Oxford Nanopore MinION Sequencing Research Articles

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

BackgroundSoil 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.ResultsCompared 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.ConclusionsOverall, 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.

Genome sequencing of the mpox virus 2022 outbreak with amplicon-based Oxford Nanopore MinION sequencing

We present an amplicon-based assay for MinION Nanopore sequencing of mpox virus (MPXV) genomes from clinical specimens, obtaining high-quality results with an average genome coverage of 99% for Ct values of up to 25, and a genome coverage of 97.1% for Ct values from 25 to 30 which are challenging to sequence. This assay is easy to implement in PCR-based workflows and provides accurate genomic data within a short time.

Whole genome sequencing and phylogenetic analysis of SARS-CoV-2 strains isolated during the COVID-19 pandemic in Nigeria

ObjectivesThe emergence and spread of SARS-CoV-2 have stimulated ongoing research into the virus transmission dynamics, circulating variants, and potential mutations. This study was conducted to understand the genomic dynamics of the epidemic in Nigeria. DesignWhole genome sequencing was conducted on SARS-CoV-2 samples collected during the first and second outbreaks using the Oxford Nanopore MinION sequencing platform. Phylogenetic analysis was conducted, and genomes were grouped into different pangolin lineages. ResultsThe study revealed four circulating SARS-CoV-2 variants. The Alpha (B.1.1.7) variant was the most prevalent (32.7%), followed by Beta (B.1 B.1.1, L.3, and B.1.1.318) (30.8%), Eta (B.1.525) (28.9%), and Delta (B.1.617, AY.1, AY.109, and AY.36) (7.7%). Phylogenetic analysis revealed three clusters with four Nextstrain clades (20I, 20B, 21D, and 21J). The Alpha lineages (B.1.1.7) clustered with references from Italy. The Beta lineages (Clade 20B) (B.11, B.11318, and L3) and sub-lineage B.11 were distinct. Sub-lineage B.11318 is clustered with references from the USA, whereas sub-lineage L3 is clustered with references from Russia, the Philippines, Australia, and Japan. The 21D and 21J, belonging to two Pango lineages, Eta (B.1525) and Delta (B.1.617 and AY.109), showed high genetic similarity. ConclusionThe phylogenetic relatedness of the lineages suggests multiple virus introduction, which could be a source of more virulent, locally adapted variants.

Genome Sequence of the Plant-Growth-Promoting Endophyte Curtobacterium flaccumfaciens Strain W004

We report the whole-genome sequences of the endophyte Curtobacterium flaccumfaciens strain W004 isolated from the seeds of winter wheat, cv. Bezostaya 100. The genome was obtained using Oxford Nanopore MinION sequencing. The bacterium has a circular chromosome consisting of 3.63 kbp with a G+C% content of 70.89%. We found that Curtobacterium flaccumfaciens strain W004 could promote the growth of spring wheat plants, resulting in an increase in grain yield of 54.3%. Sequencing the genome of this new strain can provide insights into its potential role in plant–microbe interactions.

Occurrence and characterization of NDM-5-producing Escherichia coli from retail eggs

The New Delhi Metallo-β-lactamase (NDM) producing Enterobacterales has been detected from diverse sources but has rarely been reported in retail eggs. In this study, 144 eggshell and 96 egg content samples were collected in 2022 from Guangdong province and were screened for NDM-producing strains. Four Escherichia coli strains (ST3014, ST10, ST1485, and ST14747) recovered from two (1.39%, 2 of 144) eggshells and two (2.08%, 2 of 96) egg content samples were identified as blaNDM−5-positive strains. Oxford Nanopore MinION sequencing and conjugation assays revealed that the blaNDM−5 gene was carried by IncX3 (n = 1), IncI1 (n = 1), and IncHI2 (n = 2). The IncI1-plasmid-carrying blaNDM−5 displayed high homology with one plasmid pEC6563-NDM5 from the human clinic, while the IncHI2 plasmid harboring blaNDM−5 shared highly similar structures with plasmids of animal origin. To the best of our knowledge, this is the first report on the identification of blaNDM−5-positive bacteria in retail eggs. NDM-producing E. coli could be transmitted to humans by the consumption of eggs or direct contact, which could pose a potential threat to human health.

