Application of MinION Long-Read Sequencer for Semi-targeted Detection of Foodborne Pathogens.

Foodborne pathogens remain a serious health issue in developed and developing countries. Safeness of food products has been assured for years with culture-based microbiological methods; however, these present several limitations such as turnaround time and extensive hands-on work, which have been typically address taking advantage of DNA-based methods such as real-time PCR (qPCR). These, and other similar techniques, are targeted assays, meaning that they are directed for the specific detection of one specific microbe. Even though reliable, this approach suffers from an important limitation that unless specific assays are design for every single pathogen potentially present, foods may be considered erroneously safe. To address this problem, next-generation sequencing (NGS) can be used as this is a nontargeted method; thus it has the capacity to detect every potential threat present. In this chapter, a protocol for the simultaneous detection and preliminary serotyping of Salmonella enterica serovar Enteritidis, Salmonella enterica serovar Typhimurium, Listeria monocytogenes, and Escherichia coli O157:H7 is described.

Assessing different next-generation sequencing technologies for wastewater-based epidemiology.

Wastewater-based epidemiology has proven to be an important public health asset during the COVID-19 pandemic. It can provide less biassed and more cost-effective population-level monitoring of the disease burden as compared to clinical testing. An essential component of SARS-CoV-2 wastewater monitoring is next-generation sequencing, providing genomic data to identify and quantify circulating viral strains rapidly. However, the specific choice of sequencing method influences the quality and timeliness of generated data and hence its usefulness for wastewater-based pathogen surveillance. Here, we systematically benchmarked Illumina Novaseq 6000, Element Aviti, ONT R9.4.1 MinION flow cell, and ONT R9.4.1 Flongle flow cell sequencing data to facilitate the selection of sequencing technology. Using a time series of wastewater samples from influent of six wastewater treatment plants throughout Switzerland, along with spike-in experiments, we show that higher sequencing error rates of ONT Nanopore sequencing reduce the accuracy of estimates of the relative abundance of viral variants, but the overall trend is in good concordance among all technologies. We find that the sequencing runtime for ONT Nanopore flow cells can be reduced to as little as five hours without significant impact on the quality of variant estimates. Our findings suggest that SARS-CoV-2 variant tracking is readily achievable with all tested technologies, albeit with different tradeoffs in terms of cost, timeliness and accuracy.

Eyeing DNA barcoding for species identification of fish larvae.

Identification of fish larvae based on morphology is typically limited to higher taxonomic ranks (e.g., family or order), as larvae possess few morphological diagnostic characters for precise discrimination to species. When many samples are presented at any one time, the use of morphology to identify such specimens can be laborious and time-consuming. Using a reverse workflow for specimen sorting and identification leveraging high-throughput DNA sequencing, thousands of fish larvae can be DNA barcoded and sorted into molecular operational taxonomic units (mOTUs) in a single sequencing run with the nanopore sequencing technology (e.g., MinION). This process reduces the time and financial costs of morphology-based sorting and instead deploys experienced taxonomists for species taxonomic work where they are needed most. In this study, a total of 3022 fish larval specimens from plankton tows across four sites in Singapore were collected and sorted based on this workflow. Eye tissue from individual samples was used for DNA extraction and sequencing of cytochrome c oxidase subunit I. We generated a total of 2746 barcodes after quality filtering (90.9% barcoding success), identified 2067 DNA barcodes (75.3% identification success), and delimited 256 mOTUs (146 genera, 52 families). Our analyses identified specific challenges to species assignment, such as the potential misidentification of publicly available sequences used as reference barcodes. We highlighted how the conservative application and comparison of a local sequence database can help resolve identification conflicts. Overall, this proposed approach enables and expedites taxonomic identification of fish larvae, contributing to the enhancement of reference barcode databases and potentially better understanding of fish connectivity.

Insights on the virulence and genomic features of Lactococcus garvieae isolated from giant freshwater prawn Macrobrachium rosenbergii (de Man 1879).

