High-throughput DNA Sequencing Research Articles

Metagenomic analysis of deep-sea bacterial communities in the Makassar and Lombok Straits

The extreme conditions of the deep-sea environment, including limited light, low oxygen levels, high pressure, and nutrient scarcity, create a natural habitat for deep-sea bacteria. These remarkable microorganisms have developed unique strategies to survive and adapt to their surroundings. However, research on the diversity of deep-sea bacteria, both culture-dependent and culture-independent, in Indonesian waters remains insufficient. This study focused on exploring the biodiversity of deep-sea bacteria, specifically in the Makassar and Lombok Strait, the main Indonesian throughflow pathway characterized by relatively fertile water, which serves as an important deep-sea region. High-throughput DNA sequencing of full-length 16S rRNA was employed to construct a genomic database. The results of the bioinformatic analysis revealed that two stations, 48 and 50 (Makassar Strait), exhibited a more similar community structure of deep-sea bacteria than did station 33 (Lombok Strait). Among the predominant phyla found at a depth of 1000 m, the top ten were Proteobacteria, Firmicutes, Bacteroidetes, Actinobacteria, Planctomycetes, Acidobacteria, Nitrospinae, Verrucomicrobia, Candidatus Melainabacteria, and Cyanobacteria. Furthermore, the genera Colwellia, Moritella, Candidatus Pelagibacter, Alteromonas, and Psychrobacter consistently appeared at all three stations, albeit with varying relative abundance values. These bacterial genera share common characteristics, such as psychrophilic, halophilic, and piezophilic tendencies, and are commonly found in deep-sea ecosystem. The environmental conditions at a depth of 1000 m were relatively stable, with an average pressure 10 MPa, temperature 4.68 °C, salinity 34.58 PSU, pH 8.06, chlorophyll-a 0.29 µg/L, nitrate 3.19 µmol/L, phosphate 6.32 µmol/L and dissolved oxygen (DO) 2.90 mg/L. The bacterial community structures at the three sampling stations located at the same depth (1000 m) exhibited similarities, as indicated by the closely aligned similarity index values.

Uncovering ASO-Targetable Deep Intronic AIRE Variants: Insights and Therapeutic Implications.

High-throughput DNA sequencing has accelerated the discovery of disease-causing genetic variants, yet only in 10-40% of cases yield a genetic diagnosis. Increased implementation of genome sequencing has enabled a deeper exploration of the noncoding genome and recognition of noncoding variants as major contributors to disease. In a recent study, we identified a deep intronic variant in the AutoImmune REgulator (AIRE) gene (c.1504-818 G>A) as the cause of autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED), a life-threatening monogenic autoimmune disorder most often caused by biallelic AIRE defects. This deep intronic variant disrupts normal splicing AIRE , causing pseudoexon inclusion and altered protein function. By developing an antisense oligonucleotide (ASO) targeting the pseudoexon sequence, we restored normal AIRE transcript in vitro, thereby revealing a potential genotype-specific candidate treatment. Our study illustrates key aspects of intronic variant detection, validation, and candidate ASO development. Herein, we briefly highlight the growing potential of ASO-based therapies for deep intronic variants, addressing the unmet need of personalized, genotype-specific therapies in diseases lacking curative options.

Selective expansion of motor cortical projections in the evolution of vocal novelty.

Deciphering how cortical architecture evolves to drive behavioral innovations is a long-standing challenge in neuroscience and evolutionary biology. Here, we leverage a striking behavioral novelty in the Alston's singing mouse ( Scotinomys teguina ), compared to the laboratory mouse ( Mus musculus ), to quantitatively test models of motor cortical evolution. We used bulk tracing, serial two-photon tomography, and high-throughput DNA sequencing of over 76,000 barcoded neurons to discover a specific and substantial expansion ( ∼ 200%) of orofacial motor cortical (OMC) projections to the auditory cortical region (AudR) and the midbrain periaqueductal gray (PAG), both implicated in vocal behaviors. Moreover, analysis of individual OMC neurons' projection motifs revealed preferential expansion of exclusive projections to AudR. Our results imply that selective expansion of ancestral motor cortical projections can underlie behavioral divergence over short evolutionary timescales, suggesting potential mechanisms for the evolution of enhanced cortical control over vocalizations-a crucial preadaptation for human language.

