Amplicon Libraries Research Articles

Establishment and Application of a Multiplex PCR NGS Method for the Genotyping of HLA-Class I and HPA.

Selecting compatible HLA-Class I and/or HPA platelets based on genotyping could alleviate immune platelet transfusion refractoriness (PTR). A fast and reliable method of HLA-Class I and HPA genotyping is necessary to construct a platelet donor bank with known HLA-Class I and HPA genotypes. Ten pairs of specific primers for HLA-A, HLA-B, HLA-C, HPA-1 through HPA-6w, HPA-15 and HPA-21w were designed. The appropriate fragments were optimised for amplification in a single multiplex reaction. After a cleanup step using paramagnetic beads, the amplicon library was prepared and sequenced. To validate the accuracy of the developed method, commercial NGS kits for the genotyping of HLA-A, HLA-B and HLA-C and the TaqMan real-time PCR method in-house for the genotyping of HPA-1 through HPA-6w, HPA-15 and HPA-21w were used to detect all the specimens in parallel. A total of 386 specimens were detected and the results of genotyping HLA-A, HLA-B, HLA-C and HPA-1 through HPA-6w, HPA-15 and HPA-21w were obtained simultaneously, which is 100% consistent between the two methods. Four new HLA alleles, HLA-A*11:451, HLA-A*30:01:26, HLA-B*39:201 and HLA-B*40:538, were also reconfirmed. Two novel SNVs, c.2671C > T and c.2681T > G, in the coding region of ITGA2B were detected, all of which are heterozygous in individuals. A novel NGS method based on multiplex PCR was established to detect HLA-Class I and HPA simultaneously, which is high-throughput, rapid and accurate and could be applied to build a platelet donor bank.

Generation of Barcode-Tagged Vibrio fischeri Deletion Strains and Barcode Sequencing (BarSeq) for Multiplex Strain Competitions.

Vibrio fischeri is a model mutualist for studying molecular processes affecting microbial colonization of animal hosts. We present a detailed protocol for a barcode sequencing (BarSeq) approach that combines targeted gene deletion with short-read sequencing technology to enable studies of mixed bacterial populations. This protocol includes wet lab steps to plan and produce the deletions, approaches to scale up mutant generation, protocols to prepare and conduct the strain competition, library preparation for sequencing on an Illumina iSeq 100 instrument, and data analysis with the barseq python package. Aspects of this protocol could be readily adapted for tagging wild-type V. fischeri strains with a neutral barcode for examination of population dynamics or BarSeq analyses in other species. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Production of the erm-bar DNA Basic Protocol 2: Generation of a targeted and barcoded deletion strain of V. fischeri Alternate Protocol: Parallel generation of multiple barcode-tagged V. fischeri deletion strains Basic Protocol 3: Setting up mixed populations of barcode-tagged strains Basic Protocol 4: Performing a competitive growth assay Basic Protocol 5: Amplicon library preparation and equimolar pooling Basic Protocol 6: Sequencing on Illumina iSeq 100 Basic Protocol 7: BarSeq data analysis.

Metagenomic analyses of a consortium for the bioremediation of hydrocarbons polluted soils.

A bacterial consortium was isolated from a soil in Noblejas (Toledo, Spain) with a long history of mixed hydrocarbons pollution, by enrichment cultivation. Serial cultures of hydrocarbons polluted soil samples were grown in a minimal medium using diesel (1mL/L) as the sole carbon and energy source. The bacterial composition of the Noblejas Consortium (NC) was determined by sequencing 16S rRNA gene amplicon libraries. The consortium contained around 50 amplicon sequence variants (ASVs) and the major populations belonged to the genera Pseudomonas, Enterobacter, Delftia, Stenotrophomonas, Achromobacter, Acinetobacter, Novosphingobium, Allorhizobium-Neorhizobium-Rhizobium, Ochrobactrum and Luteibacter. All other genera were below 1%. Metagenomic analysis of NC has shown a high abundance of genes encoding enzymes implicated in aliphatic and (poly) aromatic hydrocarbons degradation, and almost all pathways for hydrocarbon degradation are represented. Metagenomic analysis has also allowed the construction of metagenome assembled genomes (MAGs) for the major players of NC. Metatranscriptomic analysis has shown that several of the ASVs are implicated in hydrocarbon degradation, being Pseudomonas, Acinetobacter and Delftia the most active populations.

