Next-generation DNA Sequencing Technologies Research Articles

Variant functional assessment in Drosophila by overexpression: what can we learn?

The last decade has been highlighted by the increased use of next-generation DNA sequencing technology to identify novel human disease genes. A critical downstream part of this process is assigning function to a candidate gene variant. Functional studies in Drosophila melanogaster, the common fruit fly, have made a prominent contribution in annotating variant impact in an in vivo system. The use of patient-derived knock-in flies or rescue-based, "humanization", approaches are novel and valuable strategies in variant testing but have been recently widely reviewed. An often-overlooked strategy for determining variant impact has been GAL4/upstream activation sequence-mediated tissue-defined overexpression in Drosophila. This mini-review will summarize the recent contribution of ectopic overexpression of human reference and variant cDNA in Drosophila to assess variant function, interpret the consequence of the variant, and in some cases infer biological mechanisms.

Using Graphdiyne Nanoribbons for Molecular Electronics Spectroscopy and Nucleobase Identification: A Theoretical Investigation.

In pursuit of fast, cost-effective, and reliable DNA sequencing techniques, a variety of two-dimensional (2D) material-based nanodevices such as solid-state nanopores and nanochannels have been explored and established. Given the promising potential of graphene for the design and fabrication of nanobiosensors, other 2D carbon allotropes such as graphyne and graphdiyne have also attracted a great deal of attention as candidate materials for the development of sequencing technology. Herein, employing the 2D electronic molecular spectroscopy (2DMES) method, we investigate the capability of graphdiyne nanoribbons (GDNRs) as the building blocks of a feasible, precise, and ultrafast sequencing device. Using first-principles calculations, we study the adsorption of four canonical nucleobases (NBs), i.e., adenine (A), cytosine (C), guanine (G), and thymine (T) on an armchair GDNR (AGDNR). Our calculations reveal that compared to graphene, graphdiyne demonstrates more distinct binding energies for different NBs, indicating its more promising ability to unambiguously recognize DNA bases. Utilizing the 2DMES technique, we calculate the differential conductance (Δg) of the studied NB-AGDNR systems and show that the resulting Δg maps, unique for each NB-AGDNR complex, can be used to recognize each individual NB without ambiguity. We also investigate the conductance sensitivity of the proposed nanobiosensor and show that it exhibits high sensitivity and selectivity toward various NBs. Thus, our proposed graphdiyne-based nanodevice would hold promise for next-generation DNA sequencing technology.

Conservation genetics of Sclerocactus in Colorado: the importance of accurate taxonomy to conservation

IntroductionRecent advances in genetic data collection utilizing next-generation DNA sequencing technologies have the potential to greatly aid the taxonomic assessment of species of conservation concern, particularly species that have been difficult to describe using morphology alone. Accurate taxonomic descriptions aided by genetic data are essential to directing limited conservation resources to species most in need. Sclerocactus glaucus is a plant endemic to Western Colorado that is currently listed as Threatened under the Endangered Species Act (ESA). However, in 2023, the U. S. Fish and Wildlife Service proposed de-listing S. glaucus from the ESA due to recovery of the species. Previous research had found substantial genetic structure between populations in the northern part of the S. glaucus range relative to the majority of the species distribution.MethodsIn this study we utilized double-digest Restriction-site Associated DNA sequencing (RAD-seq) in order to better understand the genetic structure of S. glaucus.ResultsOur results indicate that S. glaucus contains two distinct evolutionary lineages that warrant recognition at the level of species, with what was previously described as S. glaucus North being recognized as Sclerocactus dawsoniae.DiscussionThe newly described S. dawsoniae has a limited estimated number of individuals, low levels of nucleotide diversity, a very narrow geographic range, and an uneven geographic distribution with most plants being found in a single management area, all of which supports continued direct conservation of this species. In contrast, S. glaucus has a large estimated minimum population size, a broad geographic range that includes numerous protected areas, and adequate levels of genetic diversity. Without further conservation action, a delisting decision for S. glaucus will simultaneously remove all Endangered Species Act protections for S. dawsoniae. The current work demonstrates the importance of having robust genetic datasets when planning conservation activities for species of concern. Moving forward, we recommend that government stakeholders prioritize supporting genetic studies of endangered species prior to making any changes to listing decisions.

Identification and Analysis of the Ankle Microbiome Using Next-Generation DNA Sequencing: An Observational Analysis.

