Generation Sequencing Approach Research Articles

Decoding Seafood: Multi-Marker Metabarcoding for Authenticating Processed Seafood

Given the recognized nutritional value of fish and shifting consumer lifestyles, processed seafood has become increasingly prevalent, comprising a significant portion of global food production. Although current European Union labeling regulations do not require species declaration for these products, food business operators often voluntarily provide this information on ingredient lists. Next Generation Sequencing (NGS) approaches are currently the most effective methods for verifying the accuracy of species declarations on processed seafood labels. This study examined the species composition of 20 processed seafood products, each labeled as containing a single species, using two DNA metabarcoding markers targeting the mitochondrial cytochrome c oxidase I (COI) and 16S rRNA genes. The combined use of these markers revealed that the majority of the products contained multiple species. Furthermore, two products were found to be mislabeled, as the declared species were not detected. These findings underscore that NGS is a robust technique that could be adopted to support routine food industry activities and official control programs, thereby enhancing the ‘From Boat to Plate’ strategy and combating fraudulent practices in the complex fisheries supply chain.

Age-correlated changes in the canine oral microbiome.

Canine oral disease has been associated with significant changes in the oral microbiome rather than the presence or absence of individual species. In addition, most studies focus on a single age group of canines and as of yet, the relationship between canine microbiomes and age is poorly understood. This study used a shotgun whole gene sequencing approach in tandem with the Aladdin Bioinformatics platform to profile the microbiomes of 96 companion dogs, with the sourmash-zymo reference database being used to perform taxonomic profiling. Findings showed significant age correlations among 19 species, including positive correlations among several Porphyromonas species and a negative correlation with C. steedae. Although a significant correlation was found between predicted and actual ages, ElasticNet Regression was unable to successfully predict the ages of younger canines based on their microbiome composition. Both microbiome samples and microbial species were successfully clustered by age group or age correlation, showing that the age-microbiome relationship survives dimensionality reduction. Three distinct clusters of microbial species were found, which were characterized by Porphyromonas, Conchiformibius, and Prevotella genera, respectively. Findings showed that the microbiomes of older dogs resembled those that previous literature attributed to dogs with periodontal disease. This suggests that the process of aging may introduce greater risks for canine oral disease.

After-sealing life in urban soils: Experimental evidence of resilience and efficiency of ectomycorrhizal inoculation

The restoration of urban sealed soils is a major stake for urban planners. In particular, characterizing their resilience from a biotic perspective is of primary importance for remobilizing these artificialized substrates in urban green spaces. Using Marseille (France) as a case study, we implemented a metabarcoding next generation sequencing approach to characterize the diversity and the composition of ectomycorrhizal (EM) fungal communities present in soils sealed for 60 years. Based on an inoculation experiment, we tested the ability of the remaining spore bank to establish EM interactions with plants. Altogether, we aimed at assessing the potential of peri-urban natural habitats to provide an efficient soil EM inoculum for the restoration of urban soils. We found that: (i) long-term sealing drastically reduced the diversity of EM fungi in urban soils, (ii) long-leaved spores of hypogeous gastroid fungi were efficient to establish EM symbioses with plants, and (iii) the efficiency of EM fungal inoculation depended on the natural habitat used as an inoculum source. Oak forests provided species-rich but poorly efficient inoculum. Contrastingly, soils collected in disturbed shrublands (garrigues) hosted moderately-rich EM communities containing droughtadapted species with high ability to colonize urban soils. Our findings highlight that peri-urban landscapes are promising candidates to feed local systems of urban soil restoration. In this perspective, poorly-considered habitats such as shrublands can be considered as valuable sources of soil biota to prevent the over-exploitation of agricultural areas.

First report of isolation of opportunistic human pathogenic gut bacteria Staphylococcus cohnii from grubs of Brahmina coriacea (Hope) (Coleoptera, Scarabaeidae, Melolonthinae) from Himachal Pradesh, India

Staphylococcus cohnii (Schleifer and Kloos) an opportunistic pathogen for humans, is reported for the first time its presence in grubs of Brahmina coriacea (Hope) (Coleoptera, Scarabaeidae, Melolonthinae). The cellulolytic gut bacteria were isolated from different populations of B. coriacea, collected from different parts of Himachal Pradesh, India. The isolated S. cohnii from the grub population of Shillaroo location recorded maximum cellulolytic index. The isolated bacteria were identified using morphological, biochemical, and 16S rRNA gene sequencing approaches.