Giant freshwater prawn (Macrobrachium rosenbergii (MR)) is a significant aquafarm species commercially cultured in Taiwan. Intensive farming practices have led to the outbreak of Lactococcus garvieae (LG), which causes Lactococcosis in MR. Recently, LG has re-emerged and the number of mortalities in prawn farms has increased in Taiwan. However, there is no preventative strategy described and a lack of knowledge on virulence factors and pathogenesis from LG in MR. The most virulent strain of L. garvieae from M. rosenbergii was screened in vivo among seven isolates selected for infectivity testing injecting 0.1 mL of 108 CFU/mL bacterial concentration. Among the seven isolates screened, L. garvieae 109-6 resulted in 100% mortality within 3 days post-infection. Furthermore, 109-6 L. garvieae LD50 dosage from in MR was found to be 106 CFU/mL. Subsequently, the most virulent strain 109-6 was sequenced using MinIon Nanopore sequencing. Results indicated that the LG genome yielded a protein-coding of 3857 with 59 tRNA and 16 rRNA and no plasmid. Interestingly, the distribution of subsystems in the annotated genome revealed genes related to virulence, defence, and disease among LG 50 genes. Altogether, the virulent strain and its genome data revealed distinctive features of LG, which hinted toward its pathogenicity and could facilitate for better preventive strategies.

Primed and ready: nanopore metabarcoding can now recover highly accurate consensus barcodes that are generally indel-free.

BACKGROUND: DNA metabarcoding applies high-throughput sequencing approaches to generate numerous DNA barcodes from mixed sample pools for mass species identification and community characterisation. To date, however, most metabarcoding studies employ second-generation sequencing platforms like Illumina, which are limited by short read lengths and longer turnaround times. While third-generation platforms such as the MinION (Oxford Nanopore Technologies) can sequence longer reads and even in real-time, application of these platforms for metabarcoding has remained limited possibly due to the relatively high read error rates as well as the paucity of specialised software for processing such reads. RESULTS: We show that this is no longer the case by performing nanopore-based, cytochrome c oxidase subunit I (COI) metabarcoding on 34 zooplankton bulk samples, and benchmarking the results against conventional Illumina MiSeq sequencing. Nanopore R10.3 sequencing chemistry and super accurate (SUP) basecalling model reduced raw read error rates to ~ 4%, and consensus calling with amplicon_sorter (without further error correction) generated metabarcodes that were ≤ 1% erroneous. Although Illumina recovered a higher number of molecular operational taxonomic units (MOTUs) than nanopore sequencing (589 vs. 471), we found no significant differences in the zooplankton communities inferred between the sequencing platforms. Importantly, 406 of 444 (91.4%) shared MOTUs between Illumina and nanopore were also found to be free of indel errors, and 85% of the zooplankton richness could be recovered after just 12-15 h of sequencing. CONCLUSION: Our results demonstrate that nanopore sequencing can generate metabarcodes with Illumina-like accuracy, and we are the first study to show that nanopore metabarcodes are almost always indel-free. We also show that nanopore metabarcoding is viable for characterising species-rich communities rapidly, and that the same ecological conclusions can be obtained regardless of the sequencing platform used. Collectively, our study inspires confidence in nanopore sequencing and paves the way for greater utilisation of nanopore technology in various metabarcoding applications.

High Arctic seawater and coastal soil microbiome co-occurrence and composition structure and their potential hydrocarbon biodegradation.

