaw period (p < .05). Soil total carbon, soil water content, and microbial biomass carbon were the primary factors regulating the abundance and compositions of the bacterial communities during spring thawing periods. Based on functional predictions, the relative abundance of nitrification and denitrification genes was higher in the freezing period than in the thawing period, while the abundance was lowest in the freeze-thawing period. The correlation results found that N2O emissions were significantly correlated with amoA and amoB in nitrification genes, indicating that nitrification may be the main process of N2O production during spring thaw. This study reveals potential microbial mechanisms of N2O emission during spring thaw and provides data support and theoretical basis for further insight into the mechanism of N2O emission during spring thaw.

Organic Farming Favors phoD-Harboring Rhizospheric Bacterial Community and Alkaline Phosphatase Activity in Tropical Agroecosystem

The bacteria harboring phoD encodes alkaline phosphatase (ALP), a secretory enzyme that hydrolyzes organic phosphorous (P) to a usable form in the soil. The impact of farming practices and crop types on phoD bacterial abundance and diversity in tropical agroecosystems is largely unknown. In this research, the aim was to study the effect of farming practices (organic vs. conventional) and crop types on the phoD-harboring bacterial community. A high-throughput amplicon (phoD gene) sequencing method was employed for the assessment of bacterial diversity and qPCR for phoD gene abundance. Outcomes revealed that soils treated for organic farming have high observed OTUs, ALP activity, and phoD population than soils managed under conventional farming with the trend of maize > chickpea > mustard > soybean vegetated soils. The relative abundance of Rhizobiales exhibited dominance. Ensifer, Bradyrhizobium, Streptomyces, and Pseudomonas were observed as dominant genera in both farming practices. Overall, the study demonstrated that organic farming practice favors the ALP activity, phoD abundance, and OTU richness which varied across crop types with maize crops showing the highest OTUs followed by chickpea, mustard, and least in soybean cropping.

DOP47 Gut microbiome contributes to the development of immune checkpoint inhibitor-related colitis

Abstract Background The gut microbiota is implicated in shaping immune checkpoint inhibitors (ICIs) efficacy, but relatively little is known about how the microbiota influences immune-related adverse events (irAEs). Our study aimed to explore the association and causal relationship between gut microbiome and immune checkpoint inhibitor-related colitis. Methods We collected faecal samples from 32 patients with advanced malignant tumor patients developing irAE after anti-PD-1 therapy from May 2020 to Aug 2021. Faecal samples were analyzed by 16s rDNA amplicon sequencing method on Illumina MiSeq (PE300) sequencing platform. We also performed faecal microbiota transplantation (FMT) for antibiotic-treated mice with samples from patients with colitic irAE and without irAE. Results We compared the microbiota between 8 colitic irAE and 24 non-colitic irAE patients. Principal coordinates analysis (PCoA) plot showed significant difference of microbiota composition by Adonis analysis (P=0.003) (Fig. 1A). LEfSe analysis detected a lower abundance of Bacteroidota and a higher abundance of Proteobacteria at the phylum level and a lower abundance of Bacteroides and Bifidobacterium and higher abundance of Enterococcus at the genus level in colitic irAE (Fig. 1B). The total abundance of major butyrate-producing bacteria was lower in colitic irAE compared with non-colitic irAE patients (P=0.018) (Fig. 1C). Functional prediction analyses revealed different abundances of environmental information processing, genetic information processing, and metabolic pathways between colitic and non-colitic irAE patients (Fig. 1D). By FMT experiments in mice, we sought to explore the causal relationship between microbiota immune-related colitis. After injection of anti-PD-1 and anti-CTLA-4, three of nine colitic-irAE-FMT mice developed fatal severe colitis, while all nine non-irAE-FMT mice survived without colitis (P=0.052). The three colitic mice showed more rapid weight loss and higher disease activity index (DAI) scores compared with other mice (Fig. 2A and B). The histological colitis score in the irAE-FMT-Sac. mouse (14 points) was higher than those in the non-irAE-FMT mice (3.89 ± 1.17 points) and colitic-irAE-FMT-survival mice (3.67 ± 1.03 points) (Fig. 2C). The mean IHC scores of irAE-FMT-Sac. mouse from high to low were CD8 3.8 ± 1.3 points, CD4 2.0 ± 1.9 points, and CD20 1.1 ± 1.3 points (Fig. 2D), which also supported the occurrence of immune-related colitis. Figure 1. The gut microbiotas of patients with colitic and non-colitic type irAEs. Figure 2. Comparison of colitis in colitic-irAE-FMT and non-irAE-FMT mice. Conclusion Gut microbiota might play important role in the development of immune-related colitis, possibly by modulating metabolic pathways.