Insights into the microbiota of raw milk from seven breeds animals distributing in Xinjiang China.

Owing to its high nutritional content, raw milk contains a rich microbiota. Thus, to study microorganisms present in raw milk available in Xinjiang China, 142 raw milk samples from seven animal breeds (cow, sheep, goat, donkey, horse, camel, and yak) and four regions (Hami, Tarbagatay, Kashgar, and Ili) were analyzed by high-throughput DNA sequencing. These microorganisms were characterized by 10 dominant phyla. Proteobacteria (68.33%) was the major phylum, followed by Firmicutes (18.80%) and Thermi (3.16%). Horse milk contained more Bacteroidetes, sheep milk contained more Gammaproteobacteria, and donkey milk contained more unclassified sequences. Camel and donkey milk contained the highest and lowest bacterial diversity compared with that contained by the remaining milk samples, respectively. Additionally, spoilage microorganisms, including Chryseobacterium, Propionibacterium, and Flavobacterium, and pathogenic bacteria, including Ochrobactrum anthropi and Sphingomonas, were more prevalent in horse and yak milk, whereas probiotic lactic acid bacteria (LAB), such as Leuconostoc, Lactococcus, or Lactobacillus, were more prevalent in goat, donkey, and camel milk. Furthermore, Moraxella was abundantly present in goat, camel, and yak milk, Acinetobacter was more abundant in camel milk, and Pseudomonas was relatively abundant in sheep and donkey milk. Overall, specific harmful microorganisms and probiotic lactic acid bacteria were found in the raw milk samples obtained from different animals, which provided a basis for preventing and controlling the growth of harmful bacteria, as well as investigating probiotic resources in raw milk.

Identification of osteoporosis genes using family studies.

Osteoporosis is a multifactorial bone disease characterised by reduced bone mass and increased fracture risk. Family studies have made significant contribution in unravelling the genetics of osteoporosis. Yet, most of the underlying molecular and biological mechanisms remain unknown prompting the need for further studies. This review outlines the proper phenotyping and advanced genetic techniques in the form of high-throughput DNA sequencing used to identify genetic factors underlying monogenic osteoporosis in a family-based setting. The steps related to variant filtering prioritisation and curation are also described. From an evolutionary perspective, deleterious risk variants with higher penetrance tend to be rare as a result of negative selection. High-throughput sequencing (HTS) can identify rare variants with large effect sizes which are likely to be missed by candidate gene analysis or genome-wide association studies (GWAS) wherein common variants with small to moderate effect sizes are identified. We also describe the importance of replicating implicated genes, and possibly variants, identified following HTS to confirm their causality. Replication of the gene in other families, singletons or independent cohorts confirms that the shortlisted genes and/or variants are indeed causal. Furthermore, novel genes and/or variants implicated in monogenic osteoporosis require a thorough validation by means of in vitro and in vivo assessment. Therefore, analyses of families can continue to elucidate the genetic architecture of osteoporosis, paving the way for improved diagnostic and therapeutic strategies.