The accelerated decline in Arctic sea-ice cover and duration is enabling the opening of Arctic marine passages and improving access to natural resources. The increasing accessibility to navigation and resource exploration and production brings risks of accidental hydrocarbon releases into Arctic waters, posing a major threat to Arctic marine ecosystems where oil may persist for many years, especially in beach sediment. The composition and response of the microbial community to oil contamination on Arctic beaches remain poorly understood. To address this, we analyzed microbial community structure and identified hydrocarbon degradation genes among the Northwest Passage intertidal beach sediments and shoreline seawater from five high Arctic beaches. Our results from 16S/18S rRNA genes, long-read metagenomes, and metagenome-assembled genomes reveal the composition and metabolic capabilities of the hydrocarbon microbial degrader community, as well as tight cross-habitat and cross-kingdom interactions dominated by lineages that are common and often dominant in the polar coastal habitat, but distinct from petroleum hydrocarbon-contaminated sites. In the polar beach sediment habitats, Granulosicoccus sp. and Cyclocasticus sp. were major potential hydrocarbon-degraders, and our metagenomes revealed a small proportion of microalgae and algal viruses possessing key hydrocarbon biodegradative genes. This research demonstrates that Arctic beach sediment and marine microbial communities possess the ability for hydrocarbon natural attenuation. The findings provide new insights into the viral and microalgal communities possessing hydrocarbon degradation genes and might represent an important contribution to the removal of hydrocarbons under harsh environmental conditions in a pristine, cold, and oil-free environment that is threatened by oil spills.

Extraction of high-molecular-weight DNA from Streptococcus spp. for nanopore sequencing in resource-limited settings.

The long-read sequencing platform MinION, developed by Oxford Nanopore Technologies, enables the sequencing of bacterial genomes in resource-limited settings, such as field conditions or low- and middle-income countries. For this purpose, protocols for extracting high-molecular-weight DNA using nonhazardous, inexpensive reagents and equipment are needed, and some methods have been developed for gram-negative bacteria. However, we found that without modification, these protocols are unsuitable for gram-positive Streptococcus spp., a major threat to fish farming and food security in low- and middle-income countries. Multiple approaches were evaluated, and the most effective was an extraction method using lysozyme, sodium dodecyl sulfate, and proteinase K for lysis of bacterial cells and magnetic beads for DNA recovery. We optimized the method to consistently achieve sufficient yields of pure high-molecular-weight DNA with minimal reagents and time and developed a version of the protocol which can be performed without a centrifuge or electrical power. The suitability of the method was verified by MinION sequencing and assembly of 12 genomes of epidemiologically diverse fish-pathogenic Streptococcus iniae and Streptococcus agalactiae isolates. The combination of effective high-molecular-weight DNA extraction and MinION sequencing enabled the discovery of a naturally occurring 15 kb low-copy number mobilizable plasmid in S. iniae, which we name pSI1. We expect that our resource-limited settings-adapted protocol for high-molecular-weight DNA extraction could be implemented successfully for similarly recalcitrant-to-lysis gram-positive bacteria, and it represents a method of choice for MinION-based disease diagnostics in low- and middle-income countries.

High Sensitivity and Specificity Platform to Validate MicroRNA Biomarkers in Cancer and Human Diseases.

We developed a technology for detecting and quantifying trace nucleic acids using a bracketing protocol designed to yield a copy number with approximately ± 20% accuracy across all concentrations. The microRNAs (miRNAs) let-7b, miR-15b, miR-21, miR-375 and miR-141 were measured in serum and urine samples from healthy subjects and patients with breast, prostate or pancreatic cancer. Detection and quantification were amplification-free and enabled using osmium-tagged probes and MinION, a nanopore array detection device. Combined serum from healthy men (Sigma-Aldrich, St. Louis, MO, USA #H6914) was used as a reference. Total RNA isolated from biospecimens using commercial kits was used as the miRNA source. The unprecedented ± 20% accuracy led to the conclusion that miRNA copy numbers must be normalized to the same RNA content, which in turn illustrates (i) independence from age, sex and ethnicity, as well as (ii) equivalence between serum and urine. miR-21, miR-375 and miR-141 copies in cancers were 1.8-fold overexpressed, exhibited zero overlap with healthy samples and had a p-value of 1.6 × 10-22, tentatively validating each miRNA as a multi-cancer biomarker. miR-15b was confirmed to be cancer-independent, whereas let-7b appeared to be a cancer biomarker for prostate and breast cancer, but not for pancreatic cancer.