Effect of incorporation of broccoli residues into soil on occurrence of verticillium wilt of spring-sowing-cotton and on rhizosphere microbial communities structure and function.

Cotton verticillium wilt (CVW) represented a typical plant soil-borne disease and resulted in widespread economic losses in cotton production. However, the effect of broccoli residues (BR) on verticillium wilt of spring-sowing-cotton was not clear. We investigated the effects of BR on CVW, microbial communities structure and function in rhizosphere of two cotton cultivars with different CVW resistance using amplicon sequencing methods. Results showed that control effects of BR on CVW of susceptible cultivar (cv. EJ-1) and resistant cultivar (cv. J863) were 58.49% and 85.96%, and the populations of V. dahliae decreased by 14.31% and 34.19%, respectively. The bacterial diversity indices significantly increased in BR treatment, while fungal diversity indices significantly decreased. In terms of microbial community composition, the abilities to recruit bacteria and fungi were enhanced in BR treatment, including RB41, Gemmatimonas, Pontibacter, Streptomyces, Blastococcus, Massilia, Bacillus, and Gibberella, Plectosphaerella, Neocosmospora, Aspergillus and Preussia. However, the relative abundances of Sphingomonas, Nocardioides, Haliangium, Lysobacter, Penicillium, Mortierella and Chaetomidium were opposite tendency between cultivars in BR treatment. According to PICRUSt analysis, functional profiles prediction showed that significant shifts in metabolic functions impacting KEGG pathways of BR treatment were related to metabolism and biosynthesis. FUNGuild analysis indicated that BR treatment altered the relative abundances of fungal trophic modes. The results of this study demonstrated that BR treatment decreased the populations of V. dahliae in soil, increased bacterial diversity, decreased fungal diversity, changed the microbial community structure and function, and increased the abundances of beneficial microorganisms.

Multi-omics methods reveal that putrescine and cadaverine cause different degrees of enrichment of high-risk resistomes and opportunistic pathogens in the water and sediment of the Yellow River

Contamination of antibiotic resistomes due to animal carcass decay has become a serious environmental concern. However, the relationship between main metabolite compounds of corpse decomposition (i.e., putrescine and cadaverine) and antibiotic resistomes remains unclear. To tackle this issue, the response of antibiotic resistance genes (ARGs) and microbiome in aquatic environment to excess putrescine, cadaverine and a mixture of both based on laboratory simulation experiment was investigated by high-throughput quantitative PCR and amplicon sequencing methods. Our results showed putrescine and cadaverine led to the increasing of TC (total carbon) and TN (total nitrogen) both in water and sediment. Under the exposure of putrescine and cadaverine, the total abundance of mobile genetic elements (MGEs) and most ARGs in water was higher than in sediment. In particular, putrescine and cadaverine caused significantly different decreases in alpha diversity of microbial community in water and sediment compared with the control group. Microbial community structures both in water and sediment were also significantly affected by cadaverine and putrescine. Furthermore, putrescine and cadaverine led to different degrees of increases of high-risk ARGs (like mecA) and opportunistic pathogens (like Delftia) in sediment, promoting the prevalence of antibiotic resistant bacteria. In conclusion, our findings revealed the influences of main metabolites of carcass decay on microbiome and resistomes, providing references for risk assessment and pollution management.

A next generation targeted amplicon sequencing method to screen for insecticide resistance mutations in Aedes aegypti populations reveals a rdl mutation in mosquitoes from Cabo Verde.