Cost-effective complete genome sequencing using the MinION platform for identification of recombinant enteroviruses.

Enteroviruses (EVs) are a group of viruses that cause various human illnesses. While the CODEHOP (COnsensus-DEgenerate Hybrid Oligonucleotide Primer) method can generate VP1 gene fragments for enterovirus genotyping, it is unable to detect recombinant strains. Recent advances in viral genome sequencing using next-generation sequencing technologies have enabled comprehensive analyses. However, the high cost poses a challenge for widespread adoption. To address this issue, this study proposes a cost-effective approach for generating complete enterovirus genome sequences using the Oxford Nanopore MinION sequencer. This protocol not only facilitates the generation of accurate genome sequences for various enterovirus strains but also allows for the differentiation of co-infections from viral isolates. In addition, the method can generate polyprotein sequences as well as peptide sequences of the upstream ORF (uORF) whose expression can impact virus infection. Through the analysis of complete enterovirus genomes, this study successfully identified seven enterovirus A71 isolates obtained during the 2018 enterovirus outbreak in Malaysia and Taiwan as recombinants between enterovirus A71 and coxsackievirus A2. Furthermore, our study has made a significant discovery by establishing a strong correlation between uORF trees and the epidemics of EVA71. This finding highlights the potential of uORF sequences as valuable indicators for monitoring and understanding the spread of EVA71 infections. We also identified notable amino acid changes in the transmembrane domain of the uORF protein within a newly identified lineage. These findings provide crucial insights into the molecular characteristics and evolutionary dynamics of EVA71, offering valuable information for future research and intervention strategies. IMPORTANCE By employing a cost-effective approach for complete genome sequencing, the study has enabled the identification of novel enterovirus strains and shed light on the genetic exchange events during outbreaks. The success rate of genome sequencing and the scalability of the protocol demonstrate its practical utility for routine enterovirus surveillance. Moreover, the study's findings of recombinant strains of EVA71 and CVA2 contributing to epidemics in Malaysia and Taiwan emphasize the need for accurate detection and characterization of enteroviruses. The investigation of the whole genome and upstream ORF sequences has provided insights into the evolution and spread of enterovirus subgenogroups. These findings have important implications for the prevention, control, and surveillance of enteroviruses, ultimately contributing to the understanding and management of enterovirus-related illnesses.

Nanopore Sequencing with GraphMap for Comprehensive Pathogen Detection in Potato Field Soil.

Early detection of causal pathogens is important to prevent crop loss from diseases. However, some diseases, such as soilborne diseases, are difficult to diagnose due to the absence of visible or characteristic symptoms. In the present study, the use of the Oxford Nanopore MinION sequencer as a molecular diagnostic tool was assessed due to its long-read sequencing capabilities and portability. Nucleotide samples (DNA or RNA) from potato field soils were sequenced and analyzed using a locally curated pathogen database, followed by identification via sequence mapping. We performed computational speed tests of three commonly used mapping/annotation tools (BLAST, BWA-BLAST, and BWA-GraphMap) and found BWA-GraphMap to be the fastest tool for local searching against our curated pathogen database. The data collected demonstrate the high potential of Nanopore sequencing as a minimally biased diagnostic tool for comprehensive pathogen detection in soil from potato fields. Our GraphMap-based MinION sequencing method could be useful as a predictive approach for disease management by identifying pathogens present in field soil prior to planting. Although this method still needs further experimentation with a larger sample size for practical use, the data analysis pipeline presented can be applied to other cropping systems and diagnostics for detecting multiple pathogens.

