Validation of Golden Gate Assemblies Using Highly Multiplexed Nanopore Amplicon Sequencing.

Golden Gate cloning has revolutionized synthetic biology. Its concept of modular, highly characterized libraries of parts that can be combined into higher order assemblies allows engineering principles to be applied to biological systems. The basic parts, typically stored in Level 0 plasmids, are sequence validated by the method of choice and can be combined into higher order assemblies on demand. Higher order assemblies are typically transcriptional units, and multiple transcriptional units can be assembled into multi-gene constructs. Higher order Golden Gate assembly based on defined and validated parts usually does not introduce sequence changes. Therefore, simple validation of the assemblies, e.g., by colony polymerase chain reaction (PCR) or restriction digest pattern analysis is sufficient. However, in many experimental setups, researchers do not use defined parts, but rather part libraries, resulting in assemblies of high combinatorial complexity where sequencing again becomes mandatory. Here, we present a detailed protocol for the use of a highly multiplexed dual barcode amplicon sequencing using the Nanopore sequencing platform for in-house sequence validation. The workflow, called DuBA.flow, is a start-to-finish procedure that provides all necessary steps from a single colony to the final easy-to-interpret sequencing report.

Influence of bisphenol A and its analogues on human gut microbiota composition and metabolic activity: Insights from an in vitro model.

Food contamination is a primary route of human exposure to bisphenols (BPs), which are known to affect gut microbiota (GM) and intestinal health. This study comprehensively assessed the impact of bisphenol A (BPA) and three of its substitutes-bisphenol S (BPS), bisphenol F (BPF), and tetramethyl bisphenol F (TMBPF, the monomer of valPure V70) - on the taxonomic and functional profile of human GM using an in vitro model. Human GM was acutely exposed to 1 mM concentrations of these BPs during a 48 h anaerobic cultivation. We first examined the effects of BPA, BPS, BPF, and TMBPF on GM taxonomic and metabolic profiles, mainly focusing on short-chain fatty acids (SCFAs) production. We then evaluated the degradation potential of these BPs by GM and its influence on their estrogenic activity. Finally, we assessed the impact of GM metabolites from BPs-exposed cultures on the viability of intestinal epithelial cells (Caco-2). BPA, BPS, and BPF severely disrupted GM taxonomic composition and metabolite profiles, significantly reducing SCFAs production. In contrast, TMBPF exhibited the least disruptive effects, suggesting it may be a safer alternative. Although the GM did not biotransform the BPs, bioadsorption occurred, with affinity correlating to hydrophobicity in the order of TMBPF > BPA > BPF > BPS. GM reduced the estrogenic activity of BPs primarily through bioadsorption. However, exposure of gut epithelial cells to Post-Culture Supernatants of BPA, BPF, and TMBPF significantly reduced Caco-2 cell viability, indicating the potential formation of harmful GM-derived metabolites and/or a depletion of beneficial GM metabolites.

Targeted deep sequencing of mycobacteria species from extrapulmonary sites not identified by routine line probe assays: A retrospective laboratory analysis of stored clinical cultures.

Objectives: Nontuberculous mycobacteria (NTM) infections present a global health challenge. This study describes unidentified mycobacteria species from extrapulmonary sites, using advanced identification and sequencing techniques. Methods: Extrapulmonary mycobacteria growth indicator tube primary cultures collected retrospectively between 2019 and 2023, featuring unidentified mycobacteria species detected by GenoType Mycobacterium line probe assays, underwent multilocus targeted next-generation sequencing using Oxford Nanopore Technology, polymerase chain reaction amplicon Sanger sequencing, and Deeplex Myc-TB analysis. Previously collected clinical and laboratory data were reported. Results: A total of 28 cultures, collected from extrapulmonary sites, each from different patients, were included. Mycobacterial mixtures were identified in 19 of 28 (68%) cultures, with four of 28 (14%) showing unidentified species based on sequencing of rpoB and hsp65 targets. Mycobacterium monacense was present in 13 of 28 (46%) of the cultures. Culturable Mycobacterium tuberculosis complex (MTBC) was identified in five extrapulmonary specimens that previously tested negative for MTBC using Xpert MTB/RIF Ultra. The comparative analysis between Sanger and targeted next-generation sequencing using Oxford Nanopore Technology sequencing (for hsp65) demonstrated 27 of 28 (96%) agreement on the predominant strain. Deeplex Myc-TB could not identify NTM-MTBC co-infections in minor subpopulations. Conclusions: This study highlights the role of advanced sequencing in identifying NTM mixtures and mycobacterial co-infections. It calls for ongoing efforts to integrate next-generation sequencing into mycobacteria testing algorithms.