Bacterial and Symbiodiniaceae communities’ variation in corals with distinct traits and geographical distribution

Coral microbiomes play crucial roles in holobiont homeostasis and adaptation. The host’s ability to populate broad ecological niches and to cope with environmental changes seems to be related to the flexibility of the coral microbiome. By means of high-throughput DNA sequencing we characterized simultaneously both bacterial (16S rRNA) and Symbiodiniaceae (ITS2) communities of four reef-building coral species (Mussismilia braziliensis, Mussismilia harttii, Montastraea cavernosa, and Favia gravida) that differ in geographic distribution and niche specificity. Samples were collected in a marginal reef system (Abrolhos, Brazil) in four sites of contrasting irradiance and turbidity. Biological filters governed by the host are important in shaping corals’ microbiome structure. More structured associated microbial communities by reef site tend to occur in coral species with broader geographic and depth ranges, especially for Symbiodiniaceae, whereas the endemic and habitat-specialist host, M. braziliensis, has relatively more homogenous bacterial communities with more exclusive members. Our findings lend credence to the hypothesis that higher microbiome flexibility renders corals more adaptable to diverse environments, a trend that should be investigated in more hosts and reef areas.

Combined Application of High-Throughput Sequencing and Metabolomics to Evaluate the Microbial Mechanisms of Plant-Growth-Promoting Bacteria in Enhancing the Remediation of Cd-Contaminated Soil by Hybrid Pennisetum

The contamination of soil with the heavy metal cadmium (Cd) is increasingly prominent and severely threatens food security in China. Owing to its low cost, suitable efficacy, and ability to address the shortcomings of plant remediation by enhancing the ability of plants to take up Cd, plant–microbe combination remediation technology has become a research hotspot in heavy metal pollution remediation. A pot experiment was performed to examine the effects of inoculation with the plant-growth-promoting bacterium Brevibacillus sp. SR-9 on the biomass, Cd accumulation, and soil nutrients of hybrid Pennisetum. The purpose of this study was to determine how Brevibacillus sp. SR-9 alleviates stress caused by heavy metal contamination. High-throughput sequencing and metabolomics were used to determine the effects of inoculation on the soil bacterial community composition and microbial metabolic functions associated with hybrid Pennisetum. The results suggest that mutation of Brevibacillus sp. SR-9 effectively alleviates Cd pollution stress, leading to increased biomass and accumulation of Cd in hybrid Pennisetum. The aboveground biomass and the root weight increased by 12.08% and 27.03%, respectively. Additionally, the accumulation of Cd in the aboveground sections and roots increased by 21.16% and 15.50%, respectively. Measurements of the physicochemical properties of the soil revealed that the strain Brevibacillus sp. SR-9 slightly increased the levels of available phosphorus, total nitrogen, total phosphorus, and available potassium. High-throughput DNA sequencing revealed that Brevibacillus sp. SR-9 implantation modified the composition of the soil bacterial community by increasing the average number of Actinobacteria and Bacillus. The total nitrogen content of the soil was positively correlated with the Actinobacteria abundance, total phosphorus level, and available phosphorus level. Metabolomic analysis revealed that inoculation affected the abundance of soil metabolites, and 59 differentially abundant metabolites were identified (p < 0.05). Among these, 14 metabolites presented increased abundance, whereas 45 metabolites presented decreased abundance. Fourteen metabolic pathways were enriched in these metabolites: the folate resistance pathway, the ABC transporter pathway, D-glutamine and D-glutamic acid metabolism, purine metabolism, and pyrimidine metabolism. The abundance of the metabolites was positively correlated with the levels of available phosphorus, total potassium, total phosphorus, and total nitrogen. According to correlation analyses, the development of hybrid Pennisetum and the accumulation of Cd are strongly associated with differentially abundant metabolites, which also impact the abundance of certain bacterial populations. This work revealed that by altering the makeup of microbial communities and their metabolic processes, bacteria that promote plant development can mitigate the stress caused by Cd. These findings reveal the microbiological mechanisms through which these bacteria increase the ability of hybrid Pennisetum to take up the Cd present in contaminated soils.

Complete genome sequence of a Circovirus pigeon strain in lymphocyte-depleted bursa of Fabricius of a Common Raven (Corvus corax).

A necropsy was performed on a Common Raven (Corvus corax) presenting an opportunistic fungal respiratory infection and a bursal lymphoid depletion with inclusion bodies, suggestive of a circovirus infection. High-throughput sequencing of circular DNA in the bursa of Fabricius revealed a complete genome sequence of a Circovirus pigeon strain.