Next-generation DNA sequencing (NGS) technologies have increased the sensitivity for detecting the bacterial presence and have been used in other areas of orthopaedics to better understand the native microbiome of various joints. This study uses NGS to determine whether (1) a unique microbiome exists in human ankle tissues, (2) if components of the ankle microbiome affect patient outcomes, and (3) whether microbes found on the skin are a normal part of the ankle microbiome. A prospective study recruited 32 patients undergoing total ankle arthroplasty (n = 23) or ankle arthrodesis (n = 9) via direct anterior approach between November 2020 and October 2021. During surgery, five layers of the ankle were swabbed: skin (n = 32), retinaculum (n = 31), tibialis anterior tendon (n = 31), joint capsule (n = 31), and distal tibia (n = 32). These swabs (N = 157) were sent to MicroGen Diagnostics (Lubbock) for NGS. Demographics, medical comorbidities, surgical indication, postoperative complications, readmission, and revision surgery rates were collected from patient records. The mean age was 60.7 (range, 19 to 85) years, and the mean follow-up duration was 10.2 (range, 4.8 to 20.6) months. Of 157 swabs sent for NGS, 19 (12.1%) indicated that bacteria were present (positive), whereas the remaining 138 (87.9%) had no bacteria present (negative). The most common organisms were Cutibacterium acnes in eight ankles (25.0%) and Staphylococcus epidermidis in two ankles (6.25%). Most bacteria were found in the retinaculum (29.6%). Complications, nonunions, infections, 90-day readmission, and revision surgery rates did not differ by NGS profile. This study found that C acnes and S epidermidis were the most common bacteria in the ankle microbiome, with C acnes being present in 25% of ankles. Complication rates did not differ between patients with or without positive bacterial DNA remnants. Thus, we concluded that a unique ankle microbiome is present in some patients, which is unique from that of the skin of the ankle. Level II, Prospective cohort study.

Identification and Analysis of the Ankle Microbiome Using Next-Generation DNA Sequencing: An Observational Analysis

Category: Ankle; Basic Sciences/Biologics Introduction/Purpose: Next-generation DNA sequencing (NGS) technologies have increased the sensitivity of bacterial cultures and has been used in other areas of orthopaedics to better understand the native microbiome of various joints. This study uses NGS to determine whether (1) a unique microbiome exists in human ankle tissues, (2) if components of the ankle microbiome impact patient outcomes, and (3) whether microbes found on the skin are a normal part of the ankle microbiome. Methods: A prospective study recruited 32 patients undergoing total ankle arthroplasty (TAA, n=23) or ankle arthrodesis via an anterior approach (n=9) during 2020-2021 with one of two fellowship-trained foot and ankle surgeons at an academic medical center. During the operation, the surgeon would swab five layers of the ankle for analysis at a CLIA-licensed molecular diagnostic laboratory: skin (n=32), retinaculum (n=31), tibialis anterior tendon (n=31), joint capsule (n=31), and distal tibia (n=32). Demographics, medical comorbidities, surgical indication, postoperative complications, and readmission and reoperation rates were collected from patient records. Results: Of the 157 swabs sent for NGS, 19 (12.1%) indicated 27 bacteria were present (positive), while the remaining 138 (87.9%) had no bacteria present (negative). The most common organisms were Cutibacterium acnes, present in 11 ankles (34.4%), and Staphylococcus epidermidis, present in 3 ankles (9.4%). The most bacteria were found in the retinaculum (29.6%). No PROM nor follow-up duration was associated with the presence of bacteria, or the number of bacteria or affected layers. Similarly, complication (positive=18.2%, negative=23.8%; p=1.000), nonunion (positive=0.0%, negative=28.6%; p=1.000), infection (positive=0.0%, negative=4.8%; p=1.000), 90-day readmission (positive=0.0%, negative=4.8%; p=1.000), and reoperation (positive=0.0%, negative=4.8%; p=1.000) rates did not differ if a subject’s NGS profile was positive or negative. Conclusion: This study found that C. acnes and S. epidermidis were most commonly found in the ankle microbiome, with C. acnes being present in 34% of ankles. There was no difference in complication rates between patient with and without positive bacterial cultures following TAA or ankle arthrodesis.