Whole genome analysis and antimicrobial resistance of Neisseria gonorrhoeae isolates from Ghana.

Gonorrhoea is a major public health concern. With the global emergence and spread of resistance to last-line antibiotic treatment options, gonorrhoea threatens to be untreatable in the future. Therefore, this study performed whole genome characterization of Neisseria gonorrhoeae collected in Ghana to identify lineages of circulating strains as well as their phenotypic and genotypic antimicrobial resistance (AMR) profiles. Whole genome sequencing (WGS) was performed on 56 isolates using both the Oxford Nanopore MinION and Illumina MiSeq sequencing platforms. The Comprehensive Antimicrobial Resistance Database (CARD) and PUBMLST.org/neisseria databases were used to catalogue chromosomal and plasmid genes implicated in AMR. The core genome multi-locus sequence typing (cgMLST) approach was used for comparative genomics analysis. In vitro resistance measured by the E-test method revealed 100%, 91.0% and 85.7% resistance to tetracycline, penicillin and ciprofloxacin, respectively. A total of 22 sequence types (STs) were identified by multilocus sequence typing (MLST), with ST-14422 (n = 10), ST-1927 (n = 8) and ST-11210 (n = 7) being the most prevalent. Six novel STs were also identified (ST-15634, 15636-15639 and 15641). All isolates harboured chromosomal AMR determinants that confer resistance to beta-lactam antimicrobials and tetracycline. A single cefixime-resistant strain, that belongs to N. gonorrhoeae multiantigen sequence type (NG-MAST) ST1407, a type associated with widespread cephalosporin resistance was identified. Neisseria gonorrhoeae Sequence Typing for Antimicrobial Resistance (NG-STAR), identified 29 unique sequence types, with ST-464 (n = 8) and the novel ST-3366 (n = 8) being the most prevalent. Notably, 20 of the 29 STs were novel, indicative of the unique nature of molecular AMR determinants in the Ghanaian strains. Plasmids were highly prevalent: pTetM and pblaTEM were found in 96% and 92% of isolates, respectively. The TEM-135 allele, which is an amino acid change away from producing a stable extended-spectrum β-lactamase that could result in complete cephalosporin resistance, was identified in 28.5% of the isolates. Using WGS, we characterized N. gonorrhoeae strains from Ghana, giving a snapshot of the current state of gonococcal AMR in the country and highlighting the need for constant genomic surveillance.

A method of large DNA fragment enrichment for nanopore sequencing in region 22q11.2.

Background: 22q11.2 deletion syndrome (22q11.2DS) is a disorder caused when a small part of chromosome 22 is missing. Diagnosis is currently established by the identification of a heterozygous deletion at chromosome 22q11.2 through chromosomal microarray analysis or other genomic analyses. However, more accurate identification of the breakpoint contributes to a clearer understanding of the 22q11.2 deletion syndrome. Methods: In this study, we present a feasible nanopore sequencing method of 22q11.2 deletion. This DNA enrichment method-region-specific amplification (RSA)-is able to analyze the 22q11.2 deletion by specific amplification of an approximately 1-Mb region where the breakpoint might exist. RSA introduces universal primers into the target region DNA by a Y-shaped adaptor ligation and a single primer extension. The enriched products, completed by amplification with universal primers, are then processed by standard ONT ligation sequencing protocols. Results: RSA is able to deliver adequate coverage (>98%) and comparable long reads (average length >1Kb) throughout the 22q11.2 region. The long nanopore sequencing reads, derived from three umbilical cord blood samples, have facilitated the identification of the breakpoint of the 22q11.2 deletion, as well as by Sanger sequencing. Conclusion: The Oxford Nanopore MinION sequencer can use RSA to sequence the target region 22q11.2; this method could also be used for other hard-to-sequence parts of the genome.

Investigation of SARS-CoV-2 lineages and mutations circulating in a university-affiliated hospital in South Korea analyzed using Oxford Nanopore MinION sequencing.