Dehydrin Client Proteins Identified Using Phage Display Affinity Selected Libraries Processed With Paired-End Phage Sequencing

The LATE EMBRYOGENESIS ABUNDANT PROTEINs (LEAPs) are a class of noncatalytic, intrinsically disordered proteins with a malleable structure. Some LEAPs exhibit a protein and/or membrane binding capacity and LEAP binding to various targets has been positively correlated with abiotic stress tolerance. Regarding the LEAPs' presumptive role in protein protection, identifying client proteins (CtPs) to which LEAPs bind is one practicable means of revealing the mechanism by which they exert their function. To this end, we used phage display affinity selection to screen libraries derived from Arabidopsis thaliana seed mRNA with recombinant orthologous LEAPs from Arabidopsis and soybean (Glycine max). Subsequent high throughput sequencing of DNA from affinity-purified phage was performed to characterize the entire sub-population of phage retained by each LEAP orthologue. This entailed cataloging in-frame fusions, elimination of false positives, and aligning the hits on the CtP scaffold to reveal domains of respective CtPs that bound to orthologous LEAPs. This approach (Paired-end PhAge Sequencing, or PEPA-Seq) revealed a subpopulation of the proteome constituting the CtP repertoire in common between the two DHNs orthologues (LEA14 and GmPm12) compared to BSA (unrelated binding control). The veracity of LEAP:CtP binding for one of the CtPs (LEA14 and GmPM12 self-association) was independently assessed using temperature related intensity change (TRIC) analysis. Moreover, LEAP:CtP interactions for four other CtPs were confirmed in planta using bimolecular fluorescence complementation (BiFC) assays. The results provide insights into the involvement of the DHN Y-segments and K-domains in protein binding.

Extreme Temperatures Reduce Copepod Performance and Change the Relative Abundance of Internal Microbiota

ABSTRACTCopepods are one of the most abundant invertebrate groups in the seas and oceans and are a significant food source for marine animals. Copepods are also particularly sensitive to elevated temperatures. However, it is relatively unknown how the internal microbiome influences copepod susceptibility to warming. We addressed this fundamental knowledge gap by assessing key life history traits (survival, development, and reproduction) and changes in the internal microbiome in the tropical calanoid copepod Acartia sp. in response to warming (26°C, 30°C, and 34°C). Copepod microbiomes were analyzed using high throughput DNA sequencing of V1–V9 of 16S rRNA hypervariable regions. Copepod performance was better at 30°C than at 26°C, as indicated by faster development, a higher growth rate, and fecundity. However, these parameters strongly decreased at 34°C. We recorded 1,262,987 amplicon sequence reads, corresponding to 392 total operational taxonomic units (OTUs) at 97% similarity. Warming did not affect OTU numbers and the biodiversity indices, but it substantially changed the relative abundance of three major phyla: Proteobacteria, Actinobacteria, and Bacteroidota. The thermophilic and opportunistic Proteobacteria and Bacteroidota increased under extreme temperatures (34°C) while Actinobacteria abundance was strongly reduced. Changes in the relative abundance of these bacteria might be related to reduced copepod growth, survival, and reproduction under extreme temperatures. Profiling the functional role of all internal bacterial groups in response to the temperature change will fundamentally advance our mechanistic understanding of the performance of tropical copepods and, more generally, marine invertebrates to a warming climate.

Use of commercial or indigenous yeast impacts the S. cerevisiae transcriptome during wine fermentation.