Identification and Analysis of The Microbiome in Patients Undergoing Removal of Hardware

Category: Basic Sciences/Biologics; Ankle Introduction/Purpose: Next-generation DNA sequencing (NGS) technologies have increased the sensitivity of bacterial cultures and helped to better understand the normal flora of different joints. Researchers have utilized NGS to identify organisms associated with reoperations in shoulder surgery, but there is very little data on the field of foot and ankle. This study uses NGS to determine whether there is a unique microbiome that exists on orthopaedic hardware in the foot and ankle. Methods: A prospective study recruited 34 patients undergoing hardware removal surgery from the foot or ankle during 2020- 2023 with one of two fellowship-trained foot and ankle surgeons at an academic medical center. Patients were excluded based on the diagnosis or suspicion of infection prior to surgery. Explanted hardware was sent for analysis at a CLIA-licensed molecular diagnostic laboratory. Demographics, medical comorbidities, surgical indication, postoperative complications, readmission, and reoperation rates were collected from patient records. The majority of patients underwent surgery for diagnosis with painful orthopaedic hardware (70.6%). The cohort was majority female (67.6%) and average age was 53.15 (range 18-78) years. Results: Of the 34 samples sent for NGS, 5 (14.7%) indicated 9 bacteria were present (positive), while the remaining 29 (85.3%) had no bacteria present (negative). The most common organisms were of the genus Staphylococcus, representing 4 bacteria (44.4%), and Corynebacterium, representing 2 bacteria (22.2%). C. acnes was only present in one sample. There was no relationship between bacterial presence and patients undergoing hardware removal for diagnosis of painful orthopaedic hardware. No demographics nor postoperative outcomes were statistically associated with the presence of bacteria on removed hardware samples. The overall rate of complication, reoperation, and readmission were 8.8%, 2.9%, and 2.9%, respectively. Conclusion: This study found that there is no common microbial community present in patients undergoing hardware removal surgery. The most commonly present bacteria were of the genus Staphylococcus, bacteria overall were isolated in 14.7% of specimens. C acnes appeared to be uncommon. Further research should be done with a larger sample to support these findings.

Integration of deep learning with Ramachandran plot molecular dynamics simulation for genetic variant classification.

Functional classification of genetic variants is a key for their clinical applications in patient care. However, abundant variant data generated by the next-generation DNA sequencing technologies limit the use of experimental methods for their classification. Here, we developed a protein structure and deep learning (DL)-based system for genetic variant classification, DL-RP-MDS, which comprises two principles: 1) Extracting protein structural and thermodynamics information using the Ramachandran plot-molecular dynamics simulation (RP-MDS) method, 2) combining those data with an unsupervised learning model of auto-encoder and a neural network classifier to identify the statistical significance patterns of the structural changes. We observed that DL-RP-MDS provided higher specificity than over 20 widely used insilico methods in classifying the variants of three DNA damage repair genes: TP53, MLH1, and MSH2. DL-RP-MDS offers a powerful platform for high-throughput genetic variant classification. The software and online application are available at https://genemutation.fhs.um.edu.mo/DL-RP-MDS/.

Nisin E, a New Nisin Variant Produced by Streptococcus equinus MDC1

Members of the genus Streptococcus inhabit a variety of sites in humans and other animals and some species are prolific producers of proteinaceous antibiotics (bacteriocins). As little is known about (i) streptococci inhabiting domestic pets, and (ii) whether novel bacteriocin-producing streptococci can be isolated from domestic pets, the aim of this study is to address these gaps in the research literature. In this study, Streptococcus equinus MDC1, isolated from a healthy dog, was found to exhibit potent antibacterial activity against Micrococcus luteus in a simultaneous antagonism assay, suggesting that strain MDC1 produces a lantibiotic bacteriocin. The inhibitory activity spectrum of S. equinus MDC1, determined using agar-based deferred antagonism assays against >70 indicator strains, was found to be similar to that of nisin U (a lantibiotic produced by Streptococcus uberis). However, the spectra of the two bacteriocins differed by 23 strains, mainly with the MDC1 bacteriocin having no inhibitory activity towards certain streptococci of human origin (e.g., Streptococcus gordonii, Streptococcus anginosus, Streptococcus salivarius). The genome of S. equinus MDC1, which was sequenced completely using single-molecule real-time (SMRT) next-generation DNA sequencing technology, comprises a single 1,936,555-basepair chromosome containing seven copies of the ribosomal RNA operon, 69 tRNA genes and nearly 1900 putative coding sequences. Analysis of the MDC1 genome sequence using the bacteriocin detection algorithms BAGEL4 and antiSMASH revealed the location of a 13,164-basepair 11-gene locus, designated nmd, which encoded a mature nisin E peptide that differed from nisin U by only two amino acids (Ile15→Ala and Leu21→Ile) and an extra C-terminal asparagine residue, and the proteins required for post-translational modification of the bacteriocin, processing, export, and producer immunity. Despite the high homology (90.6% identity, 93.8% similarity) between nisin E and nisin U, there was considerably less homology (47.4–76.3% identity, 68.4–88.8% similarity) between the other proteins encoded by their respective biosynthetic loci. This new natural variant of nisin, called nisin E, represents the first nisin variant to be reported for S. equinus; additionally, its differences with nisin U may provide some insight into the amino acids that influence bacteriocin potency and killing spectrum.