Effects of two common antibiotics on the skin microbiome of ornamental reef fishes: Implications for manipulative experiments in microbial dynamics

AbstractAn understanding of the mechanistic drivers of animal‐microbial symbiosis and associations generally requires experiments that manipulate specific symbionts or microbial communities. As part of an ongoing study of the mechanisms that drive microbial communities in coral reef fishes, and specifically the role of cleanerfish in microbial transmission, we tested the effects of the commonly used antibiotics Enrofloxacin and Nitrofurazone on the skin microbiome of three ornamental reef fish species: the four‐eyed butterflyfish, Chaetodon capistratus (Chaetodontidae), the cleanerfish neon goby Elacatinus oceanops (Gobiidae) and the beaugregory damselfish Stegastes leucostictus (Pomacentridae). Our main aim was to characterize dysbiosis prompted by the delivery of the two antibiotics and understand whether both could be used to establish good starting points for microbial transmission experiments. We bathed the fish in antibiotic (or no exposure in the controls), sampled the skin microbiota via swabbing at zero (before treatment) and 3 and 7 days during the treatments, and examined the microbial community using a 16S rRNA gene sequencing approach. Nitrofurazone reduced skin‐associated microbial diversity in all species, whereas the same effect for Enrofloxacin was only seen in S. leucostictus. Although each antibiotic had its own, unique impact in microbial community, all treatments showed positive and negative shifts in the most abundant microbial taxa over time. Moreover, soon after the delivery of both antibiotics, increases in the abundance of opportunistic bacteria or potential pathogens, such as Alteromonas and Vibrio, were observed. Although both antibiotics are effective, Nitrofurazone more successfully reduces microbial diversity and therefore may be more ideal for experiments seeking to disrupt fish microbiomes.

Next generation sequencing improves the resolution of detecting mixed host blood meal sources in field collected arboviral mosquito vectors.

Accurate knowledge of blood meal hosts of different mosquito species is critical for identifying potential vectors and establishing the risk of pathogen transmission. We compared the performance of Miseq next generation sequencing approach relative to conventional Sanger sequencing approach in identification of mosquito blood meals using genetic markers targeting the 12S rRNA and cytochrome oxidase I (COI) genes. We analysed the blood meals of three mosquito vector species (Aedes aegypti, Aedes simpsoni s.l. and Culex pipiens s.l.) collected outdoors, and compared the frequency of single- versus multiple-blood feeding. Single host blood meals were mostly recovered for Sanger-based sequencing of the mitochondrial 12S rRNA gene, whereas Miseq sequencing employing this marker and the COI marker detected both single and multiple blood meal hosts in individual mosquitoes. Multiple blood meals (two or more hosts) which mostly included humans were detected in 19%-22.7% of Ae. aegypti samples. Most single host blood meals for this mosquito species were from humans (47.7%-57.1%) and dogs (9.1%-19.0%), with livestock, reptile and rodent hosts collectively accounting for 4.7%-28.9% of single host blood meals. The frequency of two or more host blood meals in Ae. simpsoni s.l. was 26.3%-45.5% mostly including humans, while single host blood meals were predominantly from humans (31.8%-47.4%) with representation of rodent, reptile and livestock blood meals (18.2%-68.2%). Single host blood meals from Cx. pipiens s.l. were mostly from humans (27.0%-39.4%) and cows (11.5%-27.36%). Multiple blood meal hosts that mostly included humans occurred in 21.2%-24.4% of Cx. pipiens s.l. samples. Estimated human blood indices ranged from 53%-76% for Ae. aegypti, 32%-82% for Ae. simpsoni s.l. and 26%-61% for Cx. pipiens s.l. and were consistently lower for Sanger-based sequencing approach compared to Miseq-based sequencing approach. These findings demonstrate that Miseq sequencing approach is superior to Sanger sequencing approach as it can reliably identify mixed host blood meals in a single mosquito, improving our ability to understand the transmission dynamics of mosquito-borne pathogens.