Despite the introduction of vaccines, treatments, and massive diagnostic testing, the evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has continued to overcome barriers that had slowed its previous spread. As the virus evolves towards increasing fitness, it is critical to continue monitoring the occurrence of new mutations that could evade human efforts to control them. We performed whole-genome sequencing using Oxford Nanopore MinION sequencing on 58 SARS-CoV-2 isolates collected during the ongoing coronavirus disease 2019 pandemic at a tertiary hospital in South Korea and tracked the emergence of mutations responsible for massive spikes in South Korea. The differences among lineages were more pronounced in the spike gene, especially in the receptor-binding domain (RBD), than in other genes. Those RBD mutations could compromise neutralization by antibodies elicited by vaccination or previous infections. We also reported multiple incidences of Omicron variants carrying mutations that could impair the diagnostic sensitivity of reverse transcription-polymerase chain reaction-based testing. These results provide an understanding of the temporal changes of variants and mutations that have been circulating in South Korea and their potential impacts on antigenicity, therapeutics, and diagnostic escape of the virus. We also showed that the utilization of the nanopore sequencing platform and the ARTIC workf low can provide convenient and accurate SARS-CoV-2 genomic surveillance even at a single hospital.

Gut microbiota shift in layer pullets fed on black soldier fly larvae-based feeds towards enhancing healthy gut microbial community

Globally, most gut microbiota-related studies have focused on broilers due to their diverse microbial communities compared to that of layer chicken. However, in Africa few studies have been undertaken despite the increasing benefits to the poultry industry. The utilization of Insect-Based diets to improve the gastrointestinal function and gut health in poultry is increasingly gaining global attention. Here, we evaluated the potential roles of commercial black soldier fly larvae-based feeds (BSFLF) in reshaping the abundance, composition and diversity of the gut microbiota of layer chickens using high throughput Oxford nanopore Minion sequencing of the full length bacterial 16S rRNA gene. Two hundred and fifty ISA Brown layer chicks were reared in pens for a period of 20 weeks. The layer pullets were allotted 5 dietary treatments that were formulated as follows: control diet (T1): 100% FM + 0% BSFL, T2: 25% BSFL + 75% FM; T3: 50% BSFL + 50% FM; T4: 75% BSFL + 25% FM, and T5: 100% BSFL + 0% FM. Sampling was done from the eight major regions including oesophagus, crop, proventriculus, gizzard, duodenum, ileum, large intestines and ceca. Out of the 400,064 classified reads analyzed, the most dominant phyla identified across the feed treatments were Firmicutes, Bacteroidetes, Proteobacteria and Actinobacteria. The diet treatment with 100% inclusion levels of BSFL showed the highest intra-species alpha diversity and richness according to Chao1 and Shannon index. Intra-species beta diversity assessment revealed that the diet types significantly influenced the abundance of the microbiota, but differences between most abundant taxa were similar. There was increase in abundance of potentially beneficial bacteria (Lactobacillus, Bacteroides and Enterococcus) with increased inclusion levels of BSFLF in layer pullets diets. Across the different gut segments, Lactobacillus dominated all the eight regions and the ceca was the most diverse segment. Our findings unravel complex gut microbial shift in laying hen fed BSFLF and therefore underpins the potential roles of beneficial bacteria as promising prebiotics and probiotics in reshaping of the gut microbiota to maintain good gut health.

Next generation biosecurity: Towards genome based identification to prevent spread of agronomic pests and pathogens using nanopore sequencing.