PSX-14 Effect of starch supplementation on rumen bacterial abundance in Bos taurus taurus steers consuming low-quality forage

Abstract Low-quality forage provides a limited supply of ruminally available nitrogen and is generally resistant to digestion, inhibiting microbial growth in the rumen and ultimately decreasing forage utilization. Supplementation with protein is well documented to increase forage utilization by promoting microbial growth. Even when protein requirements are met, additional energy, often provided as starch, may be required to meet performance expectations. The objective of this study was to evaluate the effect of increasing starch supplementation on rumen bacterial populations in Bos taurus taurus steers supplemented protein and consuming low-quality forage. Ruminal samples were collected from ruminally-cannulated Angus steers [n = 5; body weight (BW) = 375 ± 45 kg] used in a 4 × 4 Latin square fed a basal diet of King Ranch Bluestem Hay (3.5% CP, 73% NDF) ad libitum, and supplemented with one of four isonitrogenous (130 mg N/kg BW) supplements with increasing starch (2% starch = 100% soybean meal; 20% starch = 73% soybean meal, 26.2% corn, 0.8% urea; 38% starch = 47% soybean meal, 51.6% corn, 1.4% urea; 56% starch = 19% soybean meal, 78.6% corn, 2.4% urea). Rumen content samples were collected via rumen cannula 4 h after supplementation and immediately frozen. Microbial DNA was extracted; full length 16S rRNA amplicon libraries were prepared using Kinnex kits and sequenced on the PacBio Revio platform. Reads were analyzed using DADA2 and QIIME2. Statistical analysis was performed with the GLIMMIX procedure of SAS 9.4 (SAS Inst. Inc., Cary, NC). Firmicutes and Bacteriodata were the most abundant phyla in all treatments (82% and 11%, respectively), and were not affected by starch supplementation (P ≥ 0.45). Actinobacteriota and Euryarchaeota abundance responded quadratically (P < 0.01) to increasing starch provision. Christensenellaceae R-7 group, Rikenellaceae RC9 gut group, Lachnospiraceae XPB1014 group, Butyrvibrio, Lachnospiraceae NK3A20 group, and Prevotella represented the most abundant genera for all treatments. Increasing supplemental starch resulted in a quadratic response (P < 0.01) for abundance of Christensenellaceae R-7 and Lachnospiraceae XPB1014 groups. Relative abundance of Butyrvibrio was 4.05% for steers fed 2% starch supplement and increased to 18.24 and 11.85% for 20 and 38% starch, respectively and then decreased to 4.34% for steers fed 56% starch supplement (quadratic, P < 0.01) Abundance of Pseudobutyrvibrio followed a similar but less dramatic pattern (quadratic, P < 0.01). In contrast, abundance of Streptococcus and Methanobrevibacter decreased with the first two levels of starch and then increased with 56% (quadratic; P < 0.01). In conclusion, responses to the first two increments of starch supplementation suggest increased fibrolytic and decreased methanogenic activity. These results are supported by the previously reported increase in TDOMI for 20 and 38% starch 49.0 and 48.2 g/kg MBW, respectively versus 45.7 and 44.3 g/kg MBW for 2 and 56%, respectively.

PSX-15 Effect of starch supplementation on rumen bacterial abundance in Bos taurus indicus steers consuming low-quality forage

Abstract Deficient ruminally available nitrogen caused by a basal diet of low-quality forage can limit microbial growth in the rumen and decrease forage utilization. Protein supplementation is well-documented to improve forage digestibility and performance. Additionally, starch has been evaluated to increase energy supply to grazing cattle when the forage cannot support adequate levels of production. However, most research in this area has been conducted in Bos taurus taurus, especially the response of the rumen microbiome to supplementation. Accordingly, the objective of this study was to evaluate the effects of increasing starch supplementation on rumen bacterial populations in Bos taurus indicus steers supplemented protein and consuming low-quality forage. Ruminal samples were collected from five ruminally-cannulated Brahman steers (BW 420 ± 55 kg) used in a 4×4 Latin square fed a basal diet of King Ranch Bluestem Hay (3.5% CP and 73% NDF) provided ad libitum, and one of four isonitrogenous (130 mg N/kg BW) supplements with increasing levels of starch (2% starch= 100% soybean meal; 20% starch= 73% soybean meal, 26.2% corn, 0.8% urea; 38% starch = 47% soybean meal, 51.6% corn, 1.4% urea; 56% starch= 19% soybean meal, 78.6% corn, 2.4% urea). Rumen content samples were collected via rumen cannula at 4h post-feeding. Microbial DNA was extracted; full length 16S rRNA amplicon libraries were prepared using Kinnex kits and then sequenced on the PacBio Revio platform. Reads were analyzed using DADA2 and QIIME2. Statistical analysis was performed with the GLIMMIX procedure of SAS 9.4 (SAS Inst. Inc., Cary, NC). Firmicutes, Bacteroidata, Actinobacteriota, Verrucomicrobiota, and Euryarchaeota were the most abundant phyla in all treatments (83, 10, 3, 0.4, and 1%, respectively), and were not affected by treatment (P ≥ 0.10). Christensenellaceae R-7 group, Rikenellaceae RC9 gut group, Lachnospiraceae-XPB1014-group, Butyrvibrio, Lachnospiraceae-NK3A20-group, and Prevotella represented the most abundant genera for all treatments (7, 5, 11, 9, 7, and 3%, respectively). Lachnospiraceae-XPB1014-group and Lachnospiraceae-NK3A20-group responded quadratically (P ≤ 0.02) to increasing starch with decreased abundance for the 20% and 38% starch supplement. Comparatively, relative abundance of Butyrvibrio responded quadratically (P < 0.01) with increased abundance in the 20% and 38% starch supplement. Pseudobutyrvibrio increased with supplementation of 20% starch and then decreased as starch supplementation increased (quadratic, P = 0.04). In conclusion, supplementation of starch elicited changes in microbial relative abundance at the genus level with the response in Butyrivibrio suggesting greater fibrolytic activity. Forage and total digestible NDF intake in these steers decreased with increased starch supplementation; however, TDOMI was not affected by starch provision.