Identification and Analysis of the Ankle Microbiome Using Next-Generation DNA Sequencing

Category: Ankle; Basic Sciences/Biologics Introduction/ Purpose: Next-generation DNA sequencing (NGS) technologies have made it possible to detect microbial genome sequences within human tissues with a sensitivity that has not been reached before, allowing research to better characterize “normal” host flora. Researchers have utilized NGS to identify the shoulder microbiome, but there are no reports yet on the ankle microbiome. Using NGS, this study aims to determine whether (1) a unique microbiome exists in human ankle tissues, (2) components of the ankle microbiome impact patient outcomes, and (3) microbes found on the skin make up a normal part of the ankle microbiome within the tissue. Methods: A prospective study recruited 33 patients undergoing total ankle arthroplasty (TAA, n=24) or ankle arthrodesis via an anterior approach (n=9) between November 2020 and October 2021 with one of two fellowship-trained foot and ankle surgeons at an academic medical center. During the operation, the surgeon would swab five layers of the ankle: skin (n=33), retinaculum (n=32), tibialis anterior tendon (n=32), joint capsule (n=32), and distal tibia (n=33). These swabs (N=162) were then sent to MicroGen Diagnostics [Lubbock, TX, USA] for NGS. Retrospective data was also collected from patient records to include demographics, medical comorbidities, surgical indication, postoperative complications, readmission and reoperation rates, and pre- and postoperative patient-reported outcome measures. The majority of subjects underwent surgery for ankle arthritis (97.0%). Most subjects were female (51.5%) and white (93.9%), and mean age at surgery was 60.73 (range, 19-85) years. Mean follow-up duration was 0.75 (range, 0.11-1.29) years. Results: Of the 162 swabs sent for NGS, 19 (11.7%) indicated 27 bacteria were present (positive), while the remaining 143 (88.3%) had no bacteria present (negative). The most common organisms were Cutibacterium acnes (40.7%) and Staphylococcus epidermidis (11.1%). The most bacteria were found in the retinaculum (29.6%), followed by the distal tibia (25.9%) and capsule (22.2%). No PROM nor follow-up duration was associated with the presence of bacteria, or the number of bacteria or affected layers. Similarly, complication (positive=18.2%, negative=22.7%; p=1.000), nonunion (positive=0.0%, negative=28.6%; p=1.000), infection (positive=0.0%, negative=4.5%; p=1.000), 90-day readmission (positive=0.0%, negative=4.5%; p=1.000), and reoperation (positive=0.0%, negative=4.5%; p=1.000) rates did not differ if a subject’s NGS profile was positive or negative. Conclusion: This study found that C. acnes and S. epidermidis were most commonly found in the ankle microbiome, though at a relatively low rate. While there is some evidence to suggest both S. epidermidis and Staphylococcus aureus biofilm formation are identified on explanted ankle prosthetics, no study to date has investigated whether or not these bacteria were present in the ankle joint prior to surgery. Furthermore, there is no evidence that patients presenting with these bacteria at the time of surgery are at a higher risk of failure following TAA or ankle arthrodesis.

The Long and Short of Genome Sequencing: Using a Hybrid Sequencing Strategy to Sequence Oral Microbial Genomes.