Undiagnosed West Nile virus lineage 2d infection in a febrile patient from South-west Uganda, 2018.

We report the retrospective identification and subsequent recovery of a near-complete West Nile Virus lineage 2 genomes from a hospitalized patient with acute febrile illness in Uganda, using a combination of degenerate primer polymerase chain reaction screening and a novel 1200bp nanopore-based whole-genome amplicon sequencing scheme. This represents the first West Nile virus genome to be recovered from a human in Uganda since its discovery in 1937. Basic molecular rather than serological surveillance methods could be more widely deployed in the region to better diagnose febrile infections.

Air quality in olive mill wastewater evaporation ponds: Assessment of chemical and microbiological pollutants.

Olive mill wastewater (OMW), a pollutant residue from the olive oil industry, is typically stored as sludge in evaporation ponds. This study examines the long-term emissions of OMW sludge and its impact on local air quality, analysing chemical pollutants like PM2.5, volatile organic compounds (VOCs), and trace elements (TEs), along with microbial communities (bacteria and fungi). The study also considered meteorological conditions and back-trajectories to identify sources of these elements. The ecological risk index (ERI) was found to be over 720 due to high Hg levels in the sludge (19.0 ± 0.9 ng/g) and air (0.28 ± 0.13 ng/m³), indicating a significant ecological threat. VOCs, particularly oxygenated compounds such as aldehydes and phenol, contributed to the area's strong odour. Meteorological conditions and Sahara dust intrusions influenced bioaerosol loads and seasonal bacterial diversity, whose composition is closely associated with VOC concentrations. The results could contribute to a better understanding of the environmental dynamics in the OMW sludge evaporation ponds, and they could also assist in formulating effective management strategies.

Deciphering Bordetella pertussis epidemiology through culture-independent multiplex amplicon and metagenomic sequencing.

Whooping cough (pertussis) has re-emerged despite high vaccine coverage in Australia and many other countries worldwide, partly attributable to genetic adaptation of the causative organism, Bordetella pertussis, to vaccines. Therefore, genomic surveillance has become essential to monitor circulating strains for these genetic changes. However, increasing uptake of PCR for the diagnosis of pertussis has affected the availability of cultured isolates for typing. In this study, we evaluated the use of targeted multiplex PCR (mPCR) amplicon sequencing and shotgun metagenomic sequencing for culture-independent typing of B. pertussis directly from respiratory swabs. We developed a nine-target mPCR amplicon assay that could accurately type major lineages [ptxP3/non-ptxpP3, fim3A/B, fhaB3/non-fhaB3, and epidemic lineages (ELs) 1-5] circulating in Australia. Validation using DNA from isolates and 178 residual specimens collected in 2010-2012 (n = 87) and 2019 (n = 91) showed that mPCR amplicon sequencing was highly sensitive with a limit of detection of 4.6 copies [IS481 cycle threshold (Ct) 27.3]. Shotgun metagenomic sequencing was successful in genotyping B. pertussis in 84% of clinical specimens with PCR Ct < 24 and was concordant with mPCR typing results. The results revealed an expansion of EL4 strains from 2010 to 2012 to 2019 in Australia and identified unrecognized co-circulating cases of Bordetella holmesii. This study provides valuable insight into the circulating lineages in Australia prior to the COVID-19 pandemic during which border closure and other interventions reduced pertussis cases to an all-time low, and paves the way for the genomic surveillance of B. pertussis in the era of culture-independent PCR-based diagnosis. IMPORTANCE: In this paper, we evaluated the use of targeted multiplex PCR (mPCR) amplicon sequencing and shotgun metagenomic sequencing for culture-independent typing of Bordetella pertussis directly in respiratory swabs. We first developed a novel targeted mPCR amplicon sequencing assay that can type major circulating lineages and validated its accuracy and sensitivity on 178 DNA extracts from clinical swabs. We also demonstrate the feasibility of using deep metagenomic sequencing for determining strain lineage and markers of virulence, vaccine adaptation, macrolide resistance, and co-infections. Our culture-independent typing methods applied to clinical specimens revealed the expansion of a major global epidemic lineage in Australia (termed EL4) just prior to the COVID-19 pandemic. It also detected cases of previously hidden co-infections from another Bordetella species called Bordetella holmesii. These findings offer valuable insight into the circulating pertussis lineages in Australia prior to the COVID-19 pandemic during which border closure and other interventions reduced pertussis cases to an all-time low. It also provides comparative data for future surveillance as pertussis resurgence after the COVID-19 pandemic has been reported this year in Australia and many other countries. Overall, our paper demonstrates the utility, sensitivity, and specificity of mPCR amplicon and metagenomic sequencing-based culture-independent typing of B. pertussis, which not only paves the way for culture-independent genomic surveillance of B. pertussis but also for other pathogens in the era of PCR-based diagnosis.