Understanding microbial functions during wine fermentation, particularly the role of Saccharomyces cerevisiae, is crucial for enhancing wine quality. While commercially available S. cerevisiae strains are commonly used, indigenous strains can offer unique flavors, potentially reflecting vineyard terroir. By leveraging high-throughput DNA sequencing and transcriptomic analysis, we explored the impact of vine age on the grape mycobiome and characterized microbial functions during grape fermentation. Our findings revealed that older vines harbor higher fungal loads, but fungal diversity remains similar across vine ages. Additionally, uninoculated fermentations exhibited diverse fungal dynamics, including the beneficial wine yeasts Metschnikowia and Pichia. Transcriptomic analysis uncovered significant differences in S. cerevisiae gene expression between inoculated and uninoculated fermentations, highlighting the potential of indigenous yeast to enhance wine diversity and inform winemaking practices.

An accurate haplotyping method using multiplex pyrosequencing with AS-PCR to detect ABCB1 haplotypes associated with rivaroxaban-derived hemorrhagic events

In clinical practice, owing to the comprehensive genetic insights they offer, haplotypes have attracted greater attention than individual single nucleotide polymorphisms (SNPs). Due to the long distances across SNP locations, detecting the haplotype using genomic DNA is challenging. Current haplotyping methods are either expensive and labor-intensive (high-throughput DNA sequencing), or haplotyping a single clinical sample (computational approach) is impossible. Herein, we propose using mRNA as a haplotyping target to minimize the distance among SNPs and employing allele-specific PCR (AS-PCR) to pick up a desired haplotype, followed by multiplex pyrosequencing to type the alleles at the SNP location of interest. AS-PCR was improved by combining an additional 3′-phosphorylated modified probe to achieve the specific separation of two closely similar templates. Only the sample with more than two heterozygotes needs to be haplotyped; therefore, we propose a stratification strategy to screen the samples for further haplotyping. This method was evaluated by associating ABCB1 haplotypes with the rivaroxaban-derived side effect in a cohort of 505 patients with nephrotic syndrome, focusing on the SNPs of ABCB1: rs1236C > T, rs2677G > T/A, and rs3435C > T. We successfully identified five bleeding-related haplotypes: rs1236T-rs2677T-rs3435T, rs1236C-rs2677G-rs3435T, rs1236T-rs2677G-rs3435C, rs1236C-rs2677G-rs3435C, and rs1236T-rs2677T-rs3435C. We compared the results with those from the conventional computational algorithm PHASE and observed that PHASE results dismissed the impact of rs1236C-rs2677G-rs3435C and rs1236C-rs2677G-rs3435T on bleeding risk and erroneously suggested a false positive association of rs1236C-rs2677A-rs3435T with increased bleeding risk.

A modified dual preparatory method for improved isolation of nucleic acids from laser microdissected fresh-frozen human cancer tissue specimens.

A central theme in cancer research is to increase our understanding of the cancer tissue microenvironment, which is comprised of a complex and spatially heterogeneous ecosystem of malignant and non-malignant cells, both of which actively contribute to an intervening extracellular matrix. Laser microdissection (LMD) enables histology selective harvest of cellular subpopulations from the tissue microenvironment for their independent molecular investigation, such as by high-throughput DNA and RNA sequencing. Although enabling, LMD often requires a labor-intensive investment to harvest enough cells to achieve the necessary DNA and/or RNA input requirements for conventional next-generation sequencing workflows. To increase efficiencies, we sought to use a commonplace dual preparatory (DP) procedure to isolate DNA and RNA from the same LMD harvested tissue samples. While the yield of DNA from the DP protocol was satisfactory, the RNA yield from the LMD harvested tissue samples was significantly poorer compared to a dedicated RNA preparation procedure. We determined that this low yield of RNA was due to incomplete partitioning of RNA in this widely used DP protocol. Here, we describe a modified DP protocol that more equally partitions nucleic acids and results in significantly improved RNA yields from LMD-harvested cells.

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.

Seasonal variation in the stomach microbiota of two sympatrically breeding Pygoscelis penguin species at Signy Island, South Orkney Islands.