Abstract 3770: Comparative analysis of single cell expression and digital spatial profiling in FFPE tumor tissues

Abstract Formalin Fixed Paraffin Embedded (FFPE) tissues are a staple in clinical diagnostics associated with solid tumor. Next Generation Sequencing (NGS) approaches characterize genomes, epigenomes, transcriptomes, and proteomes from a single sample source. FFPE blocks from several different tumor tissue types were serially sliced into FFPE slides. Individual slides were then utilized to explore the genome, epigenome, single cell RNA-Seq (scRNA-Seq), and Digital Spatial Profiling (DSP). Here we present a comparative analysis of two transcriptomic modalities within one FFPE tissue sample: fixed-cell scRNA-Seq of the transcriptome and digital spatial profiling of the whole transcriptome. Independent analysis of scRNA-Seq and DSP identified common trends as well as distinct differences within the same sample. Comparative analysis within sample between modalities identified statistically significant differences, driven by local tumor microenvironment, but also highlighted commonalities between methods. Cross sample comparisons showed striking differences, as indicated by tumor type. These studies highlight the strengths of each assay individually, as well as the power of a dual-assay approach for comprehensive analysis of the tumor and its microenvironment. Conclusions from this standalone analysis were further aggregated into downstream multiomics analyses, including proteomics analysis, illustrating the expanding landscape of information that can be extracted from FFPE derived tissue and the potential for novel discovery and diagnostic power integrating these complex data types holds. Citation Format: Anthony Moore, Andrea O'Hara, Pranay Vishwanath, Laure Turner, Haythem Latif, Chris Mozdzierz, Ginger Zhou. Comparative analysis of single cell expression and digital spatial profiling in FFPE tumor tissues [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3770.

Abstract 2440: Cancer driver mutations as quantitative biomarkers of cancer risk interspecies analyses using CarcSeq

Abstract Unmet needs in cancer risk assessment include the ability to predict rodent life-time tumor responses from short-term exposures and a scientific basis for rodent to human extrapolation. As an approach to address these needs, the use of hotspot cancer driver mutations (CDMs) as biomarkers of cancer risk was integrated with an error-corrected, next generation sequencing (NGS) approach. We have developed and investigated the sensitivity of a high-throughput NGS method capable of detecting a panel of the most prevalent point mutations observed in human oncogenes and tumor suppressor genes. This method, CarcSeq, involves performing multiple, high-fidelity PCR reactions to amplify the most prevalent mutations reported to occur in human tumors, tagging the amplicons with 9 bp unique identifier sequences at each end, constructing libraries using Illumina ChIP Seq kits, sequencing the libraries on an Illumina NextSeq500 platform and generating single-strand consensus sequences (SSCS), bioinformatically, as a means of error-correction. We validated CarcSeq mutant fraction measurements through observations related to known cancer driver tissue-specificities and mutation spectra for normal human breast and lung, ductal carcinoma, and lung adenocarcinoma samples. Performance of a reconstruction experiment established CarcSeq has a sensitivity of 104. The approach has now been adapted to analyze analogous conserved codons in rat and mouse. For each of the three species, the developed panel includes 13-15 amplicons encompassing known cancer driver genes (e.g., KRAS, PIK3CA, and TP53) encompasses the equivalent of 30 known human hotspot codons and has a combined target sequence length of ~1 kb. Amplicons were generated from DNA isolated from 16-week-old, untreated rodent tissue samples with known differences in spontaneous tumor incidence: female Wistar, Sprague Dawley, and F344 rat mammary samples and male and female B6C3F1 and CD-1 mouse lung samples. The rodent panel was optimized to generate ~100,000 SSCS/amplicon. CDMs were observed at conserved codons in young, untreated rodents, and results are consistent with what was expected based on strain differences in spontaneous tumorigenesis. Citation Format: Kelly L. Harris, Binsheng Gong, Barbara Parsons, Jennifer Faske, Joshua Xu. Cancer driver mutations as quantitative biomarkers of cancer risk interspecies analyses using CarcSeq [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2440.