MhGeneS: An Analytical Pipeline to Allow for Robust Microhaplotype Genotyping.

Microhaplotypes are small linked genomic regions comprising two or more single-nucleotide polymorphisms (SNPs) that are being applied in forensics and are emerging in wildlife monitoring studies and genomic epidemiology. Typically, targeted in non-coding regions, microhaplotypes in exonic regions can be designed with larger amplicons to capture functional non-synonymous sites and minimise insertion/deletion (indel) polymorphisms. Quality control is an important first step for high-confidence genotyping to counteract such false-positive variants. As genetic markers with higher polymorphism compared to biallelic SNPs, it is critical to ensure sequencing errors across the microhaplotype amplicon are filtered out to avoid introducing false-haplotypes. We developed the MhGeneS pipeline which works in tandem with Seq2Sat to help validate microhaplotype genotyping of the coding region of genes, with broader applicability to any microhaplotype profiling. We genotyped microhaplotype regions of the Zfx (≅ 160 bp) and Zfy (≅ 140 bp) genes, as well as an exon of the prion protein (Prnp) gene (≅ 370 bp) in caribou (Rangifer tarandus) using paired-end Illumina technology. As important quality metrics affecting microhaplotype calling, we identified the sequencing error rate profile related to the overlap or non-overlap of paired-end reads as well as the read depth as significant. In the case of Prnp, we achieved confident microhaplotype calling through MhGeneS by removing small sections of the 5' and 3' amplicons and using a minimum read depth of 20. Read depth and sequence trimming may be locus-specific, and validation of these parameters is recommended before the high-throughput profiling of samples.

The Ribosomal Operon Database: A Full-Length rDNA Operon Database Derived From Genome Assemblies.

Current rDNA reference sequence databases are tailored towards shorter DNA markers, such as parts of the 16/18S marker or the internally transcribed spacer (ITS) region. However, due to advances in long-read DNA sequencing technologies, longer stretches of the rDNA operon are increasingly used in environmental sequencing studies to increase the phylogenetic resolution. There is, therefore, a growing need for longer rDNA reference sequences. Here, we present the ribosomal operon database (ROD), which includes eukaryotic full-length rDNA operons fished from publicly available genome assemblies. Full-length operons were detected in 34.1% of the 34,701 examined eukaryotic genome assemblies from NCBI. In most cases (53.1%), more than one operon variant was detected, which can be due to intragenomic operon copy variability, allelic variation in non-haploid genomes, or technical errors from the sequencing and assembly process. The highest copy number found was 5947 in Zea mays. In total, 453,697 unique operons were detected, with 69,480 operon variant clusters remaining after intragenomic clustering at 99% sequence identity. The operon length varied extensively across eukaryotes, ranging from 4136 to 16,463 bp, which will lead to considerable polymerase chain reaction (PCR) bias during amplification of the entire operon. Clustering the full-length operons revealed that the different parts (i.e., 18S, 28S, and the hypervariable regions V4 and V9 of 18S) provide divergent taxonomic resolution, with 18S, the V4 and V9 regions being the most conserved. The ROD will be updated regularly to provide an increasing number of full-length rDNA operons to the scientific community.

The choice of 16S rRNA gene sequence analysis impacted characterization of highly variable surface microbiota in dairy processing environments.