The unintentional movement of agronomic pests and pathogens is steadily increasing due to the intensification of global trade. Being able to identify accurately and rapidly early stages of an invasion is critical for developing successful eradication or management strategies. For most invasive organisms, molecular diagnostics is today the method of choice for species identification. However, the currently implemented tools are often developed for certain taxa and need to be adapted for new species, making them ill-suited to cope with the current constant increase in new invasive species. To alleviate this impediment, we developed a fast and accurate sequencing tool allowing to modularly obtain genetic information at different taxonomical levels. Using whole genome amplification (WGA) followed by Oxford nanopore MinION sequencing, our workflow does not require any a priori knowledge on the investigated species and its classification. While mainly focusing on harmful plant pathogenic insects, we also demonstrate the suitability of our workflow for the molecular identification of bacteria (Erwinia amylovora and Escherichia coli), fungi (Cladosporium herbarum, Colletotrichum salicis, Neofabraea alba) and nematodes (Globodera rostochiensis). On average, the pairwise identity between the generated consensus sequences and best GenBank BLAST matches was 99.6 ± 0.6%. Additionally, assessing the generated insect genomic dataset, the potential power of the workflow to detect pesticide resistance genes, as well as arthropod-infecting viruses and endosymbiotic bacteria is demonstrated.

High-quality genome assembly of a Pestalotiopsis fungus using DIY-friendly methods

Of the millions of fungal species estimated to exist, about 100,000 have been identified, and only approximately 3000 of those have representative genome assemblies available. Here, we isolated a wild species of Pestalotiopsis from the Los Angeles area, extracted DNA in a low-cost environment (e.g., home lab), and generated a high-quality genome assembly using the low-cost Oxford Nanopore MinION sequencing platform. We found that Pestalotiopsis has a genome composed of 7 nuclear chromosomes, comprising 47.7 megabases. Using this genome, we perform a multi-locus phylogenetic analysis and finally, we discuss how this project (costing $300) demonstrates the increased accessibility of whole genome sequencing.

RespiCoV: Simultaneous identification of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) and 46 respiratory tract viruses and bacteria by amplicon-based Oxford-Nanopore MinION sequencing.

Since December 2019 the world has been facing the outbreak of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Identification of infected patients and discrimination from other respiratory infections have so far been accomplished by using highly specific real-time PCRs. Here we present a rapid multiplex approach (RespiCoV), combining highly multiplexed PCRs and MinION sequencing suitable for the simultaneous screening for 41 viral and five bacterial agents related to respiratory tract infections, including the human coronaviruses NL63, HKU1, OC43, 229E, Middle East respiratory syndrome coronavirus, SARS-CoV, and SARS-CoV-2. RespiCoV was applied to 150 patient samples with suspected SARS-CoV-2 infection and compared with specific real-time PCR. Additionally, several respiratory tract pathogens were identified in samples tested positive or negative for SARS-CoV-2. Finally, RespiCoV was experimentally compared to the commercial RespiFinder 2SMART multiplex screening assay (PathoFinder, The Netherlands).

Complete Genome Sequences of Three Staphylococcus haemolyticus Strains Isolated from the Lung of a TGFβ1 Transgenic Mouse with Lung Fibrosis

ABSTRACTWe report here the complete genome sequences of three Staphylococcus haemolyticus strains isolated from a mouse fibrotic lung tissue and exhibiting proapoptotic activity on human lung alveolar epithelial cells. The genomes were obtained from a combination of Illumina MiSeq and Oxford Nanopore MinION sequencing.

In-situ sequencing reveals the effect of storage on lacustrine sediment microbiome demographics and functionality

The sediment microbiome is a demographically diverse and functionally active biosphere. Ensuring that data acquired from sediment is truly representative of the microbiome is critical to achieving robust analyses. Sample storage and the processing and timing of nucleic acid purification after environmental sample extraction may fundamentally affect the detectable microbial community and thereby significantly alter resultant data. Direct sequencing of environmental samples is increasingly commonplace due to the advent of the portable Oxford Nanopore MinION sequencing device. Here we demonstrate that storing sediment subsamples at − 20 °C or storing the cores at 4 °C for 10 weeks prior to analysis, has a significant effect on the sediment microbiome analysed using sedimentary DNA (sedDNA), especially for Alpha-, Beta- and Deltaproteobacteria species. Furthermore, these significant differences are observed regardless of sediment type. We show that the taxa which are predominantly affected by storage are Proteobacteria, and therefore recommend on-site purifications are performed to ensure an accurate representation of these taxa are observed in the microbiome. Comparisons of sedimentary RNA (sedRNA) analyses, revealed substantial differences between samples purified and sequenced immediately on-site, samples that were frozen before transportation, and cores that were stored at 4 °C prior to analysis. Our data therefore suggest that a more accurate representation of the sediment microbiome demography and functionality may be achieved by environmental sequencing as rapidly as possible to minimise confounding effects of storage.