Since our chapter on genome sequencing using the GS-FLX pyrosequencer in the First Edition of this book, significant advances have been made in next-generation DNA sequencing (NGS) technology. Not only has the GS-FLX become extinct, but the more recent introduction and establishment of the so-called third-generation DNA sequencers by Pacific Biosciences (PacBio) and Oxford Nanopore Technologies (ONT) has revolutionized genomics yet again by generating ultra-long (>100,000 basepair) sequence reads concomitant with an incredible reduction in cost per sequenced basepair. Unfortunately, the ultra-high sequence yields of third-generation sequencers are compromised by their inherent sequencing error rates, prompting an alternative sequencing strategy, i.e., a hybrid sequencing strategy, which combines PacBio/ONT primary datasets with complementary datasets generated by mainstream short-read NGS platforms, e.g., Illumina or Ion Torrent. Although the concept of a hybrid sequencing strategy is not new, existing yields and accuracy of ultra-long and short-read sequencing technologies makes such a strategy achievable, resulting in complete genome sequences in one hit. In this chapter, we describe our updated laboratory and bioinformatic protocols that will allow the average research group to obtain complete oral microbial genome sequences assembled from a combination of DNA sequence data generated by NGS and third-generation platforms.

Bioinformatic Analysis of Natively Paired VH:VL Antibody Repertoires for Antibody Discovery.

Next-generation DNA sequencing (NGS) of human antibody repertoires has been extensively implemented to discover novel antibody drugs, to analyze B-cell developmental features, and to investigate antibody responses to infectious diseases and vaccination. Because the antibody repertoire encoded by human B cells is highly diverse, NGS analyses of antibody genes have provided a new window into understanding antibody responses for basic immunology, biopharmaceutical drug discovery, and immunotherapy. However, many antibody discovery protocols analyze the heavy and light chains separately due to the short-read nature of most NGS technologies, whereas paired heavy and light chain data are required for complete antibody characterization. Here, we describe a computational workflow to process millions of paired antibody heavy and light chain DNA sequence reads using the Illumina MiSeq 2x300 NGS platform. In this workflow, we describe raw NGS read processing and initial quality filtering, the annotation and assembly of antibody clonotypes relating to paired heavy and light chain antibody lineages, and the generation of complete heavy+light consensus sequences for the downstream cloning and expression of human antibody proteins.

Biosynthesis-guided discovery reveals enteropeptins as alternative sactipeptides containing N-methylornithine.

The combination of next-generation DNA sequencing technologies and bioinformatics has revitalized natural product discovery. Using a bioinformatic search strategy, we recently identified ∼600 gene clusters in otherwise overlooked streptococci that code for ribosomal peptide natural products synthesized by radical S-adenosylmethionine enzymes. These grouped into 16 subfamilies and pointed to an unexplored microbiome biosynthetic landscape. Here we report the structure, biosynthesis and function of one of these natural product groups, which we term enteropeptins, from the gut microbe Enterococcus cecorum. We show three reactions in the biosynthesis of enteropeptins that are each catalysed by a different family of metalloenzymes. Among these, we characterize the founding member of a widespread superfamily of Fe-S-containing methyltransferases, which, together with an Mn2+-dependent arginase, installs N-methylornithine in the peptide sequence. Biological assays with the mature product revealed bacteriostatic activity only against the producing strain, extending an emerging theme of fratricidal or self-inhibitory metabolites in microbiome firmicutes.

The genetic and molecular features of the intronic pentanucleotide repeat expansion in spinocerebellar ataxia type 10.

Spinocerebellar ataxia type 10 (SCA10) is characterized by progressive cerebellar neurodegeneration and, in many patients, epilepsy. This disease mainly occurs in individuals with Indigenous American or East Asian ancestry, with strong evidence supporting a founder effect. The mutation causing SCA10 is a large expansion in an ATTCT pentanucleotide repeat in intron 9 of the ATXN10 gene. The ATTCT repeat is highly unstable, expanding to 280–4,500 repeats in affected patients compared with the 9–32 repeats in normal individuals, one of the largest repeat expansions causing neurological disorders identified to date. However, the underlying molecular basis of how this huge repeat expansion evolves and contributes to the SCA10 phenotype remains largely unknown. Recent progress in next-generation DNA sequencing technologies has established that the SCA10 repeat sequence has a highly heterogeneous structure. Here we summarize what is known about the structure and origin of SCA10 repeats, discuss the potential contribution of variant repeats to the SCA10 disease phenotype, and explore how this information can be exploited for therapeutic benefit.

Clinical application of advanced multi-omics tumor profiling: Shaping precision oncology of the future.