Metabarcoding using nanopore sequencing enables identification of diverse and zoonotic vector-borne pathogens from neglected regions: A case study investigating dogs from Bhutan.

The diversity and prevalence of canine vector-borne pathogens (VBPs) in Bhutan have to date remained unexplored, whilst recent epidemiological surveys in other South Asian nations have found diseases caused by VBPs to be rife in local dog populations. Importantly, many of such VBPs can infect people as well, with a building body of evidence identifying potentially zoonotic rickettsial organisms infecting humans in Bhutan. Given the lack of data on canine pathogens in Bhutan we employed a suite of deep-sequencing metabarcoding methods using Oxford Nanopore Technologies' MinION™ device to holistically characterise the bacterial, apicomplexan and filarial worm blood-borne pathogens of dogs in the country's south. Of the 95 stray, owned and community dogs sampled 78% (95% CI = 69%-85%) were infected with at least one VBP. Pathogen species detected were highly diverse including the bacteria Mycoplasma haemocanis in 16% (95% CI: 10-24%), Ehrlichia canis in 4% (95% CI: 2-10%), Anaplasma platys in 2% (95% CI: 0.5-7%) of dogs as well as the zoonotic species Bartonella clarridgeiae in 1% (95% CI: 0.1-6%), a potentially novel Bartonella spp. and an Ehrlichia chaffeensis-like bacterium, both in 1% (95% CI: 0.1-6%) of dogs. The apicomplexan haemoparasites Hepatozoon canis in 62% (95% CI: 52-71%), Babesia gibsoni in 45% (95% CI: 36-55%) and Babesia vogeli in 3% (95% CI: 1-9%) of dogs were also detected. Finally, 5% (95% CI: 2-12%) of dogs were found to be infected with the filarioid Acanthocheilonema reconditum and 1% (95% CI: 0.1-6%) with zoonotic Dirofilaria sp. hongkongensis. One canine was found positive to the filarioid Setaria tundra, a species normally found infecting cervids. The elucidated diversity of VBP communities highlights the strength of assumption-free diagnostics, such as metabarcoding, in detecting rare, novel, and unexpected pathogens. This approach to identifying pathogen diversity is of critical importance when investigating regions and populations that have thus far been neglected, with the findings aiding the development of future One Health informed strategies for disease control.

High-quality genome assembly and annotation of five bacteria isolated from the Abu Dhabi sabkha-shore region.

OBJECTIVES: Sabkhas represent polyextreme environments characterized by elevated salinity levels, intense ultraviolet (UV) radiation exposure, and extreme temperature fluctuations. In this study, we present the complete genomes of five bacterial isolates isolated from the sabkha-shore region and investigate their genomic organization and gene annotations. A better understanding of the bacterial genomic organization and genetic adaptations of these bacteria holds promise for engineering microbes with tailored functionalities for diverse industrial and agricultural applications, including bioremediation and promotion of plant growth under salinity stress conditions. DATA DESCRIPTION: We present a comprehensive genome sequencing and annotation of five bacteria (kcgeb_sa, kcgeb_sc, kcgeb_sd, kcgeb_S4, and kcgeb_S11) obtained from the shores of the Abu Dhabi Sabkha region. Initial bacterial identification was conducted through 16 S rDNA amplification and sequencing. Employing a hybrid genome assembly technique combining Illumina short reads (NovaSeq 6000) and Oxford Nanopore long reads (MinION), we obtained complete annotated high-quality gap-free genome sequences. The genome sizes of the kcgeb_sa, kcgeb_sc, kcgeb_sd, kcgeb_S4, and kcgeb_S11 isolates were determined to be 2.4 Mb, 4.1 Mb, 2.9 Mb, 5.05 Mb, and 4.1 Mb, respectively. Our analysis conclusively assigned the bacterial isolates as Staphylococcus capitis (kcgeb_sa), Bacillus spizizenii (kcgeb_sc and kcgeb_S11), Pelagerythrobacter marensis (kcgeb_sd), and Priestia aryabhattai (kcgeb_S4).