Influence of Bacterial Fertilizers on the Structure of the Rhizospheric Fungal Community of Cereals South of Western Siberia

The general lack of knowledge on the conditions of Western Siberia (Omsk region) and the taxonomic diversity of zonal soils determines the relevance of these studies. The research was carried out in order to study the effect of complex biologics on the taxonomic diversity of the fungal component of the microbiome of the rhizosphere of cereals and the phytosanitary condition of crops in the southern forest-steppe (meadow-chernozem soil) and subtaiga (gray forest soil) zones of the Omsk Irtysh region (Western Siberia). This work was carried out in 2022–2023, using laboratory studies in combination with field experiments and metagenomic and statistical analyses. The objects of research were varieties of cereals and grain forage crops of Omsk selection: soil microorganisms. The scheme of the experiment involved the study of the following options: varieties of cereals (factor A): spring soft wheat—Omsk 42, Omsk 44, Tarskaya 12; durum wheat—Omsk coral; barley—Omsk 101; oats—Siberian hercules; bacterial preparation for seed inoculation (factor B) without the drug—Mizorin and Flavobacterin. The sampling of the plant rhizosphere for metagenomic analysis was carried out during the earing phase (July). For the first time, the taxonomic composition of the fungal community was determined based on the analysis of amplicon libraries of fragments of ribosomal operons of ITS2 fungi during colonization of crop roots by nitrogen-fixing bacteria in various soil and climatic zones of the Omsk region. The fungal component of the microbiome was analyzed in two zones of the Omsk region (southern forest-steppe and subtaiga). The five dominant phyla of soil fungi were located in the following decreasing series: Ascomycota (about 70%) > Mortierellomycota (about 7%) > Basidiomycota (about 5%) > Mucoromycota (3%) > Chytridiomycota (1%). The five main genera of fungi inhabiting the rhizosphere of cereals are located in a decreasing row: Giberella (6.9%) > Mortierella (6.6%) > Chaetomium (4.8%) > Cladosporium (3.8%) > Rhizopus (3.3%). The predominantly positive effect of biologics of associative nitrogen fixation on the fungal community of the soil (rhizosphere) of experimental sites located in different soil and climatic zones has been established. During seed bacterization, the growth of saprotrophic fungal genera was noted in relation to the control variants Pseudogymnoascus, Chloridium, Clonostachys, Trihoderma, etc., and the fungicidal properties of bacterial strains introduced into the soil were actively manifested relative to phytopathogenic fungi of the genera Alternaria, Blumeria, Fusarium, etc. According to the results of determining the number of infectious structures of Rhizoctonia solani, it was found that the population of the soil with viable cells of this pathogen was 1–3 pcs/g (below the threshold of harmfulness, PV 20 pcs/g of soil), which indicates a favorable phytosanitary situation with respect to the pathogen. The fungicidal effect of the applied bacterial fertilizers on Rhizoctonia solani could not be detected. The number of Bipolaris sorokiniana varied depending on the drug used. In the conditions of the southern forest-steppe zone of the Omsk region (meadow-chernozem soil), the greatest fungicidal effect was noted in Flavobacterin application variants on wheat of the Omsk 42 variety, durum wheat of the Omsk coral variety, and barley; the decrease in conidia relative to the control was 73, 35, and 29%, respectively. In the subtaiga zone of the Omsk Irtysh region (gray forest soil), as in the southern forest-steppe zone, pre-sowing bacterization of seeds with Flavobacterin led to a decrease in Bipolaris sorokiniana in the rhizosphere of wheat of the Omsk 42 variety by 18%, and oats by 27%, to control. The use of the drug Mizorin in some variants of the experiment led to an insignificant decrease in the harmful fungus or had no effect at all.

Dynamics of Cellulose Degradation by Soil Microorganisms from Two Contrasting Soil Types.