Accurate knowledge of the microbiota collected from surfaces in food processing environments is important for food quality and safety. This study assessed discrepancies in taxonomic composition and alpha and beta diversity values generated from eight different bioinformatic workflows for the analysis of 16S rRNA gene sequences extracted from the microbiota collected from surfaces in dairy processing environments. We found that the microbiota collected from environmental surfaces varied widely in density (0-9.09 log10 CFU/cm2) and Shannon alpha diversity (0.01-3.40). Consequently, depending on the sequence analysis method used, characterization of low-abundance genera (i.e., below 1% relative abundance) and the number of genera identified (114-173 genera) varied considerably. Some low-abundance genera, including Listeria, varied between the amplicon sequence variant (ASV) and operational taxonomic unit (OTU) methods. Centered log-ratio transformation inflated alpha and beta diversity values compared to rarefaction. Furthermore, the ASV method also inflated alpha and beta diversity values compared to the OTU method (P < 0.05). Therefore, for sparse, uneven, low-density data sets, the OTU method and rarefaction are better for taxonomic and ecological characterization of surface microbiota.IMPORTANCECulture-dependent environmental monitoring programs are used by the food industry to identify foodborne pathogens and spoilage biota on surfaces in food processing environments. The use of culture-independent 16S rRNA amplicon sequencing to characterize this surface microbiota has been proposed as a tool to enhance environmental monitoring. However, there is no consensus on the most suitable bioinformatic analyses to accurately capture the diverse levels and types of bacteria on surfaces in food processing environments. Here, we quantify the impact of different bioinformatic analyses on the results and interpretation of 16S rRNA amplicon sequences collected from three cultured dairy facilities in New York State. This study provides guidance for the selection of appropriate 16S rRNA analysis procedures for studying environmental microbiota in dairy processing environments.

Barcode 100K Specimens: In a Single Nanopore Run.

It is a global priority to better manage the biosphere, but action must be informed by comprehensive data on the abundance and distribution of species. The acquisition of such information is currently constrained by high costs. DNA barcoding can speed the registration of unknown animal species, the most diverse kingdom of eukaryotes, as the BIN system automates their recognition. However, inexpensive sequencing protocols are critical as the census of all animal species is likely to require the analysis of a billion or more specimens. Barcoding involves DNA extraction followed by PCR and sequencing with the last step dominating costs until 2017. By enabling the sequencing of highly multiplexed samples, the Sequel platforms from Pacific BioSciences slashed costs by 90%, but these instruments are only deployed in core facilities because of their expense. Sequencers from Oxford Nanopore Technologies provide an escape from high capital and service costs, but their low sequence fidelity has, until recently, constrained adoption. However, the improved performance of its latest flow cells (R10.4.1) erases this barrier. This study demonstrates that a MinION flow cell can characterise an amplicon pool derived from 100,000 specimens while a Flongle flow cell can process one derived from several thousand. At $0.01 per specimen, DNA sequencing is now the least expensive step in the barcode workflow.

Microbial source tracking and identification of fecal contamination patterns in saltwater estuaries between base- and storm-flow events using dual genome-based approaches.

Fecal contamination from natural and anthropogenic sources poses significant threats to saltwater estuaries, particularly after storms or heavy rainfall. Monitoring fecal contamination is essential for protecting these vulnerable ecosystems having important ecological and economic values. In this study, we investigated the abundance, sources, and potential causes of fecal contamination at three marine and seven freshwater stations across Vaughn Bay (WA, USA), a shellfish growing district, during base- and storm-flow events. Additionally, we evaluated the performance of fecal indicator bacteria (FIB) quantification, optical brightener assessment, and qPCR analysis for fecal contamination quantification. We compared the effectiveness of qPCR-based microbial source tracking (MST), which targeted a broad range of hosts including, such as humans, birds, cows, horses, ruminants, dogs, and pigs, with sequencing-based MST in identifying fecal contamination sources. Both MST analysis approaches identified birds and humans as the primary sources of fecal contamination. For marine water stations, freshwater creeks VBU001, VBU002, and VB047, along with drain VB007, were identified as the main sources of human-derived fecal contamination in Vaughn Bay, based on Kendall's tau analysis (τ: 0.58-0.97). This information indicates that the septic systems in the catchment areas of these creeks and drains require further investigation to achieve effective fecal contamination control. Optical brightener, FIB enumeration and qPCR quantification results were generally higher during storm-flow events, although they showed poor correlation with each other (Pearson r < 0.40), likely due to physiological and phylogenetic differences among the target organisms of these methods. However, the sequencing-based method faces challenges in precise quantitative identification of differences in fecal contamination between base- and storm-flow events. Due to its high-throughput and cost-effectiveness, we recommend using sequencing-based analysis for large-scale identification of the primary sources of fecal contamination in water environments, followed by targeted qPCR quantification of MST markers for more precise assessments.