Rapid genotyping of Toxoplasma gondii isolates via Nanopore-based multi-locus sequencing.

Toxoplasma gondii is an obligate intracellular parasite associated with severe disease, especially in the immunosuppressed. It is also a cause of congenital malformation and abortion in both animals and humans and is considered one of the most important foodborne pathogens worldwide with different strains showing variable distribution and differing pathogenicity. Thus, strain-level differentiation of T. gondii isolates is an essential asset in the understanding of parasite's diversity, geographical distribution, epidemiology and health risk. Here, we designed and implemented an Oxford Nanopore MinION protocol to analyse genomic sequence variation including single nucleotide polymorphisms (SNPs) and insertion/deletion polymorphisms (InDel's) of four different genomic loci, part of protein coding genes SAG2, SAG3, ROP17 and ROP21. This method provided results with the sequencing depth necessary for accurate differentiation of T. gondii strains and represents a rapid approach compared to conventional techniques which we further validated against environmental samples isolated from wild wood mice. In summary, multi-locus sequence typing (MLST) of both highly conserved and more polymorphic areas of the genome, provided robust data for strain classification in a platform ready for further adaption for other strains and pathogens.

Nanopore sequencing of forensic short tandem repeats using QNome of Qitan Technology.

Devices of nanopore sequencing can be highly portable and of low cost. Thus, nanopore sequencing is promising in in-field forensic applications. Previous investigations have demonstrated that nanopore sequencing is feasible for genotyping forensic short tandem repeats (STRs) by using sequencers of Oxford Nanopore Technologies. Recently, Qitan Technology launched a new portable nanopore sequencer and became the second supplier in the world. Here, for the first time, we assess the QNome (QNome-3841) for its accuracy in nanopore sequencing of STRs and compare with MinION (MinION Mk1B). We profile 54 STRs of 21 unrelated individuals and 2800M standard DNA. The overall accuracy for diploid STRs and haploid STRs were 53.5% (378 of 706) and 82.7% (134 of 162), respectively, by using QNome. The accuracies were remarkably lower than those of MinION (diploid STRs, 84.5%; haploid, 90.7%), with a similar amount of sequencing data and identical bioinformatics analysis. Although it was not reliable for diploid STRs typing by using QNome, the haploid STRs were consistently correctly typed. The majority of errors (58.8%) in QNome-based STR typing were one-repeat deviations of repeat units in the error from true allele, related with homopolymers in repeats of STRs.

First Report of Melon Severe Mosaic Orthotospovirus infecting cucurbits in the United States.

Cucurbits (family Cucurbitaceae) includes globally important fruit and vegetable crops. Virus diseases pose a serious threat to cucurbits, limiting crop quality and yield (Regina et al. 2021). In fall 2023, leaf and fruit samples from two squash plants with chlorotic mosaic symptoms and fruit distortion from Monroe and Pope counties in Arkansas were received for diagnosis at the University of Arkansas Division of Agriculture Plant Clinic. Based on symptoms, samples were assessed for melon severe mosaic orthotospovirus (MeSMV) using the ImmunoStrip® developed for detection of the virus (Agdia® Inc., Elkhart, Indiana). The presence of MeSMV was also confirmed by RT-PCR using the Agdia Tospovirus group PCR primers. An amplicon was sequenced and showed 91% sequence identity to the MESMV type isolate (NC_033834, VE440-A). To further verify the results, nucleic acids from a squash sample from Pope County were extracted as described by Poudel et al. (2013), DNase treated, and sequenced on an Oxford Nanopore MinION as described by Liefting et al. (2021). A total of 25,914 raw reads were analyzed using VirFind (Ho and Tzanetakis 2014), which identified 112 reads mapping to the three segments of MeSMV. Primers for all three RNAs were developed and amplified 638, 650, and 1153 nt of the S, M, and L segments of the virus respectively. The amplicons were sequenced bidirectionally and show 89-93% identity to the type isolate from Mexico (GenBank accessions PP301332-4). MeSMV has only been identified in Mexico and can cause significant losses to honeydew melon, zucchini, and cucumber (Ciuffo et al. 2009). Thus, this is the first report of MeSMV outside Mexico. Given the severity of the symptoms observed in cucurbit crops, the virus poses a potential threat to the cucurbit industry in the United States. Growers should be aware of this virus and take the necessary precautions to prevent its spread in the field.