The gut microbiomes of Antarctic penguins are important for the fitness of the host birds and their chicks. The compositions of microbial communities in Antarctic penguin guts are strongly associated with the birds' diet, physiological adaptation and phylogeny. Whilst seasonal changes in food resources, distribution and population parameters of Antarctic penguins have been well addressed, little research is available on the stability or variability of penguin stomach microbiomes over time. Here, we focused on two Pygoscelis penguin species breeding sympatrically in the maritime Antarctic and analysed their stomach contents to assess whether penguin gut microbiota differed over three austral summer breeding seasons. We used a high-throughput DNA sequencing approach to study bacterial diversity in stomach regurgitates of Adélie (Pygoscelis adeliae) and chinstrap (Pygoscelis antarctica) penguins that have a similar foraging regime on Signy Island (South Orkney Islands). Our data revealed significant differences in bacterial alpha and beta diversity between the study seasons. We also identified bacterial genera that were significantly associated with specific breeding seasons, diet compositions, chick-rearing stages and sampling events. This study provides a baseline for establishing future monitoring of penguin gut microbiomes in a rapidly changing environment.

Seasonal variation in airborne microbial communities of the Akiyoshido Cave: Lampenflora dispersed by phototrophic bioaerosols

Lampenflora, such as phototrophic microorganisms, multiply on speleothem surfaces under the show-cave lighting, causing deterioration of the natural and cultural heritages of the caves. Speleothems change color from white to green through microbial photosynthesis and are destroyed by microbial metabolisms. Aerosol transmission are suspected to disperses photosynthetic microorganisms. However, the mechanism underlying the process remains unclear, because the complexity of air ventilation affects the cave atmosphere. In the present study, we collected aerosol samples from the Akiyoshido Cave, which is a special natural monument in Japan and possesses various types of speleothems, to analyze microbial concentrations and communities in the cave atmosphere. Under a fluorescence microscope, phototrophic and heterotrophic microorganisms were observed in the aerosols with in the cave. The aerosol concentrations showed seasonal changes depending on air-flow variations in cave ventilation. High-throughput DNA sequencing targeting 16S rRNA genes revealed that the airborne bacterial communities inside the cave were dominated by members of the phyla Actinomycetota, Bacillota, and Pseudomonadota, originating from the external terrestrial, phyllosphere or freshwater environments, as well as its interior (guano). Cyanobacteria showed small relative abundances (from 1.0 to 10%) randomly in the aerosol samples at several locations in the cave, suggesting that the lampenflora-derived cyanobacteria were dispersed throughout the cave by the ventilation. Additionally, phototrophic microorganisms closely related to the relatives of the genera Leptolyngbya, Calothrix, and Chroococcidiopsis from the phylum Cyanobacteria were isolated from the aerosol samples. These results confirm that Cyanobacteria are one of the candidate microorganisms responsible for lampenflora dispersion and are “alive and airborne” in caves.

Bacterial cell surface characterization by phage display coupled to high-throughput sequencing

The remarkable capacity of bacteria to adapt in response to selective pressures drives antimicrobial resistance. Pseudomonas aeruginosa illustrates this point, establishing chronic infections during which it evolves to survive antimicrobials and evade host defenses. Many adaptive changes occur on the P. aeruginosa cell surface but methods to identify these are limited. Here we combine phage display with high-throughput DNA sequencing to create a high throughput, multiplexed technology for surveying bacterial cell surfaces, Phage-seq. By applying phage display panning to hundreds of bacterial genotypes and analyzing the dynamics of the phage display selection process, we capture important biological information about cell surfaces. This approach also yields camelid single-domain antibodies that recognize key P. aeruginosa virulence factors on live cells. These antibodies have numerous potential applications in diagnostics and therapeutics. We propose that Phage-seq establishes a powerful paradigm for studying the bacterial cell surface by identifying and profiling many surface features in parallel.