The search for active cellulolytic consortia among soil microorganisms is of significant applied interest, but the dynamics of the formation of such communities remain insufficiently studied. To gain insight into the formation of an active cellulolytic community, the experiment was designed to examine the colonization of a sterile substrate (cellulose) by microorganisms from two soil types: sod-podzolic and chernozem. To achieve this, the substrate was placed in the soil and incubated for six months. To assess microbiome dynamics, the experiment employed sequencing of 16S rRNA gene fragment and ITS2 amplicon libraries at four time points. It was demonstrated that, from the second month of the experiment, the prokaryotic component of the communities reached a state of stability, with a community composition specific to each soil type. The results demonstrated no relationship between changes in community diversity and soil respiration. There also was no significant shift in the community diversity throughout the chronosequence. Furthermore, the taxonomic composition of the community shifted towards a decrease in the proportion of Pseudomonadota and an increase in representatives of the Bacteroidota, Bacillota, and Verrucomicrobiota phyla. The network analysis of the community demonstrated that, in contrast to sod-podzolic soil, chernozem is distinguished by a higher modularity, with the formation of taxon-specific groups of microorganisms at each stage of the chronoseries. These differences are attributed to the alterations in the eukaryotic component of the community, particularly in the prevalence of nematodes and predatory fungi, which in turn influenced the cellulolytic community.

Identification of 8 candidate microsatellite instability loci in colorectal cancer and validation of the ACVR2A mechanism in the tumor progression

This study probes the utility of biomarkers for microsatellite instability (MSI) detection and elucidates the molecular dynamics propelling colorectal cancer (CRC) progression. We synthesized a primer panel targeting 725 MSI loci, informed by The Cancer Genome Atlas (TCGA) and ancillary databases, to construct an amplicon library for next-generation sequencing (NGS). K-means clustering facilitated the distillation of 8 prime MSI loci, including activin A receptor type 2A (ACVR2A). Subsequently, we explored ACVR2A’s influence on CRC advancement through in vivo tumor experiments and hematoxylin–eosin (HE) staining. Transwell assays gauged ACVR2A’s role in CRC cell migration and invasion, while colony formation assays appraised cell proliferation. Western blotting illuminated the impact of ACVR2A suppression on CRC’s PI3K/AKT/mTOR pathway protein expressions under hypoxia. Additionally, ACVR2A’s influence on CRC-induced angiogenesis was quantified via angiogenesis assays. K-means clustering of NGS data pinpointed 32 MSI loci specific to tumor and DNA mismatch repair deficiency (dMMR) tissues. ACVR2A emerged as a pivotal biomarker, discerning MSI-H tissues with 90.97% sensitivity. A curated 8-loci set demonstrated 100% sensitivity and specificity for MSI-H detection in CRC. In vitro analyses corroborated ACVR2A’s critical role, revealing its suppression of CRC proliferation, migration, and invasion. Moreover, ACVR2A inhibition under CRC-induced hypoxia markedly escalated MMP3, CyclinA, CyclinD1, and HIF1α protein expressions, alongside angiogenesis, by triggering the PI3K/AKT/mTOR cascade. The 8-loci ensemble stands as the optimal marker for MSI-H identification in CRC. ACVR2A, a central element within this group, deters CRC progression, while its suppression amplifies PI3K/AKT/mTOR signaling and angiogenesis under hypoxic stress.

Determining the footprint of breeding in the seed microbiome of a perennial cereal

BackgroundSeed endophytes have a significant impact on plant health and fitness. They can be inherited and passed on to the next plant generation. However, the impact of breeding on their composition in seeds is less understood. Here, we studied the indigenous seed microbiome of a recently domesticated perennial grain crop (Intermediate wheatgrass, Thinopyrum intermedium L.) that promises great potential for harnessing microorganisms to enhance crop performance by a multiphasic approach, including amplicon and strain libraries, as well as molecular and physiological assays.ResultsIntermediate wheatgrass seeds harvested from four field sites in Europe over three consecutive years were dominated by Proteobacteria (88%), followed by Firmicutes (10%). Pantoea was the most abundant genus and Pantoea agglomerans was identified as the only core taxon present in all samples. While bacterial diversity and species richness were similar across all accessions, the relative abundance varied especially in terms of low abundant and rare taxa. Seeds from four different breeding cycles (TLI C3, C5, C704, C801) showed significant differences in bacterial community composition and abundance. We found a decrease in the relative abundance of the functional genes nirK and nifH as well as a drop in bacterial diversity and richness. This was associated with a loss of amplicon sequence variants (ASVs) in Actinobacteria, Alphaproteobacteria, and Bacilli, which could be partially compensated in offspring seeds, which have been cultivated at a new site. Interestingly, only a subset assigned to potentially beneficial bacteria, e.g. Pantoea, Kosakonia, and Pseudomonas, was transmitted to the next plant generation or shared with offspring seeds.ConclusionOverall, this study advances our understanding of the assembly and transmission of endophytic seed microorganisms in perennial intermediate wheatgrass and highlights the importance of considering the plant microbiome in future breeding programs.