CURRENT STATUS OF METATRANSCRIPTOMIC AND RELATED STUDIES IN HEMATOPHAGOUS DISEASE-TRANSMITTING VECTORS

The incidence of numerous vector-borne diseases (VBDs) has recently increased alarmingly due to various widespread factors, including unplanned urbanization, greater human mobility, environmental changes, vector resistance to insecticides, and evolving pathogens. In this context, the World Health Organization (WHO) has repositioned effective and sustainable vector control as a key approach to prevent and eliminate VBDs. It has been shown that the microbiome influences development, nutrition, and pathogen defense in disease-transmitting vectors such as mosquitoes, sandflies, tsetse flies, triatomine bugs, and ticks. Consequently, understanding the endogenous regulation of vector biology can aid in developing effective approaches for vector control. In this respect, a metatranscriptomic approach analyzes all the expressed RNAs in an environmental sample (meta-RNAs) and can thus reveal how the metabolic activities of the microbiome influence vector biology. This review includes an extensive analysis of available literature on microbial and viral studies for some of the major hematophagous disease-transmitting arthropods, with a focus on studies that used next generation sequencing (NGS) approaches. Since a consensus terminology for these “meta-sequencing analyses” has not yet been established, a definition of these terms is presented here to provide the framework for systematically sorting the available information for each of the VBDs analyzed here to single out metatranscriptomic analyses. Finally, key gaps in knowledge were identified for some of these hematophagous disease-transmitting arthropods which will prove very useful for driving future studies.

An insight into the prokaryotic diversity from a polymetallic nodule-rich region in the Central Indian Ocean Basin using next generation sequencing approach.

Deep sea is a vast, dark, and difficult-to-access terrain and is now looked upon as a unique niche harboring diverse microorganism. We used a metataxonomic approach to decipher the microbial diversity present in the water column (surface to near bottom), water overlaying the sediments, and the deep-sea sediments (up to 35 cm) from the Indian Contract Region (ICR) in the Central Indian Ocean Basin (CIOB). Samples were collected from #IRZ (Impact Reference Zone), #PRZ (Potential Reference Zone), and #BC20 (Control site, outside potential mining area) with an average water depth of 5,200 m. 16S rRNA (V3-V4 region) amplicon sequencing on the MiSeq platform resulted in 942,851 ASVs across 65 water and sediment samples. Higher prokaryotic diversity was observed below 200 m in the water column to the seafloor. Proteobacteria was the most dominant bacterial phylum among all the water samples while Firmicutes, Actinobacteria and, Bacteroidota dominated the sediments. Sediment (below 10 cm) was co-dominated by Firmicutes. Thermoplasmata was the dominant archaeal group in the water column while Crenarchaeota was in the sediments. BC20 was less diverse than IRZ and PRZ. Deep Sea microorganisms could play a vital role in the mineralization processes, nutrient cycling, and also different biogeochemical cycles.

Family analysis and literature study of hereditary hypophosphatemic rickets with hypercalciuria