MISIP: a data standard for the reuse and reproducibility of any stable isotope probing-derived nucleic acid sequence and experiment.

DNA/RNA-stable isotope probing (SIP) is a powerful tool to link in situ microbial activity to sequencing data. Every SIP dataset captures distinct information about microbial community metabolism, process rates, and population dynamics, offering valuable insights for a wide range of research questions. Data reuse maximizes the information derived from the labor and resource-intensive SIP approaches. Yet, a review of publicly available SIP sequencing metadata showed that critical information necessary for reproducibility and reuse was often missing. Here, we outline the Minimum Information for any Stable Isotope Probing Sequence (MISIP) according to the Minimum Information for any (x) Sequence (MIxS) framework and include examples of MISIP reporting for common SIP experiments. Our objectives are to expand the capacity of MIxS to accommodate SIP-specific metadata and guide SIP users in metadata collection when planning and reporting an experiment. The MISIP standard requires 5 metadata fields-isotope, isotopolog, isotopolog label, labeling approach, and gradient position-and recommends several fields that represent best practices in acquiring and reporting SIP sequencing data (e.g., gradient density and nucleic acid amount). The standard is intended to be used in concert with other MIxS checklists to comprehensively describe the origin of sequence data, such as for marker genes (MISIP-MIMARKS) or metagenomes (MISIP-MIMS), in combination with metadata required by an environmental extension (e.g., soil). The adoption of the proposed data standard will improve the reuse of any sequence derived from a SIP experiment and, by extension, deepen understanding of in situ biogeochemical processes and microbial ecology.

Discovery of Gibellula floridensis from Infected Spiders and Analysis of the Surrounding Fungal Entomopathogen Community.

Characterization of fungal spider pathogens lags far behind their insect counterparts. In addition, little to nothing is known concerning the ecological reservoir and/or fungal entomopathogen community surrounding infection sites. Five infected spider cadavers were identified in the neo-tropical climate of north-central Florida, USA, from three of which viable cultures were obtained. Multi-locus molecular phylogenetic and morphological characterization identified one isolate as a new Gibellula species, here named, Gibellula floridensis, and the other isolates highly similar to Parengyodontium album. The fungal entomopathogen community surrounding infected spiders was sampled at different habitats/trophic levels, including soil, leaf litter, leaf, and twig, and analyzed using ITS amplicon sequencing. These data revealed broad but differential distribution of insect-pathogenic fungi between habitats and variation between sites, with members of genera belonging to Metarhizium and Metacordyceps from Clavicipitaceae, Purpureocillium and Polycephalomyces from Ophiocordyceps, and Akanthomyces and Simplicillium from Cordycipitaceae predominating. However, no sequences corresponding to Gibellula or Parengyodontium, even at the genera levels, could be detected. Potential explanations for these findings are discussed. These data highlight novel discovery of fungal spider pathogens and open the broader question regarding the environmental distribution and ecological niches of such host-specific pathogens.

The Cultivation of Halophilic Microalgae Shapes the Structure of Their Prokaryotic Assemblages.

The influence of microalgae on the formation of associated prokaryotic assemblages in halophilic microbial communities is currently underestimated. The aim of this study was to characterize shifts in prokaryotic assemblages of halophilic microalgae upon their transition to laboratory cultivation. Monoalgal cultures belonging to the classes Chlorodendrophyceae, Bacillariophyceae, Trebouxiophyceae, and Chlorophyceae were isolated from habitats with intermediate salinity, about 100 g/L, nearby Elton Lake (Russia). Significant changes were revealed in the structure of algae-associated prokaryotic assemblages, indicating that microalgae supported sufficiently diverse and even communities of prokaryotes. Despite some similarities in their prokaryotic assemblages, taxon-specific complexes of dominant genera were identified for each microalga species. These complexes were most different among Alphaproteobacteria, likely due to their close association with microalgae. Other taxon-specific bacteria included members of phylum Verrucomicrobiota (Coraliomargarita in assemblages of Navicula sp.) and class Gammaproteobacteria (Salinispirillum in microbiomes of A. gracilis). After numerous washings of algal cells, only alphaproteobacteria Marivibrio remained in all assemblages of T. indica, likely due to a firm attachment to the microalgae cells. Our results may be useful for further efforts to develop technologies applied for industrial cultivation of halophilic microalgae and for developing approaches to obtain new prokaryotes with a microalgae-associated lifestyle.