Microbiome Modulation in Acne Patients and Clinical Correlations.

The imbalance of skin microbiota in acne can induce changes leading to induction or to aggravation of chronic inflammatory lesions; complex mechanisms are involved. Cutibacterium acnes (C. acnes) ribotypes RT4 and RT5 express more biofilm and are associated with inflammatory acne lesions. C. acnes RT6 is a non-acne ribotype, beneficial for the skin. In an open clinical trial, acne adults were included and assessed clinically at baseline and at month 2 using the Investigator Global Assessment of Acne (IGA) score. A topical emulsion was applied twice daily for 2 months (M2) in each included patient. In the same series of acne patients, skin swab samples were collected from acne patients at baseline and M2 from lesional and non-lesional skin; skin swabs were collected for the metagenomic long-read analysis of microbiota. Acne patients with a gravity score IGA of >1<3 were included in this pilot study. An emulsion of O/W formulated with vegetal extract of Umbelliferae associated with a polysaccharide at 1% was applied twice daily for 2 months. At baseline and M2 clinical assessments were made; skin swab samples were also taken for microbiota analysis from lesional and non-lesional skin in each included patient. Extractions of genomic DNA (gDNA) from swab samples from baseline and from M2 were made, followed by full-length (V1-V9) amplification of the 16S rDNA and sequencing of amplicon libraries for strain-level bacterial community profiling. In a series of 32 adult acne patients, the mean initial IGA scale was 3.1; at M2 the IGA scale was 1.5 (p < 0.001). The mean decrease in acne lesions was by 63%. Microbiome metagenomic long-read analysis in these series was mainly dominated by C. acnes followed by Staphylococcus epidermidis (S. epidermidis). The density of C. acnes ribotypes RT6 (non-acne strain) was increased at M2 compared to baseline and the density of ribotypes C. acnes RT1 to RT5 was decreased at M2, compared to baseline (p < 0.0001). S. epidermidis ribotypes (1 to 36) were non significantly increased at M2, compared to baseline (p < 0.1). In a series of 32 acne patients that applied an emulsion based on vegetal extract of Umbelliferae and a polysaccharide at 1% twice daily, a significant clinical improvement in IGA scale for acne lesions was seen at M2, compared to baseline (p < 0.0001). The clinical improvement was correlated with an improvement in skin microbiome at M2 compared to baseline, indicated by the increase in the relative abundance of non-acne strain of C. acnes ribotype 6 and of the decrease in the relative abundance of acne strains ribotypes C. acnes RT1 to RT5.

VAMPirus: A versatile amplicon processing and analysis program for studying viruses.

Amplicon sequencing is an effective and increasingly applied method for studying viral communities in the environment. Here, we present vAMPirus, a user-friendly, comprehensive, and versatile DNA and RNA virus amplicon sequence analysis program, designed to support investigators in exploring virus amplicon sequencing data and running informed, reproducible analyses. vAMPirus intakes raw virus amplicon libraries and, by default, performs nucleotide- and amino acid-based analyses to produce results such as sequence abundance information, taxonomic classifications, phylogenies and community diversity metrics. The vAMPirus analytical framework leverages 16 different opensource tools and provides optional approaches that can increase the ratio of biological signal-to-noise and thereby reveal patterns that would have otherwise been masked. Here, we validate the vAMPirus analytical framework and illustrate its implementation as a general virus amplicon sequencing workflow by recapitulating findings from two previously published double-stranded DNA virus datasets. As a case study, we also apply the program to explore the diversity and distribution of a coral reef-associated RNA virus. vAMPirus is streamlined within Nextflow, offering straightforward scalability, standardization and communication of virus lineage-specific analyses. The vAMPirus framework is designed to be adaptable; community-driven analytical standards will continue to be incorporated as the field advances. vAMPirus supports researchers in revealing patterns of virus diversity and population dynamics in nature, while promoting study reproducibility and comparability.