BackgroundHereditary hypophosphatemia rickets with hypercalciuria (HHRH) is a rare autosomal recessive disorder characterised by reduced renal phosphate reabsorption leading to hypophosphataemia, rickets and bone pain. Here, we present a case of HHRH in a Chinese boy.Case presentationWe report a 11-year-old female proband, who was admitted to our hospital with bilateral genuvarum deformity and short stature. Computed Tomography (CT) showed kidney stones, blood tests showed hypophosphatemia, For a clear diagnosis, we employed high-throughput sequencing technology to screen for variants. Our gene sequencing approach encompassed whole exome sequencing, detection of exon and intron junction regions, and examination of a 20 bp region of adjacent introns. Flanking sequences are defined as ±50 bp upstream and downstream of the 5′ and 3′ ends of the coding region.The raw sequence data were compared to the known gene sequence data in publicly available sequence data bases using Burrows-Wheeler Aligner software (BWA, 0.7.12-r1039), and the pathogenic variant sites were annotated using Annovar. Subsequently, the suspected pathogenic variants were classified according to ACMG’s gene variation classification system. Simultaneously, unreported or clinically ambiguous pathogenic variants were predicted and annotated based on population databases. Any suspected pathogenic variants identified through this analysis were then validated using Sanger sequencing technology. At last, the proband and her affected sister carried pathogenic homozygous variant in the geneSLC34A3(exon 13, c.1402C > T; p.R468W). Their parents were both heterozygous carriers of the variant. Genetic testing revealed that the patient has anLRP5(exon 18, c.3917C > T; p.A1306V) variant of Uncertain significance, which is a rare homozygous variant.ConclusionThis case report aims to raise awareness of the presenting characteristics of HHRH. The paper describes a unique case involving variants in both theSLC34A3andLRP5genes, which are inherited in an autosomal recessive manner. This combination of gene variants has not been previously reported in the literature. It is uncertain whether the presence of these two mutated genes in the same individual will result in more severe clinical symptoms. This report shows that an accurate diagnosis is critical, and with early diagnosis and correct treatment, patients will have a better prognosis.

Early-Onset Dystonia and Visual Impairment Preceding Epileptic Encephalopathy Associated with PIGA Gene Mutation.

The association between dystonia and early-onset epileptic encephalopathy (EOEE) may have a genetic basis. Phosphatidylinositol glycan biosynthesis class A protein (PIGA) germline mutations have been described in the last decade and associated with refractory EOEEs. Dysmorphisms and visceral abnormalities have also been reported. Here, we present the case of a now 8-month-old child who was evaluated for dystonia, visual impairment, and developmental delay at 2 months of age, followed by refractory focal seizures when he was 4 months old. The remaining examination was normal, besides an accelerated linear growth. His brain magnetic resonance and an extensive metabolic investigation failed to show any abnormalities. At 7 months of age, the exome sequencing found a hemizygous PIGA pathogenic variant-c.1352T > C (p.(Ile451Thr). Seizures improved after the association of carbamazepine with levetiracetam and the beginning of the ketogenic diet. To our knowledge, this is the first time the phenotype associated with this specific mutation is described. Our patient had the singularity of manifesting with remarkable dystonia, over 2 months before the onset of seizures. We also point to the utility of the gene sequencing approach in the diagnosis of patients with dystonia and EOEEs, since identification of the genetic cause may help in patient's management and families' empowerment.

Combined DNA Analysis from Stool and Blood Samples Improves Tumor Tracking and Assessment of Clonal Heterogeneity in Localized Rectal Cancer Patients.

Objectives: In this study, stool samples were evaluated for tumor mutation analysis via a targeted next generation sequencing (NGS) approach in a small patient cohort suffering from localized rectal cancer. Introduction: Colorectal cancer (CRC) causes the second highest cancer-related death rate worldwide. Thus, improvements in disease assessment and monitoring that may facilitate treatment allocation and allow organ-sparing "watch-and-wait" treatment strategies are highly relevant for a significant number of CRC patients. Methods: Stool-based results were compared with mutation profiles derived from liquid biopsies and the gold standard procedure of tumor biopsy from the same patients. A workflow was established that enables the detection of de-novo tumor mutations in stool samples of CRC patients via ultra-sensitive cell-free tumor DNA target enrichment. Results: Notably, only a 19% overall concordance was found in mutational profiles across the compared sample specimens of stool, tumor, and liquid biopsies. Conclusion: Based on these results, the analysis of stool and liquid biopsy samples can provide important additional information on tumor heterogeneity and potentially on the assessment of minimal residual disease and clonal tumor evolution.

Performance assessment of the Bruker Biotyper MALDI-TOF MS for the identification of difficult-to-identify viridans group streptococci.