High-Throughput Sequencing Methods for the Detection of Two Strawberry Viruses in Post-Entry Quarantine.

High-throughput sequencing (HTS) technologies may be a useful tool for testing imported plant germplasm for multiple pathogens present in a sample, offering strain-generic detection not offered by most PCR-based assays. Metatranscriptomics (RNAseq) and tiled amplicon PCR (TA-PCR) were tested as HTS-based techniques to detect viruses present in low titres. Strawberry mottle virus (SMoV), an RNA virus, and strawberry vein banding virus (SVBV), a DNA virus, were selected for comparison of RNAseq and TA-PCR with quantitative PCR assays. RNAseq of plant ribosomal RNA-depleted samples of low viral titre was used to obtain datasets from 3 M to 120 M paired-end (PE) reads. RNAseq demonstrated PCR-like sensitivity, able to detect as few as 10 viral copies/µL when 60 million (M) PE reads were generated. The custom TA-PCR primer panels designed for each virus were successfully used to recover most of the reference genomes for each virus. Single- and multiple-target TA-PCR allowed the detection of viruses in samples with around 10 viral copies/µL with a minimum continuous sequence length recovery of 500 bp. The limit of detection of the HTS-based protocols described here is comparable to that of quantitative PCR assays. This work lays the groundwork for an increased flexibility in HTS detection of plant viruses.

Improvement in Microbiota Recovery Using Cas-9 Digestion of Manuka Plastid and Mitochondrial DNA.

Understanding host-microbe interactions in planta is an expanding area of research. Amplicon sequencing of the 16S rRNA gene is a powerful and common method to study bacterial communities associated with plants. However, the co-amplification of mitochondrial and plastid 16S rRNA genes by universal primers impairs the sensitivity and performance of 16S rRNA sequencing. In 2020, a new method, Cas-16S-seq, was reported in the literature to remove host contamination for profiling the microbiota in rice, a well-studied domestic plant, by engineering RNA-programmable Cas9 nuclease in 16S rRNA sequencing. For the first time, we tested the efficiency and applicability of the Cas-16S-seq method on foliage, flowers, and seed of a non-domesticated wild plant for which there is limited genomic information, Leptospermum scoparium (manuka). Our study demonstrated the efficiency of the Cas-16S-seq method for L. scoparium in removing host contamination in V4-16S amplicons. An increase of 46% in bacterial sequences was found using six guide RNAs (gRNAs), three gRNAs targeting the mitochondrial sequence, and three gRNAs targeting the chloroplast sequence of L. scoparium in the same reaction. An increase of 72% in bacterial sequences was obtained by targeting the mitochondrial and chloroplast sequences of L. scoparium in the same sample at two different steps of the library preparation (DNA and 1st step PCR). The number of OTUs (operational taxonomic units) retrieved from soil samples was consistent when using the different methods (Cas-16S-seq and 16S-seq) indicating that the Cas-16S-seq implemented for L. scoparium did not introduce bias to microbiota profiling. Our findings provide a valuable tool for future studies investigating the bacterial microbiota of L. scoparium in addition to evaluating an important tool in the plant microbiota research on other non-domesticated wild species.

Distributions of MICA and MICB Alleles Typed by Amplicon-Based Next-Generation Sequencing in South Koreans.

Major histocompatibility complex class I chain-related genes A and B (MICA and MICB) play a role as ligands in activating the NKG2D receptor expressed in natural killer cells, γδ T-cells and αß CD8 T-cells and have been defined in human diseases and haematopoietic stem cell transplantation (HSCT). MICA and MICB alleles were genotyped at the three-field level by amplicon-based next-generation sequencing (NGS) using a MiSeqDx system and compared with the results from previous studies in healthy South Korean donors. Exons 2-5 of MICA and exons 2-4 of MICB were amplified using a multiplex polymerase chain reaction (PCR). Sequence reads of ≥ 51 depth counts were consistently obtained for each sample exon, and target exons were determined to match reference sequences contained in the IPD-IMGT/HLA database. MICA and MICB alleles were tested using exon combinations. The program was designed to recognise specific sequences and discriminate between the MICA*008:01:01/*027 alleles. A total of 22 alleles were found in MICA and MICB. We observed 1 HLA-C ~ HLA-B ~ MICA ~ MICB ~ HLA-DRB1 haplotype with significant linkage disequilibrium between alleles at all neighbouring HLA loci. These results are consistent with previous microarray results. Genotyping of MICA and MICB was possible using 11-loci HLA genes. We updated the distribution of MICA and MICB based on three-field allele and haplotype frequencies containing linkage disequilibrium in South Koreans using amplicon-based NGS. These data suggest that high-resolution MICA and MICB typing data obtained using NGS may aid in performing HSCT and disease association studies.