PSV-12 Microbial community structure of dairy cattle is stable through oscillating crude protein inclusion

Abstract Diet has an important role in shaping the structure of rumen microbial communities. However, little is known about the interaction between dietary crude protein (CP) level and CP feeding pattern on the rumen microbiome of dairy cows. Here, the objective was to evaluate the influence of CP level (low protein [LP], 13.8%; high protein [HP], 15.5% of dry matter [DM] basis) and CP feeding pattern (OF = oscillating, SF = static) on the composition and diversity of the rumen microbiome of lactating dairy cows using 16S rRNA gene amplicon sequencing. Rumen-cannulated Holstein cows (n = 8) in mid- to late-lactation were assigned to a 2 x 2 factorial experiment consisting of 4 experimental periods of 28 d. Each experimental period had an adaptation phase to the diet (24 d) and a sampling phase (d 25 to 28). The treatments with oscillating feeding alternated diets every 48 h to vary CP above and below the mean CP level (OF-LP = 13.8 ± 1.8%; OF-HP = 15.5 ± 1.8% CP of DM) whereas diets were constant in the static feeding (SF-LP = 13.8%; SF-HP = 15.5% CP of DM). According to the NASEM (2021) model predictions the LP diet supplied 94% of the microbial protein requirement while the HP diet exceeded (104%) requirement. Rumen samples were collected 4 h after feeding through a rumen cannula and amplicon libraries were generated targeting the V4 region of the 16S rRNA gene. The amplicon sequences were analyzed in qiime2. Sequences where trimmed based on their quality and then grouped in ASVs using DADA2. The ASVs were used to analyze the composition and diversity of the rumen microbiome in each treatment. Alpha diversity of the rumen microbiome did not change across treatments (Kruskal Wallis, P &amp;gt; 0.05), but Pielou’s evenness increased by 0.01 units (Kruskal Wallis, P = 0.04). These results support the hypothesis that the rumen microbiome is stable to oscillating CP feeding patterns and compensates for the changes in crude protein content in the diet. Further research is required to understand the dynamics of this compensation and what other metabolic processes in the ruminant contribute to maintaining production under an oscillating diet.

High-throughput DNA extraction and cost-effective miniaturized metagenome and amplicon library preparation of soil samples for DNA sequencing.

Reductions in sequencing costs have enabled widespread use of shotgun metagenomics and amplicon sequencing, which have drastically improved our understanding of the microbial world. However, large sequencing projects are now hampered by the cost of library preparation and low sample throughput, comparatively to the actual sequencing costs. Here, we benchmarked three high-throughput DNA extraction methods: ZymoBIOMICS™ 96 MagBead DNA Kit, MP BiomedicalsTM FastDNATM-96 Soil Microbe DNA Kit, and DNeasy® 96 PowerSoil® Pro QIAcube® HT Kit. The DNA extractions were evaluated based on length, quality, quantity, and the observed microbial community across five diverse soil types. DNA extraction of all soil types was successful for all kits, however DNeasy® 96 PowerSoil® Pro QIAcube® HT Kit excelled across all performance parameters. We further used the nanoliter dispensing system I.DOT One to miniaturize Illumina amplicon and metagenomic library preparation volumes by a factor of 5 and 10, respectively, with no significant impact on the observed microbial communities. With these protocols, DNA extraction, metagenomic, or amplicon library preparation for one 96-well plate are approx. 3, 5, and 6 hours, respectively. Furthermore, the miniaturization of amplicon and metagenome library preparation reduces the chemical and plastic costs from 5.0 to 3.6 and 59 to 7.3 USD pr. sample. This enhanced efficiency and cost-effectiveness will enable researchers to undertake studies with greater sample sizes and diversity, thereby providing a richer, more detailed view of microbial communities and their dynamics.

Microbial Community Changes in Silkworms Suspected of Septicemia and Identification of Serratia sp.

Diseases that occur in silkworms include soft rot, hardening disease, digestive diseases, and sepsis. However, research on the causes of bacterial diseases occurring in silkworms and the resulting changes in the microbial community is lacking. Therefore, we examined the morphological characteristics of sepsis and changes in the microbial community between silkworms that exhibit a unique odor and healthy silkworms; thus, we established a relationship between disease-causing microorganisms and sepsis. After producing a 16S rRNA amplicon library for samples showing sepsis, we obtained information on the microbial community present in silkworms using next-generation sequencing. Compared to that in healthy silkworms, in silkworms with sepsis, the abundance of the Firmicutes phylum was significantly reduced, while that of Proteobacteria was increased. Serratia sp. was dominant in silkworms with sepsis. After bacterial isolation, identification, and reinfection through the oral cavity, we confirmed this organism as the disease-causing agent; its mortality rate was 1.8 times higher than that caused by Serratia marcescens. In summary, we identified a new causative bacterium of silkworm sepsis through microbial community analysis and confirmed that the microbial community balance was disrupted by the aberrant proliferation of certain bacteria.

Low cycle number multiplex PCR: A novel strategy for the construction of amplicon libraries for next-generation sequencing.