Genome Sequences of SARS-CoV-2 Sublineage B.1.617.2 Strains from 12 Children in Chattogram, Bangladesh

ABSTRACTWe announce the complete genome sequences of 12 severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) sublineage B.1.617.2 strains (Delta variant) obtained from nasopharyngeal and oropharyngeal swab samples from 12 pediatric patients in Chittagong, Bangladesh, displaying COVID-19 symptoms. Oxford Nanopore MinION sequencing technology was used to generate the genomic sequences.

Complete Genome Sequencing of Field Isolates of Peste des Petits Ruminants Virus from Tanzania Revealed a High Nucleotide Identity with Lineage III PPR Viruses.

Simple SummaryPeste des petits ruminants virus (PPRV) causes a highly devastating disease, peste des petits ruminants (PPR), in sheep and goats, which is targeted for global control and eradication. However, in many developing countries, access to expensive sequencing technologies is limited and is compounded by difficulties in transporting clinical samples across international borders. Oxford nanopore MinION is a relatively cheap sequencing technology using portable devices that require minimal supporting laboratory infrastructure or technical expertise for sample preparation and rapid sequencing. In this study, Oxford nanopore MinION sequencing was carried out to generate complete genomes of PPRV from archived PPRV-positive samples collected from PPR outbreaks in goats in Ngorongoro and Momba districts in Tanzania during 2016 and 2018, respectively. Complete genomes of PPRV of 15,948 nucleotides long were generated within four hours of sequencing. The phylogenetic analysis of the complete genomes revealed a high nucleotide identity (96.19–99.24%) with lineage III PPR viruses currently circulating in East Africa, indicating a common origin. The Oxford nanopore MinION sequencer can be deployed to overcome diagnostic and surveillance challenges in developing countries in the PPR Global Control and Eradication program. However, the coverage depth was uneven across the genome and amplicon dropout was observed between the matrix (M) and fusion (F) genes. Thus, larger field studies are needed to allow the collection of sufficient data to assess the robustness of nanopore sequencing technology.Peste des petits ruminants virus (PPRV) causes a highly devastating disease of sheep and goats that threatens food security, small ruminant production and susceptible endangered wild ruminants. With policy directed towards achieving global PPR eradication, the establishment of cost-effective genomic surveillance tools is critical where PPR is endemic. Genomic data can provide sufficient in-depth information to identify the pockets of endemicity responsible for PPRV persistence and viral evolution, and direct an appropriate vaccination response. Yet, access to the required sequencing technology is low in resource-limited settings and is compounded by the difficulty of transporting clinical samples from wildlife across international borders due to the Convention on International Trade in Endangered Species (CITES) of Wild Fauna and Flora, and Nagoya Protocol regulations. Oxford nanopore MinION sequencing technology has recently demonstrated an extraordinary performance in the sequencing of PPRV due to its rapidity, utility in endemic countries and comparatively low cost per sample when compared to other whole-genome (WGS) sequencing platforms. In the present study, Oxford nanopore MinION sequencing was utilised to generate complete genomes of PPRV isolates collected from infected goats in Ngorongoro and Momba districts in the northern and southern highlands of Tanzania during 2016 and 2018, respectively. The tiling multiplex polymerase chain reaction (PCR) was carried out with twenty-five pairs of long-read primers. The resulting PCR amplicons were used for nanopore library preparation and sequencing. The analysis of output data was complete genomes of PPRV, produced within four hours of sequencing (accession numbers: MW960272 and MZ322753). Phylogenetic analysis of the complete genomes revealed a high nucleotide identity, between 96.19 and 99.24% with lineage III PPRV currently circulating in East Africa, indicating a common origin. The Oxford nanopore MinION sequencer can be deployed to overcome diagnostic and surveillance challenges in the PPR Global Control and Eradication program. However, the coverage depth was uneven across the genome and amplicon dropout was observed mainly in the GC-rich region between the matrix (M) and fusion (F) genes of PPRV. Thus, larger field studies are needed to allow the collection of sufficient data to assess the robustness of nanopore sequencing technology.