Aedes mosquito vectors transmit many viruses of global health concern, including dengue, chikungunya and Zika. These vector-borne viral diseases have a limited number of treatment options, and vaccines vary in their effectiveness. Consequently, integrated vector management is a primary strategy for disease control. However, the increasing emergence and spread of insecticide resistance is threatening the efficacy of vector control methods. Identifying mutations associated with resistance in vector populations is important to monitor the occurrence and evolution of insecticide resistance and inform control strategies. Rapid and cost-effective genome sequencing approaches are urgently needed. Here we present an adaptable targeted amplicon approach for cost-effective implementation within next generation sequencing platforms. This approach can identify single nucleotide polymorphisms (SNPs) and small insertions and deletions (indels) in genes involved in insecticide resistance in Aedes aegypti mosquitoes. We designed and tested eleven amplicons, which included segments of the ace-1 (carbamate target), the Voltage-Gated Sodium Channel (vgsc; pyrethroids, DDT and organochlorines), and rdl (dieldrin) genes; thereby covering established knockdown resistance (kdr) mutations (e.g., S989P, I1011M/V, V1016G/I and F1534C), with the potential to identify novel ones. The amplicon assays were designed with internal barcodes, to facilitate multiplexing of large numbers of mosquitoes at low cost, and were sequenced using an Illumina platform. Our approach was evaluated on 152 Ae. aegypti mosquitoes collected in Cabo Verde, an archipelago with a history of arbovirus outbreaks. The amplicon sequence data revealed 146 SNPs, including four non-synonymous polymorphisms in the vgsc gene, one in ace-1 and the 296S rdl mutation previously associated with resistance to organochlorines. The 296S rdl mutation was identified in 98% of mosquitoes screened, consistent with the past use of an organochlorine compound (e.g., DDT). Overall, our work shows that targeted amplicon sequencing is a rapid, robust, and cost-effective tool that can be used to perform high throughput monitoring of insecticide resistance.

Promotion Effects of Taxus chinensis var. mairei on Camptotheca acuminata Seedling Growth in Interplanting Mode

Wild Camptotheca acuminata Decne (C. acuminata) resources are becoming endangered and face poor growth. Preliminary investigation results found that the growth of C. acuminata in an artificial mixed forest of Taxus chinensis var. mairei (Lemee et Levl.), Cheng et L. K. Fu (T. chinensis var. mairei) and C. acuminata was significantly higher than that in pure forests. Understanding the reasons for the above differences can help create a mixed forest of T. chinensis var. mairei and C. acuminata to solve the problem of depleting C. acuminata resources. In this study, the growth and soil indexes under two different modes (C. acuminata/T. chinensis var. mairei interplanted and monocultured C. acuminata seedlings) were compared. The results showed that plant height, basal diameter, photosynthesis rate and chlorophyll content of C. acuminata under the interplanting mode were higher than those under monoculture. The growth rates of plant height and basal diameter that were calculated from interplanted specimens increased by 25% and 40%, respectively, compared with those from specimens that were monocultured. Photosynthetic rates from different light intensities under interplanting were higher than those in seedlings under monoculture. The contents of chlorophylls a and b and total chlorophyll under interplanting were 1.50, 1.59, and 1.47 times higher than those under monoculture, respectively. The numbers of bacteria and fungi in the interplanted culture were higher than those in the monoculture. Furthermore, the differences in microbial diversity under different planting modes were analyzed via the amplicon sequencing method. Soil enzyme activities increased under interplanting compared with that in the monoculture. Taxane allelochemicals were detected in the range of 0.01–0.67 μg/g in the interplanting mode from April to September. T. chinensis var. mairei may increase the establishment and productivity of C. acuminata seedlings under interplanting mode through improvements in enzyme activity, changes in microorganism population structure, and release of allelochemicals.

Highly specific qPCR and amplicon sequencing method for detection of quarantine citrus pathogen Phyllosticta citricarpa applicable for air samples

AbstractThe fungus Phyllosticta citricarpa is a quarantine pathogen in the EU and is of high economic importance in many parts of the world where favourable climate conditions drive the development of citrus black spot (CBS) disease. Disease symptoms include necrotic lesions on leaves and fruits. Low disease pressure can reduce crop marketability, while higher disease pressure can cause premature fruit drop, significantly increasing crop losses. The wind‐dispersed spores of P. citricarpa are especially problematic for rapid pathogen dispersal, but also provide an opportunity for early detection of the disease spreading into a new area. In this study we have developed and validated a quantitative PCR (qPCR) assay based on the TEF1‐α sequence. Specificity testing demonstrated that it is currently the only qPCR assay that does not cross‐react with closely related Phyllosticta species. The assay is sensitive and can detect a single copy of the TEF1 gene in a reaction, it is highly repeatable and reproducible and can be used for testing of the sticky tapes from spore traps as well as citrus fruit samples. High‐throughput sequencing (HTS) of the DNA barcodes ITS1 and TEF1 was also explored for the detection and discrimination of P. citricarpa. The limit of detection of the HTS was 1000 spores on a daily spore trap tape. This study makes an important improvement to the diagnostics of the CBS and the methods developed can also be applied to improve the surveillance and early detection of the pathogen when linked to spore samplers in the field.