Application of hsp60 amplicon sequencing to characterize microbial communities associated with juvenile and adult Euprymna scolopes squid.

The symbiotic relationship between Vibrio (Aliivibrio) fischeri and the Hawaiian bobtail squid, Euprymna scolopes, serves as a key model for understanding host-microbe interactions. Traditional culture-based methods have primarily isolated V. fischeri from the light organs of wild-caught squid, yet culture-independent analyses of this symbiotic microbiome remain limited. This study aims to enhance species-level resolution of bacterial communities associated with E. scolopes using hsp60 amplicon sequencing. We validated our hsp60 sequencing approach using pure cultures and mixed bacterial populations, demonstrating its ability to distinguish V. fischeri from other closely-related vibrios and the possibility of using this approach for strain-level diversity with further optimization. This approach was applied to whole-animal juvenile squid exposed to either seawater or a clonal V. fischeri inoculum, as well as ventate samples and light organ cores from wild-caught adults. V. fischeri accounted for the majority of the identifiable taxa for whole-animal juvenile samples and comprised 94%-99% of amplicon sequence variants (ASVs) for adult light organ core samples, confirming that V. fischeri is the dominant, if not sole, symbiont typically associated with E. scolopes light organs. In one ventate sample, V. fischeri comprised 82% of reads, indicating the potential for non-invasive community assessments using this approach. Analysis of non-V. fischeri ASVs revealed that Bradyrhizobium spp. and other members of the Rhodobacterales order are conserved across juvenile and adult samples. These findings provide insight into the presence of additional microbial associations with the squid host tissue outside of the light organ that have not been previously detected through traditional culture methods.

Integrating targeted genetic markers to genotyping-by-sequencing for an ultimate genotyping tool.

New selection methods, using trait-specific markers (marker-assisted selection (MAS)) and/or genome-wide markers (genomic selection (GS)), are becoming increasingly widespread in breeding programs. This new era requires innovative and cost-efficient solutions for genotyping. Reduction in sequencing cost has enhanced the use of high-throughput low-cost genotyping methods such as genotyping-by-sequencing (GBS) for genome-wide single-nucleotide polymorphism (SNP) profiling in large breeding populations. However, the major weakness of GBS methodologies is their inability to genotype targeted markers. Conversely, targeted methods, such as amplicon sequencing (AmpSeq), often face cost constraints, hindering genome-wide genotyping across a large cohort. Although GBS and AmpSeq data can be generated from the same sample, an efficient method to achieve this is lacking. In this study, we present the Genome-wide & Targeted Amplicon (GTA) genotyping platform, an innovative way to integrate multiplex targeted amplicons into the GBS library preparation to provide an all-in-one cost-effective genotyping solution to breeders and research communities. Custom primers were designed to target 23 and 36 high-value markers associated with key agronomical traits in soybean and barley, respectively. The resulting multiplex amplicons were compatible with the GBS library preparation enabling both GBS and targeted genotyping data to be produced efficiently and cost-effectively. To facilitate data analysis, we have introduced Fast-GBS.v3, a user-friendly bioinformatic pipeline that generates comprehensive outputs from data obtained following sequencing of GTA libraries. This high-throughput low-cost approach will greatly facilitate the application of DNA markers as it provides required markers for both MAS and GS in a single assay.

Progress in malaria genomic surveillance using long-read sequencing.

Recent studies by Girgis et al. and de Cesare et al. promise to advance malaria genomic surveillance using inexpensive and portable long-read amplicon-sequencing technologies. These technologies allow rapid characterization of drug-resistance markers, antigenic diversity, and diagnostic target loci from dried blood spots, providing new tools for surveillance in endemic regions and informing interventions to combat malaria more effectively.