Differentiating Streptococcus pneumoniae among nonpneumococcal viridans group streptococci (VGS) is challenging in conventional laboratories. Therefore, we aimed to evaluate the performance of the latest Bruker Biotyper matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) system in identifying VGS by comparing the results to those of the specific gene sequencing approach. Clinical isolates were initially identified using the BD Phoenix system to identify Streptococcus species. The optochin test was used to distinguish nonpneumococcal VGS from S. pneumoniae. The species of individual reference strains and clinical isolates were determined by comparing the sequences of the 16S rDNA, gyrB, sodA, groESL, or coaE genes with those in the GenBank sequence databases. We evaluated the performance of the Bruker Biotyper MALDI-TOF MS in VGS identification using two different machines with three databases. We collected a total of 103 nonpneumococcal VGS and 29 S. pneumoniae blood isolates at a medical center in northern Taiwan. Among these isolates, only seven could not be identified at the species level by the specific gene sequencing approach. We found that none of the nonpneumococcal VGS isolates were misidentified as pneumococci by the latest Biotyper system, and vice versa. However, certain strains, especially those in the mitis and bovis groups, could still not be correctly identified. The latest Bruker Biotyper 4.1 (DB_10833) showed significant improvement in identifying VGS strains. However, a specific gene sequencing test is still needed to precisely differentiate the species of strains in the mitis and bovis groups.

The Potential of Intertwining Gene Diagnostics and Surgery for Mitral Valve Prolapse

Mitral valve prolapse (MVP) is common among heart valve disease patients, causing severe mitral regurgitation (MR). Although complications such as cardiac arrhythmias and sudden cardiac death are rare, the high prevalence of the condition leads to a significant number of such events. Through next-generation gene sequencing approaches, predisposing genetic components have been shown to play a crucial role in the development of MVP. After the discovery of the X-linked inheritance of filamin A, autosomal inherited genes were identified. In addition, the study of sporadic MVP identified several genes, including DZIP1, TNS1, LMCD1, GLIS1, PTPRJ, FLYWCH, and MMP2. The early screening of these genetic predispositions may help to determine the patient population at risk for severe complications of MVP and impact the timing of reconstructive surgery. Surgical mitral valve repair is an effective treatment option for MVP, resulting in excellent short- and long-term outcomes. Repair rates in excess of 95% and low complication rates have been consistently reported for minimally invasive mitral valve repair performed in high-volume centers. We therefore conceptualize a potential preventive surgical strategy for the treatment of MVP in patients with genetic predisposition, which is currently not considered in guideline recommendations. Further genetic studies on MVP pathology and large prospective clinical trials will be required to support such an approach.

Identification of 17 Distinct IDH1/IDH2 Mutations from Acute Myeloid Leukemia Peripheral Blood Using a Multiplex Real-Time PCR Closed Cartridge System