Establishment and Validation of a New Analysis Strategy for the Study of Plant Endophytic Microorganisms.

Amplicon sequencing of bacterial or fungal marker sequences is currently the main method for the study of endophytic microorganisms in plants. However, it cannot obtain all types of microorganisms, including bacteria, fungi, protozoa, etc., in samples, nor compare the relative content between endophytic microorganisms and plants and between different types of endophytes. Therefore, it is necessary to develop a better analysis strategy for endophytic microorganism investigation. In this study, a new analysis strategy was developed to obtain endophytic microbiome information from plant transcriptome data. Results showed that the new strategy can obtain the composition of microbial communities and the relative content between plants and endophytic microorganisms, and between different types of endophytic microorganisms from the plant transcriptome data. Compared with the amplicon sequencing method, more endophytic microorganisms and relative content information can be obtained with the new strategy, which can greatly broaden the research scope and save the experimental cost. Furthermore, the advantages and effectiveness of the new strategy were verified with different analysis of the microbial composition, correlation analysis, inoculant content test, and repeatability test.

Changes in soil fungal communities after onset of wheat yellow mosaic virus disease.

Rhizosphere-associated microbes have important implications for plant health, but knowledge of the association between the pathological conditions of soil-borne virus-infected wheat and soil microbial communities, especially changes in fungal communities, remains limited. We investigated the succession of fungal communities from bulk soil to wheat rhizosphere soil in both infected and healthy plants using amplicon sequencing methods, and assessed their potential role in plant health. The results showed that the diversity of fungi in wheat rhizosphere and bulk soils significantly differed post wheat yellow mosaic virus disease onset. The structure differences in fungal community at the two wheat health states or two compartment niches were evident, soil physicochemical properties (i.e., NH4 +) contribute to differences in fungal community structure and alpha diversity. Comparison analysis showed Mortierellomycetes and Dothideomycetes as dominant communities in healthy wheat soils at class level. The genus Pyronemataceae and Solicoccozyma were significantly are significantly enriched in rhizosphere soil of diseased plant, the genus Cystofilobasidium, Cladosporium, Mortierella, and Stephanonectria are significantly enriched in bulk soil of healthy plant. Co-occurrence network analysis showed that the fungi in healthy wheat soil has higher mutual benefit and connectivity compared with diseased wheat. The results of this study demonstrated that the occurrence of wheat yellow mosaic virus diseases altered both fungal community diversity and composition, and that NH4 + is the most important soil physicochemical factor influencing fungal diversity and community composition.

Methods for the detection of tumor-specific single nucleotide somatic mutations in plasma cDNA samples

Introduction . Liquid biopsy is considered as a minimally invasive method of molecular genetic analysis that can be used for early diagnosis, prognosis of disease development, monitoring of residual disease or treatment outcomes, and selection of optimal drug therapy schemes for a patient. Along with the development of tests based on the study of panels of oncologically significant genes or their regions, for various forms of genetically heterogeneous tumors a promising approach could be the use as an object of liquid biopsy of an individual spectrum of somatic mutations of a particular patient that can be detected on the basis of high-throughput sequencing of tumor tissue. Aim . To determine the applicability of different methods for detecting single-nucleotide somatic mutations detected in tumor tissue of a particular patient in cDNA preparations from blood plasma obtained before surgical removal of the tumor and to evaluate the possibility of quantifying the proportion of the alternative variant in the total pool of cDNA. Materials and methods. We used normal and tumor tissue, as well as blood plasma samples from patients with hepatocellular carcinoma, and various methods for detecting single-nucleotide somatic mutations: real-time polymerase chain reaction (PCR) with intercalating dye or with TaqMan probes, droplet digital PCR and high-throughput sequencing of target amplicons. Results . Using the example of a somatic mutation in the TLN1 gene detected in tumor tissue of a patient with hepatocellular carcinoma, methods were developed and tested, each of which allows specific detection of the mutant variant in small amounts (2 ng) of cDNA from the blood plasma of the same patient. The use of droplet PCR and target amplicon sequencing methods allowed us to quantify the proportion of the mutant variant in the total cDNA pool, which was 19.7 and 23.5 %, respectively. Conclusion . Among the methods investigated, droplet digital PCR and targeted amplicon sequencing allow not only reliable detection of mutant variants in small amounts of cDNA, but also adequate quantification, which is particularly important for the development of ways to monitor tumor growth during treatment. The close values of the proportion of mutant variants in cDNA detected by these methods indicate the accuracy of quantitative analysis and the possibility of their use for cross-validation of the results obtained.