Next-generation DNA sequencing technology has dramatically advanced clinical oncology through the identification of therapeutic targets and molecular biomarkers, leading to the personalization of cancer treatment with significantly improved outcomes for many common and rare tumor entities. More recent developments in advanced tumor profiling now enable dissection of tumor molecular architecture and the functional phenotype at cellular and subcellular resolution. Clinical translation of high-resolution tumor profiling and integration of multi-omics data into precision treatment, however, pose significant challenges at the level of prospective validation and clinical implementation. In this review, we summarize the latest advances in multi-omics tumor profiling, focusing on spatial genomics and chromatin organization, spatial transcriptomics and proteomics, liquid biopsy, and exvivo modeling of drug response. We analyze the current stages of translational validation of these technologies and discuss future perspectives for their integration into precision treatment.

A Unique Mitochondrial Gene Block Inversion in Antarctic Trematomin Fishes: A Cautionary Tale.

Many Antarctic notothenioid fishes have major rearrangements in their mitochondrial (mt) genomes. Here, we report the complete mt genomes of 3 trematomin notothenioids: the bald notothen (Trematomus (Pagothenia) borchgrevinki), the spotted notothen (T. nicolai), and the emerald notothen (T. bernacchii). The 3 mt genomes were sequenced using next-generation Illumina technology, and the assemblies verified by Sanger sequencing. When compared with the canonical mt gene order of the Antarctic silverfish (Pleuragramma antarctica), we found a large gene inversion in the 3 trematomin mt genomes that included tRNAIle, ND1, tRNALeu2, 16S, tRNAVal, 12S, tRNAPhe, and the control region. The trematomin mt genomes contained 3 intergenic spacers, which are thought to be the remnants of previous gene and control region duplications. All control regions included the characteristic conserved regulatory sequence motifs. Although short-read next-generation DNA sequencing technology has allowed the rapid and cost-effective sequencing of a large number of complete mt genomes, it is essential in all cases to verify the assembly in order to prevent the publication and use of erroneous data.

Challenges for precision public health communication in the era of genomic medicine

Although still in the early stages of development, the advent of fast, high-output, and cost-effective next-generation DNA sequencing technology is moving precision medicine into public health. Before this shift toward next-generation sequencing in public health settings, individual patients met geneticists after showing symptoms and through limited family screening. In the new era of precision public health, everyone is a possible participant in genetic sequencing, simply by being born (newborn screening), by donating blood (biobanking), or through population screening. These initiatives are increasingly offered to individuals throughout their life and more individuals are encountering opportunities to use DNA sequencing. This article raises awareness of these growing areas and calls for different models of public engagement and communication about genomics, including screening asymptomatic populations, obtaining consent for unspecified and unforeseen future uses of genomic data, and managing variants of uncertain significance. Given that such communication challenges loom large, established norms of practice in genomic medicine and research should be reconsidered.

Comparison of ear canal microbiome in rabbits with and without otitis externa using next generation DNA sequencing

Contemporary research has increasingly explored the clinical applicability of next-generation DNA sequencing (NGS) technologies in veterinary medicine, which has provided new and practical opportunities for rabbit otitis externa clinical interventions. The objectives of this study were to characterize the normal external ear canal microbiome of clinically healthy rabbits compared to otitis externa presenting rabbits, and to assess the diagnostic viability of NGS in aural veterinary medicine. Swabs from the external ear canal of 34 clinically healthy rabbits and 16 rabbits diagnosed with otitis externa were collected. Alongside bioinformatic analysis, library preparation was performed targeting the V1-V3 region of the 16S rRNA bacterial gene and the ITS-2 region for fungal DNA analysis. In the clinically healthy group, the bacterial species with the highest relative abundances were an uncharacterized Phytoplasma from the family Acholeplasmataceae (8.74%) and Staphylococcus epidermidis (5.56%), while in the otitis group the species with the highest relative abundances were Staphylococcus aureus (12.59%), Corynebacterium lactis (9.27%), and Corynebacterium mastitidis (7.92%). Fungal species with the highest relative abundances in the healthy group were a species from the genus Cladosporium (14.46%), while in the otitis externa group the fungal species with the highest relative abundances were a species from the genus Cladosporium (9.89%) and Malassezia restricta (4.76%). Additionally, there was a significantly higher number of different bacterial and fungal species in the clinically healthy group compared to the otitis group ( P = 0.00 and P = 0.00, respectively). This study provided evidence that the rabbit aural microbiome profile is distinctly different between a clinically healthy and an otitis state. It also highlighted new bacterial and fungal organisms of note in rabbits diagnosed with otitis externa compared to those previously thought to be the primary disease-causing organisms. Understanding the microbial population dynamics in the rabbit aural microbiome is particularly critical for helping clinicians recognize, prevent, or revert the progression of otitis.