Multiplex PCR is a critical step when preparing amplicon library for next-generation sequencing. However, there are several challenges related to multiplex PCR including poor uniformity, nonspecific amplification, and primer-dimers. To address these issues, we propose a novel solution strategy that involves using a low cycle number (<10 cycles) in multiplex PCR and then employing carrier DNAs and magnetic beads for the selection of targeted products. This technique improves the amplicon uniformity while also reducing primer-dimers and PCR artifacts. To evaluate our technique, we initially utilized 120 DNA fragments from mouse genome containing single nucleotide polymorphism (SNP) sites. Sequencing results demonstrated that with only 7 cycles of multiplex PCR, 95.8% of the targeted SNP sites were mapped, with a coverage of at least 1×. The average sequencing depth of all amplicons was 1705.79±1205.30×; 87% of them reached a coverage depth that exceeded 0.2-fold of the average sequencing depth. Our method had a greater uniformity (87%) when compared to Hi-Plex PCR (53.3%). Furthermore, we validated our strategy by randomly selecting 90 primer pairs twice from the initial set of 120 primer-pairs. Next, we used the same protocol to prepare amplicon libraries. The two groups had an average sequencing depth of 1013.30±585.57× and 219.10±158.27×, respectively; over 84% of the amplicons had a sequencing depth that exceeded 0.2-fold of average depth. These results suggest that the use of a low cycle number in multiplex PCR is a cost-effective and efficient approach for the preparation of amplicon libraries.

ASV (Amplicon Sequence Variant) taxonomic affiliation analysis of &lt;i&gt;Cryptosporidium scrofarum&lt;/i&gt; species in pigs in the Vologda Region, the Northwestern Federal District of the Russian Federation

The purpose of the research is isolation, identification, and analysis of ASV (Amplicon Sequence Variant) types of Cryptosporidia spp. in pigs in the Vologda Region of the Russian Federation.Materials and methods. The research has been conducted in the Russian Federation for the first time. The research was conducted on pig farms in the Vologda Region of the Northwestern Federal District of the Russian Federation from January to October 2023. Feces were taken from piglets of various age groups, as well as milking sows. The samples were studied using the equipment of the resource center “Genomic Technologies, Proteomics and Cell Biology” of ARRIAM. Species of the genus Cryptosporidia were identified in fecal samples using high-throughput sequencing of 18S rRNA gene fragment amplicon libraries as obtained from nested PCR followed by “denoising”, sequence combining, and restoring the original phylotypes (ASV, (Amplicon Sequence Variant)).Results and discussion. Cryptosporidia spp. species were identified in each age group studied. As a result of high-throughput sequencing of the libraries using the Illumina technology, 20 to 100 thousand nucleotide sequences (reads) were obtained for each sample after processing of which a total of 2,372 ASVs were identified. The analysis of the ASV taxonomic affiliation performed with phylogenetic analysis supplemented by an analysis using the blastn algorithm in the GenBank database showed that, in total, 10 ASVs were only present in all studied samples that had high similarity to sequences deposited in the GenBank as 18S rRNA gene fragments of Cryptosporidium scrofarum. Eight ASV types were unique and did not repeat from farm to farm. Probably, these sequences belong to local populations of C. scrofarum subspecies. Of interest is the discovery of a unique Cryptosporidium sequence of ASV8 type which is only 91.47% similar to the closest relative of the genus, which may indicate a rather distant taxonomic relationship. This type of nucleotide sequence can be further described as a new species. All identified unique ASV nucleotide sequences were deposited in GenBank.

Alpha Diversity Indices as Indicators of the Variability of Gut Microbiota in Obese Adolescents of Different Ethnicities.

We compared alpha diversity indices of the intestinal microbiota in adolescents with obesity and normal body weight, taking into account their ethnicity. Intestinal biocenosis was studied by metasequencing of amplicon libraries of V3-V4 fragments of the 16S rRNA gene. The alpha diversity of the microbiota was assessed using classical and alternative indices. Statistically significant differences in intestinal microbiota were observed between Russians with obesity and Buryats with normal body weight, as well as between Russians with obesity and Buryats with obesity when assessing the Shannon-Weaver, Chao1 indices, Faith phylogenetic diversity index, ACE, Fisher, Gini coefficient, Margalef, and Menkhinik indices. It was shown that alpha diversity indices can be used to assess significance of differences and variability of the intestinal microbiota in multifactorial diseases such as obesity in adolescents; however, the scope of application of the criteria should be considered.