Long-read HiFi sequencing of NUDT15: Phased full-gene haplotyping and pharmacogenomic allele discovery.

To determine the phase of NUDT15 sequence variants for more comprehensive star (*) allele diplotyping, we developed a novel long-read single-molecule real-time HiFi amplicon sequencing method. A 10.5 kb NUDT15 amplicon assay was validated using reference material positive controls and additional samples for specimen type and blinded accuracy assessment. Triplicate NUDT15 HiFi sequencing of two reference material samples had nonreference genotype concordances of >99.9%, indicating that the assay is robust. Notably, short-read genome sequencing of a subset of samples was unable to determine the phase of star (*) allele-defining NUDT15 variants, resulting in ambiguous diplotype results. In contrast, long-read HiFi sequencing phased all variants across the NUDT15 amplicons, including a *2/*9 diplotype that previously was characterized as *1/*2 in the 1000 Genomes Project v3 data set. Assay throughput was also tested using 8.5 kb amplicons from 100 Ashkenazi Jewish individuals, which identified a novel NUDT15 *1 suballele (c.-121G>A) and a rare likely deleterious coding variant (p.Pro129Arg). Both novel alleles were Sanger confirmed and assigned as *1.007 and *20, respectively, by the PharmVar Consortium. Taken together, NUDT15 HiFi amplicon sequencing is an innovative method for phased full-gene characterization and novel allele discovery, which could improve NUDT15 pharmacogenomic testing and subsequent phenotype prediction.

Effects of a Carnobacterium maltaromaticum strain at natural contamination levels on the microbiota of vacuum-packaged beef steaks during chilled storage

We investigated the impact of a Carnobacterium maltaromaticum strain at a natural contamination level on the dynamics of microbiota and survival of Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium on chilled vacuum-packaged (VP) beef. C. maltaromaticum A5 up to 2 log CFU/cm2 was spiked onto beef steaks with or without E. coli O157:H7 and S. Typhimurium. The steaks were vacuum-packaged and stored at 2 °C for 12 weeks. The dynamics of the microbiota were determined using plating and 16S rRNA gene amplicon sequencing methods. The predominant bacterial species in the final microbiota were determined using whole metagenome and genome sequencing. C. maltaromaticum on inoculated meat peaked at week 2 (>80%) even though the initial relative abundance was as low as <1%. The relative abundance of indigenous Latilactobacillus was 0.1–0.5%, but gradually increased to 75.7–100% at week 12, displacing Carnobacterium. The predominant species in the final microbiota was identified as Latilactobacillus sakei subsp. carnosus, which harbored gene clusters encoding Sakacin G and a putative class IIc bacteriocin. Neither of the two pathogens was affected by C. maltaromaticum A5. Taken together, this study indicates that the initial microbial composition plays an important role in the fate of an introduced LAB strain.

The Potential Use of Endophyte-Free inebrians as Sheep Feed Evaluated with In Vitro Fermentation