Sterile sentinels and MinION sequencing capture active soil microbial communities that differentiate crop rotations.

BACKGROUND: Soil microbial communities are difficult to measure and critical to soil processes. The bulk soil microbiome is highly diverse and spatially heterogeneous, which can make it difficult to detect and monitor the responses of microbial communities to differences or changes in management, such as different crop rotations in agricultural research. Sampling a subset of actively growing microbes should promote monitoring how soil microbial communities respond to management by reducing the variation contributed by high microbial spatial and temporal heterogeneity and less active microbes. We tested an in-growth bag method using sterilized soil in root-excluding mesh, "sterile sentinels," for the capacity to differentiate between crop rotations. We assessed the utility of different incubation times and compared colonized sentinels to concurrently sampled bulk soils for the statistical power to differentiate microbial community composition in low and high diversity crop rotations. We paired this method with Oxford Nanopore MinION sequencing to assess sterile sentinels as a standardized, fast turn-around monitoring method. RESULTS: Compared to bulk soil, sentinels provided greater statistical power to distinguish between crop rotations for bacterial communities and equivalent power for fungal communities. The incubation time did not affect the statistical power to detect treatment differences in community composition, although longer incubation time increased total biomass. Bulk and sentinel soil samples contained shared and unique microbial taxa that were differentially abundant between crop rotations. CONCLUSIONS: Overall, compared to bulk soils, the sentinels captured taxa with copiotrophic or ruderal traits, and plant-associated taxa. The sentinels show promise as a sensitive, scalable method to monitor soil microbial communities and provide information complementary to traditional soil sampling.

CRISPR/Cas9 gene targeting plus nanopore DNA sequencing with the plasmid pBR322 in the classroom.

Both nanopore-based DNA sequencing and CRISPR/Cas-based gene editing represent groundbreaking innovations in molecular biology and genomics, offering unprecedented insights into and tools for working with genetic information. For students, reading, editing, and even writing DNA will be part of their everyday life. We have developed a laboratory procedure that includes (i) the biosynthesis of a guide RNA for, (ii) targeting Cas9 to specifically linearize the pBR322 plasmid, and (iii) the identification of the cutting site through nanopore DNA sequencing. The protocol is intentionally kept simple and requires neither living organisms nor biosafety laboratories. We divided the experimental procedures into separate activities to facilitate customization. Assuming access to a well-equipped molecular biology laboratory, an initial investment of approximately $2,700 is necessary. The material costs for each experiment group amount to around $130. Furthermore, we have developed a freely accessible website (https://dnalesen.hs-mittweida.de) for sequence read analysis and visualization, lowering the required computational skills to a minimum. For those with strong computational skills, we provide instructions for terminal-based data processing. With the presented activities, we aim to provide a hands-on experiment that engages students in modern molecular genetics and motivates them to discuss potential implications. The complete experiment can be accomplished within half a day and has been successfully implemented by us at high schools, in teacher training, and at universities. Our tip is to combine CRISPR/Cas gene targeting with nanopore-based DNA sequencing. As a tool, we provide a website that facilitates sequence data analysis and visualization.

A full-length 18S ribosomal DNA metabarcoding approach for determining protist community diversity using Nanopore sequencing.