Is amplification bias consequential in transposon sequencing (TnSeq) assays? A case study with a Staphylococcus aureus TnSeq library subjected to PCR-based and amplification-free enrichment methods.

As transposon sequencing (TnSeq) assays have become prolific in the microbiology field, it is of interest to scrutinize their potential drawbacks. TnSeq data consist of millions of nucleotide sequence reads that are generated by PCR amplification of transposon-genomic junctions. Reads mapping to the junctions are enumerated thus providing information on the number of transposon insertion mutations in each individual gene. Here we explore the possibility that PCR amplification of transposon insertions in a TnSeq library skews the results by introducing bias into the detection and/or enumeration of insertions. We compared the detection and frequency of mapped insertions when altering the number of PCR cycles, and when including a nested PCR, in the enrichment step. Additionally, we present nCATRAs - a novel, amplification-free TnSeq method where the insertions are enriched via CRISPR/Cas9-targeted transposon cleavage and subsequent Oxford Nanopore MinION sequencing. nCATRAs achieved 54 and 23% enrichment of the transposons and transposon-genomic junctions, respectively, over background genomic DNA. These PCR-based and PCR-free experiments demonstrate that, overall, PCR amplification does not significantly bias the results of TnSeq insofar as insertions in the majority of genes represented in our library were similarly detected regardless of PCR cycle number and whether or not PCR amplification was employed. However, the detection of a small subset of genes which had been previously described as essential is sensitive to the number of PCR cycles. We conclude that PCR-based enrichment of transposon insertions in a TnSeq assay is reliable, but researchers interested in profiling putative essential genes should carefully weigh the number of amplification cycles employed in their library preparation protocols. In addition, nCATRAs is comparable to traditional PCR-based methods (Kendall’s correlation=0.896–0.897) although the latter remain superior owing to their accessibility and high sequencing depth.

A draft genome assembly for the eastern fox squirrel, Sciurus niger.

The eastern fox squirrel, Sciurus niger, exhibits marked geographic variation in size and coat color, is a model organism for studies of behavior and ecology, and a potential model for investigating physiological solutions to human porphyrias. We assembled a genome using Illumina HiSeq, PacBio SMRT, and Oxford Nanopore MinION sequencing platforms. Together, the sequencing data resulted in a draft genome of 2.99 Gb, containing 32,830 scaffolds with an average size of 90.9 Kb and N50 of 183.8 Kb. Genome completeness was estimated to be 93.78%. A total of 24,443 protein-encoding genes were predicted from the assembly and 23,079 (94.42%) were annotated. Repeat elements comprised an estimated 38.49% of the genome, with the majority being LINEs (13.92%), SINEs (6.04%), and LTR elements. The topology of the species tree reconstructed using maximum-likelihood phylogenetic analysis was congruent with those of previous studies. This genome assembly can prove useful for comparative studies of genome structure and function in this rapidly diversifying lineage of mammals, for studies of population genomics and adaptation, and for biomedical research. Predicted amino acid sequence alignments for genes affecting heme biosynthesis, color vision, and hibernation showed point mutations and indels that may affect protein function and ecological adaptation.