Background: Acute myeloid leukemia (AML) is characterized by clonal expansion of poorly differentiated “blast cells” in peripheral blood and bone marrow causing ineffective erythropoiesis and bone marrow failure. It has been demonstrated that mutations in isocitrate dehydrogenase 1 and 2 (IDH1/IDH2) are associated with disease relapse and reduced survival. There are 17 AML-related IDH1/IDH2 mutations located at R100, R132, R140, and R172 that are targeted by different IDH1/IDH2 selective inhibitors. The sensitivity of detecting IDH1/IDH2 needs to be improved for early cancer detection. Current PCR-based assays cannot detect all 17 IDH1/IDH2 mutations, and next generation sequencing approaches take longer, use complicated workflows, and require costly reagents as well as substantial data analysis. Therefore, the rapid identification of patients with AML who harbor IDH1/IDH2 mutations could improve the efficient selection of targeted inhibitors for these patients. Aim: To create a multiplex PCR assay that detects 17 IDH1/IDH2 mutations from peripheral blood with high sensitivity and specificity. Method: We will highlight five technical aspects of this qPCR assay that contribute to its high sensitivity and specificity: 1. the “anchor - bridge - foot” design of SuperSelective (SS) primers that can detect different mutations within the same codon. 2. the amplification-refractory mutation system (ARMS) primer design strategy was also tested which has comparable effects to SS primer. 3. a two-step nested PCR to reduce non-specific binding in products due to the amplification of unexpected primer binding sites. 4. a short (20-25nt) blocking oligonucleotide (wild-type blocker, WTB) to reduce SS and ARMS primer binding at the wild-type codon site. 5. four probes carrying different fluorescence reporters were used to identify the four IDH1/IDH2 mutation codons. Results: We synthesized 17 plasmid DNA (pDNA) templates and 14 CRISPR-engineered cell lines, each carrying one IDH1 or IDH2 mutation and the housekeeping gene ABL. SS and ARMS primer were screened with 5·10 4 cps/test of mutation template in a whole blood background. 6 mutations of R132 Codon (R132C, G, H, L, S, V), R100Q, 4 mutations of R140 (R140Q, L, G, W), and 6 mutations of R172 (R172K, M, G, S1 [516G>T], S2 [516G>C], W) can successfully be identified from whole blood lysate at the LoD level (1%) as shown in Figure 1 in red. Moreover, adding WTB to the PCR reaction improved the specificity with a reduced background signal. Importantly, in addition to the plasmid, cell line genomic DNA (gDNA) that was spiked into blood lysate at the LoD level (1%) also performed similarly to plasmid DNA as shown in Figure 1 in blue. Summary/Conclusion: We present a prototype assay with design intention to detect 17 mutations of IDH1/IDH2 from the peripheral blood of AML patients with excellent sensitivity and specificity and a strong potential to outperform any products on the market. Utilizing our Xpert Instrument System a fully automated, single-use cartridge product with pre-loaded sample-processing reagents, the assay aims to report results in less than 3 hours, including hands-on time.

Genetic and morphological insights into the Carpobrotus hybrid complex around the world

The genus Carpobrotus N.E.Br. comprises between 12 and 25 species, most of which are native to South Africa. Some Carpobrotus species are considered among the most damaging invasive species in coastal dune systems worldwide. In their introduced areas, these species represent a serious threat to native species and significantly impact soil conditions and geochemical processes. Despite being well studied, the taxonomy of Carpobrotus remains problematic, as the genus comprises a complex of species that hybridize easily and are difficult to distinguish from each other. To explore the population genetic structure of invasive Carpobrotus species (i.e., C. acinaciformis and C. edulis) across a significant part of their native and non-native ranges, we sampled 40 populations across Argentina, Italy, New Zealand, Portugal, South Africa, Spain, and the USA. We developed taxon-specific microsatellite markers using a Next Generation Sequencing approach to analyze the population genetic structure and incidence of hybridization in native and non-native regions. We identified three genetically distinct clusters, which are present in both the native and non-native regions. Based on a set of selected morphological characteristics, we found no clear features to identify taxa morphologically. Our results suggest that the most probable sources of global introductions of Carpobrotus species are the Western Cape region of South Africa and the coastline of California. We suggest that management actions targeting Carpobrotus invasions globally should focus on preventing additional introductions from the east coast of South Africa, and on searching for prospective biocontrol agents in the Western Cape region of South Africa.

SARS-CoV-2 primed platelets–derived microRNAs enhance NETs formation by extracellular vesicle transmission and TLR7/8 activation