Alterations in the gut microbiome of whiteleg shrimp (Penaeus vannamei) postlarvae following exposure to an AHPND-causing strain of Vibrio parahaemolyticus

Acute hepatopancreatic necrosis disease (AHPND), attributed to the production of PirA/PirB toxins by certain Vibrio sp. strains, poses a significant threat to global shrimp aquaculture, causing substantial mortality and economic losses. To enhance our understanding of this disease within a closed culture system on the northern coast of Peru, we conducted a comparative analysis of the gut microbiomes between healthy and diseased postlarvae. Diseased postlarvae were obtained through exposure to an AHPND-causing strain of Vibrio parahaemolyticus. Five healthy and five diseased postlarvae were randomly sampled from experimental rearing tanks, and their medial guts were extracted. High-throughput sequencing targeting the V4 region of the 16S rRNA gene was employed for amplicon library construction, and assessments of alpha and beta diversities and taxonomic composition were conducted. Our results revealed reduced diversity and distinct compositional profiles in the gut microbiomes of diseased postlarvae. The order Rhodobacteriales was dominant in the gut microbiomes of healthy postlarvae. In contrast, the order Vibrionales (including an unassigned genus within Vibrionales, Vibrio, and Pseudoalteromonas) exhibited the highest abundance in diseased postlarvae. In conclusion, exposure to an AHPND-causing strain of V. parahaemolyticus induces significant dysbiosis in the gut microbiome of whiteleg shrimp postlarvae.

Dead in the water – Role of relic DNA and primer choice for targeted sequencing surveys of anaerobic sewage sludge intended for biological monitoring

DNA-based monitoring of microbial communities that are responsible for the performance of anaerobic digestion of sewage wastes has the potential to improve resource recoveries for wastewater treatment facilities. By treating sludge with propidium monoazide (PMA) prior to amplicon sequencing, this study explored how the presence of DNA from dead microbial biomass carried over with feed sludge may mislead process-relevant biomarkers, and whether primer choice impacts such assessments. Four common primers were selected for amplicon preparation, also to determine if universal primers have sufficient taxonomic or functional coverage for monitoring ecological performance; or whether two domain-specific primers for Bacteria and Archaea are necessary. Anaerobic sludges of three municipal continuously stirred-tank reactors in Victoria, Australia, were sampled at one time-point. A total of 240 amplicon libraries were sequenced on a Miseq using two universal and two domain-specific primer pairs. Untargeted metabolomics was chosen to complement biological interpretation of amplicon gene-based functional predictions. Diversity, taxonomy, phylogeny and functional potentials were systematically assessed using PICRUSt2, which can predict community wide pathway abundance. The two chosen universal primers provided similar diversity profiles of abundant Bacteria and Archaea, compared to the domain-specific primers. About 16 % of all detected prokaryotic genera covering 30 % of total abundances and 6 % of PICRUSt2-estimated pathway abundances were affected by PMA. This showed that dead biomass in the anaerobic digesters impacted DNA-based assessments, with implications for predicting active processes, such as methanogenesis, denitrification or the identification of organisms associated with biological foams. Hence, instead of running two sequencing runs with two different domain-specific primers, we propose conducting PMA-seq with universal primer pairs for routine performance monitoring. However, dead sludge biomass may have some predictive value. In principal component analysis the compositional variation of 239 sludge metabolites resembled that of 'dead-plus-alive' biomass, suggesting that dead organisms contributed to the potentially process-relevant sludge metabolome.

High-throughput analysis using 16S rRNA gene of bacterial communities present in selected bivalves and gastropods species from Bayug Island, Iligan City, Philippines

Abstract. Albarido NA, Tabugo SR. 2024. High-throughput analysis using 16S rRNA gene of bacterial communities present in selected bivalves and gastropods species from Bayug Island, Iligan City, Philippines. Biodiversitas 25: 431-438. Seashells, which include bivalves and gastropods, have global recognition for their significant contributions to the economy, ecology, and medicine. They hold value as a food source and are highly regarded as effective biological indicators. The objective of this study is to identify the bacterial communities present in selected edible species of bivalves (Pinctada margaritifera Linnaeus, 1758 and Anadara granosa Linnaeus, 1758) and gastropods (Canarium urceus Linnaeus, 1758 and Conus stercusmuscarum Linnaeus, 1758), through high-throughput sequencing metabarcoding. Bacterial samples were collected via a swabbing technique on the surface and inside parts of selected mollusc species, which were then placed on sterilized seawater for DNA extraction. Genomic DNA was isolated from the samples, and the V3-V4 region of the 16S rRNA gene was amplified and sequenced using the Illumina MiSeq platform. Four amplicon libraries were generated, representing the two bivalve and two gastropod species in the study area. Data analysis was conducted using the Parallel Meta Suite software. Upon quality control and processing, 173,489 amplicon sequence variants (ASVs) were obtained. Within the bacterial community, the most abundant genera included Stenotrophomonas, Vibrio, Serratia, Photobacterium, and Shewanella. The assessment of alpha diversity, using the Shannon index, indicated a higher diversity in A. granosa. Furthermore, the analysis using the PICRUSt algorithm within the Parallel Meta Suite unveiled the involvement of specific bacteria found in the selected gastropod and bivalve species in various functions. These functions encompass protein production, xenobiotic metabolism, biodegradation, and other metabolism-related processes, supporting these organisms' ecological and physiological roles.