Endophytic fungal infection is the major reason for intoxication of animals caused by drunken horse grass. Fortunately, it has been established that seed detoxification techniques and isolation of endophytic fungi infect non-endophytic fungi populations with the same genetic background as endophyte-infected Achnatherum inebrians. Moreover, sheep can use endophyte-free Achnatherum inebriants (EF) without obvious toxicity symptoms. The present study selected EF as a representative grass, consisting of five different replacement levels, EF0, EF25, EF50, EF75, and EF100, corresponding to 0%, 25%, 50%, 75%, and 100% of the fermentation substrate, respectively. Simultaneously, in vitro fermentation and the 16S rRNA amplicon sequencing method was used to explore the effect of EF on sheep ruminal fermentation and microbial diversity. The results revealed that EF100 had the highest values for pH, acetate: propionate, the Patescibacteria, Kiritimatiellaeota, and Synergistetes phylum levels, Ruminococcaceae, Prevotellaceae, and Saccharofermentans genus levels than the other treatments (p &lt; 0.05). In contrast, EF25 was associated with higher levels of abundance-based coverage estimator (ACE), Chaol index of the phyla Synergistetes and Bacteroidetes, and of the genus Erysipelotrichaceae, Rikenellaceae, and Prevotella as compared with other treatments (p &lt; 0.05). EF50 resulted in the greatest values for the genus Christensenellaceae and Lachnospiraceae as compared with other treatments (p &lt; 0.05). EF75 resulted in the greatest values for the Shannon index as compared with other treatments (p &lt; 0.05). EF0 resulted in the greatest values for gas production (GP), ammonia nitrogen (NH3-N), total volatile fatty acid (TVFA), acetate, propionate, butyrate, valerate, isobutyrate, isovalerate, and the phyla Firmicutes, Proteobacteria, and Spirochaetes, and the genus Succiniclasticum, Ruminobacter, Family_XIII and Treponema as compared with other treatments (p &lt; 0.05). PICRUSt2 analysis indicated that most of the functional prediction pathways were involved in Carbohydrate metabolism and, Amino acid metabolism. Therefore, the recommended ratio of EF in sheep diet should range from 25% to 50%, and the maximum proportion should not exceed 75%.

Profiling Fusobacterium infection at high taxonomic resolution reveals lineage-specific correlations in colorectal cancer

The bacterial genus Fusobacterium promotes colorectal cancer (CRC) development, but an understanding of its precise composition at the species level in the human gut and the relevant association with CRC is lacking. Herein, we devise a Fusobacterium rpoB amplicon sequencing (FrpoB-seq) method that enables the differentiation of Fusobacterium species and certain subspecies in the microbiota. By applying this method to clinical tissue and faecal samples from CRC patients, we detect 62 Fusobacterium species, including 45 that were previously undescribed. We additionally reveal that Fusobacterium species may display different lineage-dependent functions in CRC. Specifically, a lineage (designated L1) including F. nucleatum, F. hwasookii, F. periodonticum and their relatives (rather than any particular species alone) is overabundant in tumour samples and faeces from CRC patients, whereas a non-enriched lineage (designated L5) represented by F. varium and F. ulcerans in tumours has a positive association with lymphovascular invasion.

Amplicon Sequencing ofFusariumTranslation Elongation Factor 1α Reveals that Soil Communities ofFusariumSpecies Are Resilient to Disturbances Caused by Crop and Tillage Practices

The fungal genus Fusarium contains plant pathogens, mutualists, and commensalists. Because nonpathogenic Fusarium strains can contribute to disease suppression in soil by competition or induced plant resistance, identifying factors that influence the abundance of nonpathogenic strains could lead to strategies that enhance plant-beneficial interactions and deter pathogen invasion. We developed a Fusarium-specific high-throughput translation elongation factor 1α (EF-1α) amplicon-sequencing method with subspecies resolution. Newly designed primers enabled reads to be joined for improved quality metrics after Illumina MiSeq sequencing. By including the ‘oligotyping’ program in our bioinformatic workflow, we reconstructed Fusarium EF-1α haplotypes with 100% accuracy. We conducted simulated crop rotation experiments in soil infested with the strawberry pathogen Fusarium oxysporum f. sp. fragariae ( Fof) and the following treatments: strawberry, blackberry, raspberry, lettuce, broccoli, and fallow. We profiled root Fusarium communities with EF-1α amplicons and observed that nonpathogenic strains of F. oxysporum did not colonize crops uniformly; some strains exhibited asymptomatic host preferences. In susceptible strawberry roots, &gt;90% of sequences corresponded to Fof. Bulk soil communities of Fusarium were remarkably stable and had minimal treatment-dependent effects after 1 year. Similarly, in a long-term agricultural research experiment, 18 years of conservation tillage and cover cropping did not lead to bulk soil Fusarium communities that were significantly different than controls. Although the roots of rotation crops can augment the abundance of nonpathogenic F. oxysporum strains, their abundance in bulk soil is unlikely to be altered after a single year. Climate and soil physicochemical properties may have a greater effect on Fusarium community structure.[Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Optimization and Validation of Nanopore Based Sequencing Method for Molecular Testing of CNS Tumours