Protist diversity studies are frequently conducted using DNA metabarcoding methods. Currently, most studies have utilized short read sequences to assess protist diversity. One limitation of using short read sequences is the low resolution of the markers. For better taxonomic resolution longer sequences of the 18S rDNA are required because the full-length has both conserved and hypervariable regions. In this study, a new primer pair combination was used to amplify the full-length 18S rDNA and its efficacy was validated with a test community and then validated with field samples. Full-length sequences obtained with the Nanopore MinION for protist diversity from field samples were compared with Illumina MiSeq V4 and V8-V9 short reads. Sequences generated from the high-throughput sequencers are Amplicon Sequence Variants (ASVs). Metabarcoding results show high congruency among the long reads and short reads in taxonomic annotation at the major taxonomic group level; however, not all taxa could be successfully detected from sequences. Based on the criteria of ≥95% similarity and ≥1000 bp query length, 298 genera were identified by all markers in the field samples, 250 (84%) were detected by 18S, while only 226 (76%) by V4 and 213 (71%) by V8-V9. Of the total 85 dinoflagellate genera observed, 19 genera were not defined by 18S dinoflagellate ASVs compared to only three among the total 52 diatom genera. The discrepancy in this resolution is due to the lack of taxonomically available 18S reference sequences in particular for dinoflagellates. Overall, this preliminary investigation demonstrates that application of the full-length 18S rDNA approach can be successful in field studies.

Bioaugmentation of anaerobic digesters with the enriched lignin-degrading microbial consortia through a metagenomic approach.

The recalcitrance of lignin impedes the efficient utilization of lignocellulosic biomass, hindering the efficient production of biogas and value-added materials. Despite the emergence of anaerobic digestion as a superior alternative to the aerobic method for lignin processing, achieving its feasibility requires thorough characterization of lignin-degrading anaerobic microorganisms, assessment of their biomethane production potential, and a comprehensive understanding of the degradation pathway. This study aimed to address the aforementioned necessities by bioaugmenting seed sludge with three distinct enriched lignin-degrading microbial consortia at both 25 °C and 37 °C. Enhanced biomethane yields was detected in the bioaugmented digesters, while the highest production was observed as 188 mLN CH4 gVS-1 in digesters operated at 37 °C. Moreover, methane yield showed a significant improvement in the samples at 37 °C ranging from 110% to 141% compared to the control, demonstrating the efficiency of the enriched lignin-degrading microbial community. Temperature and substrate were identified as key factors influencing microbial community dynamics. The observation that microbial communities tended to revert to the initial state after lignin depletion, indicating the stability of the overall microbiota composition in the digesters, is a promising finding for large-scale studies. Noteworthy candidates for lignin degradation, including Sporosarcina psychrophila, Comamonas aquatica, Shewanella baltica, Pseudomonas sp. C27, and Brevefilum fermentans were identified in the bioaugmented samples. PICRUSt2 predictions suggest that the pathway and specific proteins involved in anaerobic lignin degradation might share similarities with those engaged in the degradation of aromatic compounds.

First transcriptome sequencing, assembly, and annotation dataset for the freshwater angelfish, pterophyllum scalare.

Cichlids are relevant to biological research for their craniofacial variations that are analogous to human structure and associated congenital anomalies. However, only a limited number of cichlids have genetic information available. Investigating cichlids and adding to the body of knowledge about them may provide better insights into studying developmental biology and craniofacial structure. The angelfish, Pterophyllum scalare, is one cichlid for which we lack genetic information including a draft transcriptome assembly. This work is the first to provide a draft transcriptome and annotation using long-read Nanopore sequencing for P. scalare. Total RNA was extracted from angelfish tissue, and a cDNA-PCR library was prepared. Sequencing was performed on a singular R.9.4.1 MinION flow cell for 84 h. Various bioinformatic tools were then employed to assemble the sequencing reads into a transcriptome. The transcriptome was then annotated against various databases. 23 million sequencing reads were collected totalling 21.9 Gb. The N50 sequencing read length was 1255 bp and the mean read length was 938. The data had an initial mean Phred score of 10.04. After assembly, the final transcriptome consists of 98,125 transcripts with a mean length of 1552 and N50 length of 2277. The transcriptome has a completeness of 80.5% as assessed by BUSCO. Functional annotation revealed pathways related to signal transduction, carbohydrate metabolism, and transcription are the most annotated in the transcriptome.