Allelic Polymorphisms of Killer Immunoglobulin-Like Receptor Genes in Malay and Orang Asli Populations of Peninsular Malaysia

Next-generation DNA sequencing (NGS) technology advancements provide new insight into the level of variation in killer immunoglobulin-like receptor (KIR) genes. High resolution allele genotyping of seven KIR genes was conducted among 94 unrelated Malay and Orang Asli (OA) individuals of Peninsular Malaysia. A manual bioinformatics analysis is performed and optimised by Sanger sequencing method. The Malays expressed a total of 22 alleles, as compared to only 15 alleles in the OA population. In total, 12 centromeric and 9 telomeric allelic haplotypes were identified in the Malays, whereas 8 centromeric and 5 telomeric allelic haplotypes were identified in the OA. The KIR2DL1, KIR2DL3, and KIR2DS4 genes exhibited a high degree of variation and balanced distribution in the Malay and OA populations. On the other hand, KIR2DL4, KIR3DL1, KIR3DL2 and KIR3DL3 genes exhibited a high degree of conservation, with less number of alleles identified and the dominance of a single allele at high frequency. High-resolution KIR allele genotyping has revealed unique sequence variations and allelic haplotypes between individuals and populations. The distributions of KIR alleles and haplotypes are useful for genetic population studies and serve as a baseline for future transplantation matching and disease association research.

Complete mitochondrial genome of cylicocyclus auriculatus: molecular structure and phylogenetic analysis

Cylicocyclus spp. (Nematoda: Strongylida: Cyathostominae) are the common and important parasitic nematodes found in horses and donkeys worldwide. In this study, the complete mitochondrial genome of Cylicocyclus auriculatus Looss 1900, a representative member of this genus from the donkey in Southwest China was determined using the next-generation DNA sequencing technology. The genome was 13,851 bp in size and consisted of 36 genes including 12 protein-coding genes (atp6, cox1-3, cytb, nad1-6 and nad4L), 22 transfer RNA genes and two ribosomal RNA genes (rrnL and rrnS), as well as two non-coding regions. Phylogenetic analysis showed that C. auriculatus and Cylicocyclus insigne Boulenger 1917 were closely related, and then both grouped with other congeneric species and formed a monophyletic relationship with either species of Cyathostomum, Coronocyclus, Cyathostomum, Cylicostephanus or Cylicodontophorus, demonstrating their phylogenetic stability within Cyathostominae. These cumulative mitochondrial DNA data provide novel and useful genetic markers for molecular diagnostic, systematic and evolutionary biological studies of Cyathostominae nematodes.

MetEx, a Metabolomics Explorer Application for Natural Product Discovery.

Advances in next-generation DNA sequencing technologies, bioinformatics, and mass spectrometry-based metabolite detection have ushered in a new era of natural product discovery. Microbial secondary metabolomes are complex, especially when otherwise silent biosynthetic genes are activated, and there is therefore a need for data analysis software to explore and map the resulting multidimensional datasets. To that end, we herein report the Metabolomics Explorer (MetEx), a publicly available web application for the analysis of parallel liquid chromatography-coupled mass spectrometry (LC-MS)-based metabolomics data. MetEx is a highly interactive application that facilitates visualization and analysis of complex metabolomics datasets, consisting of retention time, m/z, and MS intensity features, as a function of hundreds of conditions or elicitors. The software enables prioritization of leads from three-dimensional maps, extraction of two-dimensional slices from various higher order plots, organization of datasets by elicitor chemotypes, customizable library-based dereplication, and automatically scored lead selection. We describe the application of MetEx to the first UPLC-MS-guided high-throughput elicitor screen in which Burkholderia gladioli was challenged with 750 elicitors, and the resulting profiles were interrogated by UPLC-Qtof-MS and subsequently analyzed with the app. We demonstrate the utility of MetEx by reporting elicitors for several cryptic metabolite groups and by uncovering new natural products that remain to be characterized. MetEx is available at https://mo.princeton.edu/MetEx/.