BackgroundThe World Health Organization (WHO) introduced molecular identifiers for the diagnosis and prognosis of CNS tumors including the mutational status of isocitrate dehydrogenase or IDHgenes in glial tumors. Currently used immunohistochemistry (IHC) is not capable of detecting the non‐canonical mutations, and sequencing is often required as a follow‐up. Current next‐generation sequencing (NGS) technologies used in tumor molecular marker detection introduce key challenges including high capital cost, complex infrastructure requirements, and long turnaround times. These challenges considerably limit the ability to perform NGS testing in many pathology laboratories. In this study, we aimed to use third generation nanopore sequencing technology to resolve these limitations. The Oxford Nanopore MinION, a pocket‐sized nanopore sequencing device, has minimal capital costs and infrastructural requirements, and shorter turnaround times. However, the nanopore technology has not been validated in clinical practice and has not been optimized on formalin‐fixed paraffin‐embedded (FFPE) tissue.MethodsDNA extraction of selective tumor areas was performed from the corresponding FFPE tissue blocks from a cohort of gliomas with confirmed IDH1and IDH2gene statuses (n=65). A PCR amplicon‐based approach was used to amplify hot spots of the IDH1and IDH2genes, starting with 30ng DNA material. The amplicon libraries were sequenced for 2 hours in multiplex on the MinION device and IDHSNPs were called with the Nanopolish software. ASIP Abstract Mashiat MimosaResults26 IDH mutant samples were identified: 21 IDH1R132H, 2 IDH1R132G, 2 IDH2R172G, and 1 IDH2D177H. All cases showed concordant IDHmutational status when compared to the reference methods (IHC or NGS) and both analytical sensitivity and specificity were 100%. Precision analysis of variant allele frequency (VAF) showed the coefficient of variation was less than 5% (both inter and intra runs), and the limit of detection for VAF was 3%. The range of read depth obtained was 882X to 43,000x with an average of 20,000x. This assay revealed a $50‐$100 material cost per sample, and the time taken from extracted nucleic acid to final result generation was 1‐2 business days.ConclusionThis project is the first to optimize and validate an approach to detect SNP mutations in FFPE samples using nanopore technology. It has demonstrated the feasibility and efficacy of the nanopore amplicon sequencing method in cancer FFPE tissue with excellent test performance characteristics, significantly shorter turnaround times at considerably lower costs and without any infrastructural needs. Thus, it can be used to circumvent challenges to current NGS testing platforms and can be the milestone that would make cancer NGS testing available for every laboratory.

The invasive tree Piper aduncum alters soil microbiota and nutrient content in fallow land following small scale slash-and-burn farming in tropical lowland forest in Papua New Guinea

Piper aduncum, a tree species native to the Neotropics, has been introduced to other tropical regions and successfully invades secondary forest in fallow land after small scale slash-and-burn agriculture in Papua New Guinea (PNG). However, the effect of P. aduncum invasion on soil chemical properties as well as soil biota remains poorly understood. To fill this knowledge gap, we compared soil chemistry, soil microbiota and soil fauna between sites invaded by P. aduncum and sites with secondary native vegetation, where P. aduncum absent. Soils under P. aduncum had significantly lower P content at 0–5 cm depth, lower NO3− at 5–10 cm depth and lower N and C content at both depths compared to soil under secondary vegetation where P. aduncum absent. Sites invaded by P. aduncum also harbored lower microbial biomass measured by phospholipid fatty acid (PLFA) analysis, especially at 5–10 cm depth. The composition of microbial communities, based on PLFA and amplicon sequencing methods, also differed between soils invaded and uninvaded by P. aduncum, while soil macrofauna did not show any significant difference in the density of various faunal groups between invaded and uninvaded sites. Furthermore, we studied soil chemistry and foliar nitrogen in food gardens established after clearing of secondary vegetation in sites where P. aduncum had been experimentally excluded for five years. These gardens did not differ in soil chemistry compared to the control gardens where P. aduncum was not cleared. However, P. aduncum removal was associated with a higher N content in the leaves of sweet potatoes (Ipomoea batatas), but not bananas (Musa spp.). Our results suggest that P. aduncum has negative effect on soil microbiota and may slowdown nutrient turnover and availability as well as growth of plants on invaded soil. This finding requires further attention and may have practical implication for food gardening in tropical rainforests.