Scalable, Cost-Effective, and Decentralized DNA Barcoding with Oxford Nanopore Sequencing.

DNA barcodes are useful in biodiversity research, but sequencing barcodes with dye termination methods ("Sanger sequencing") has been so time-consuming and expensive that DNA barcodes are not as widely used as they should be. Fortunately, MinION sequencers from Oxford Nanopore Technologies have recently emerged as a cost-effective and efficient alternative for barcoding. MinION barcodes are now suitable for large-scale species discovery and enable specimen identification when the target species are represented in barcode databases. With a MinION, it is possible to obtain 10,000 barcodes from a single flow cell at a cost of less than 0.10 USD per specimen. Additionally, a Flongle flow cell can be used for small projects requiring up to 300 barcodes (0.50 USD per specimen). We here describe a cost-effective laboratory workflow for obtaining tagged amplicons, preparing ONT libraries, sequencing amplicon pools, and analyzing the MinION reads with the software ONTbarcoder. This workflow has been shown to yield highly accurate barcodes that are 99.99% identical to Sanger barcodes. Overall, we propose that the use of MinION for DNA barcoding is an attractive option for all researchers in need of a cost-effective and efficient solution for large-scale species discovery and specimen identification.

Multiplex PCR method for MinION sequencing of Bagaza virus isolated from wild caught mosquitoes in South Africa.

Bagaza virus (BAGV) is a mosquito-borne orthoflavivirus known to occur in regions of southern Europe, Africa, India and the Middle East. The virus has been associated with neurological disease and fatalities in various wild bird species. Association with human disease is not confirmed although limited serological evidence has suggested human infection. Surveillance programs for screening mosquitoes for evidence of arbovirus infection play an important role in providing information regarding the circulation and spread of viruses in specific regions. BAGV was detected in a mosquito pool during surveillance of mosquitoes collected in central South Africa between November 2019 and March 2023. Homogenized mosquito pools were screened for flaviviral RNA using conventional RT-PCR and virus isolation was attempted on positive samples. BAGV was detected and subsequently isolated using cell culture. A multiplex tiling PCR method for targeted enrichment using a PCR based or amplicon sequencing approach of the complete genome of BAGV was developed and optimized. Primers were designed using alignment of complete genome sequence data retrieved from GenBank to identify suitable primer sites that would generate overlapping fragments spanning the complete genome. Six forward primers and eight reverse primers were identified that target the complete genome and amplified nine overlapping fragments, that ranged in length from 1954 to 2039 with an overlap ranging from 71 to 711 base pairs. The design strategy included multiple forward and reverse primer pairs for the 5' and 3' ends. Phylogenetic analysis with other isolates was performed and BAGV isolate VBD 74/23/3 was shown to share high similarity with previous BAGV isolates from all regions, with genetic distance ranging from 0.026 to 0.083. VBD 74/23/3 was most closely related to previous isolates from southern Africa, ZRU96/16/2 isolated from a post-mortem sample from a pheasant in 2016 and MP-314-NA-2018 isolated from mosquitoes in northwestern Namibia with genetic distance 0.0085 and 0.016 respectively. Currently there is limited complete genome sequence data available for many of the arboviruses circulating in Africa. The multiplex tiling method provided a simple and cost-effective method for obtaining complete genome sequence. This method can be readily applied to other viruses using sequence data from publicly available databases and would have important application facilitating genomic surveillance of arboviruses in low resource countries.