BackgroundHyperactive neutrophil extracellular traps (NETs) formation plays a key role in the pathogenesis of severe COVID-19. Extracellular vesicles (EVs) are vehicles which carry cellular components for intercellular communication. The association between COVID-19 patients-derived EVs and NETs formation remains elusive.MethodsWe explored the roles of EVs in NETs formation from 40 COVID-19 patients with different disease severities as well as 30 healthy subjects. The EVs-carried microRNAs profile was analyzed using next generation sequencing approach which was validated by quantitative reverse transcription PCR. The regulatory mechanism of EVs on NETs formation was investigated by using an in vitro cell-based assay, including immunofluorescence assay, flow cytometry, and immunoblotting.ResultsCOVID-19 patient–derived EVs induced NETs formation by endocytosis uptake. SARS-CoV-2 spike protein-triggered NETs formation was significantly enhanced in the presence of platelet–derived EVs (pEVs) and this effect was Toll-like receptor (TLR) 7/8- and NADPH oxidase-dependent. Increased levels of miR-21/let-7b were revealed in EVs from COVID-19 patients and were associated with disease severity. We demonstrated that the spike protein activated platelets directly, followed by the subsequent intracellular miR-21/let-7b upregulation and then were loaded into pEVs. The pEVs-carried miR-21 interacted with TLR7/8 to prime p47phox phosphorylation in neutrophils, resulting in NADPH oxidase activation to promote ROS production and NETs enhancement. In addition, miR-21 modulates NF-κB activation and IL-1β/TNFα/IL-8 upregulation in neutrophils upon TLR7/8 engagement. The miR-21 inhibitor and TLR8 antagonist could suppress efficiently spike protein-induced NETs formation and pEVs primed NETs enhancement.ConclusionsWe identified SARS-CoV-2 triggered platelets–derived GU-enriched miRNAs (e.g., miR-21/let-7b) as a TLR7/8 ligand that could activate neutrophils through EVs transmission. The miR-21-TLR8 axis could be used as a potential predisposing factor or therapeutic target for severe COVID-19.

Lipidomic diversity and proxy implications of archaea from cold seep sediments of the South China Sea

Cold seeps on the continental margins are characterized by intense microbial activities that consume a large portion of methane by anaerobic methanotrophic archaea (ANME) through anaerobic oxidation of methane (AOM). Although ANMEs are known to contain unique ether lipids that may have an important function in marine carbon cycling, their full lipidomic profiles and functional distribution in particular cold-seep settings are still poorly characterized. Here, we combined the 16S rRNA gene sequencing and lipidomic approaches to analyze archaeal communities and their lipids in cold seep sediments with distinct methane supplies from the South China Sea. The archaeal community was dominated by ANME-1 in the moderate seepage area with strong methane emission. Low seepage area presented higher archaeal diversity covering Lokiarchaeia, Bathyarchaeia, and Thermoplasmata. A total of 55 core lipids (CLs) and intact polar lipids (IPLs) of archaea were identified, which included glycerol dialkyl glycerol tetraethers (GDGTs), hydroxy-GDGTs (OH-GDGTs), archaeol (AR), hydroxyarchaeol (OH-AR), and dihydroxyarchaeol (2OH-AR). Diverse polar headgroups constituted the archaeal IPLs. High concentrations of dissolved inorganic carbon (DIC) with depleted δ13CDIC and high methane index (MI) values based on both CLs (MICL) and IPLs (MIIPL) indicate that ANMEs were active in the moderate seepage area. The ANME-2 and ANME-3 clades were characterized by enhanced glycosidic and phosphoric diether lipids production, indicating their potential role in coupling carbon and phosphurus cycling in cold seep ecosystems. ANME-1, though representing a smaller proportion of total archaea than ANME-2 and ANME-3 in the low seepage area, showed a positive correlation with MIIPL, indicating a different mechanism contributing to the IPL-GDGT pool. This also suggests that MIIPL could be a sensitive index to trace AOM activities performed by ANME-1. Overall, our study expands the understanding of the archaeal lipid composition in the cold seep and improves the application of MI using intact polar lipids that potentially link to extent ANME activities.

Evaluation of next generation sequencing approaches for SARS-CoV-2

Within public health control strategies for SARS-CoV-2, whole genome sequencing (WGS) is essential for tracking viral spread and monitoring the emergence of variants which may impair the effectiveness of vaccines, diagnostic methods, and therapeutics. In this manuscript different strategies for SARS-CoV-2 WGS including metagenomic shotgun (SG), library enrichment by myBaits® Expert Virus-SARS-CoV-2 (Arbor Biosciences), nCoV-2019 sequencing protocol, ampliseq approach by Swift Amplicon® SARS-CoV-2 Panel kit (Swift Biosciences), and Illumina COVIDSeq Test (Illumina Inc.), were evaluated in order to identify the best approach in terms of results, labour, and costs. The analysis revealed that Illumina COVIDSeq Test (Illumina Inc.) is the best choice for a cost-effective, time-consuming production of consensus sequences.