Assembly and annotation of the first complete mitochondrial genome of Phenacoccus solenopsis Tinsley (Hemiptera: Pseudococcidae)

The cotton mealybug, Phenacoccus solenopsis Tinsley (Hemiptera: Pseudococcidae), is a significant economic pest with a global distribution. Despite the increasing volume of literature on mitochondrial genome sequencing, complete mitochondrial genome sequences have not been reported for P. solenopsis yet. Here, we assembled the complete mitochondrial genome of P. solenopsis using high throughput DNA sequencing technology. The genome is 14,831 bp in length and is comprised of 37 genes: 13 protein-coding genes (PCGs), 22 transfer RNA (tRNA) genes, and two ribosomal RNA (rRNA) genes. The total length of all PCGs is 9,678 bp, accounting for 65.2% of the overall mitogenome. Among the PCGs, atp8 is the smallest gene (99 bp) and nad5 is the largest (1,593 bp). Of the 13 PCGs, seven (nad3, cox3, atp6, atp8, cox2, cox1, nad2) are encoded on the majority strand (J-strand), while six (nad1, nad6, cob, nad4L, nad4, nad5) are on the minority strand (N-strand). The analysis revealed a predominant A+T base composition, making up 90.3% of the total genome. However, the non-coding regulatory control region (CR) is missing due to the non-overlapping endpoints of the linear assembly. This assembly provides comprehensive information for investigating the evolutionary relationships between scale insects and for the precise identification of insects.

A novel approach for in vivo DNA footprinting using short double-stranded cell-free DNA from plasma.

Here, we present a method for enrichment of double-stranded cfDNA with an average length of ∼40 bp from cfDNA for high-throughput DNA sequencing. This class of cfDNA is enriched at gene promoters and binding sites of transcription factors or structural DNA-binding proteins, so that a genome-wide DNA footprint is directly captured from liquid biopsies. In short double-stranded cfDNA from healthy individuals, we find significant enrichment of 203 transcription factor motifs. Additionally, short double-stranded cfDNA signals at specific genomic regions correlate negatively with DNA methylation, positively with H3K4me3 histone modifications and gene transcription. The diagnostic potential of short double-stranded cell-free DNA (cfDNA) in blood plasma has not yet been recognized. When comparing short double-stranded cfDNA from patient samples of pancreatic ductal adenocarcinoma with colorectal carcinoma or septic with postoperative controls, we identify 136 and 241 differentially enriched loci, respectively. Using these differentially enriched loci, the disease types can be clearly distinguished by principal component analysis, demonstrating the diagnostic potential of short double-stranded cfDNA signals as a new class of biomarkers for liquid biopsies.

Breaking the Cycle: A Yeast Mannan-Rich Fraction Beneficially Modulates Egg Quality and the Antimicrobial Resistome Associated with Layer Hen Caecal Microbiomes under Commercial Conditions.

Antibiotics and antibiotic growth promoters have been extensively employed in poultry farming to enhance growth performance, maintain bird health, improve nutrient uptake efficiency, and mitigate enteric diseases at both sub-therapeutic and therapeutic doses. However, the extensive use of antimicrobials in poultry farming has led to the emergence of antimicrobial resistance (AMR) in microbial reservoirs, representing a significant global public health concern. In response, non-antibiotic dietary interventions, such as yeast mannan-rich fraction (MRF), have emerged as a promising alternative to modulate the gut microbiota and combat the AMR crisis. This study investigated whether a yeast mannan-rich fraction containing feed supplement impacted the performance of laying hens, their microbiomes, and the associated carriage of antimicrobial resistance genes under commercial conditions. High-throughput DNA sequencing was utilised to profile the bacterial community and assess changes in the antibiotic resistance genomes detected in the metagenome, the "resistome", in response to MRF supplementation. It was found that supplementation favourably influenced laying hen performance and microbial composition. Notably, there was a compositional shift in the MRF supplemented group associated with a lower relative abundance of pathobionts, e.g., Escherichia, Brachyspira and Trueperella, and their AMR-encoded genes, relative to beneficial microbes. Overall, the findings further demonstrate the ability of prebiotics to improve laying hen performance through changes associated with their microbiome and resistome.