Species Typing of Trypanosomatids Using Amplicon-Based Sequencing.

BackgroundTrypanosomatids are among the most critical parasites for public health due to their impact on human, animal, and plant health. Diseases associated with these pathogens manifest mainly in poor and vulnerable populations, where social, environmental, and biological factors modulate the case incidence and geographical distribution.MethodsWe used Sanger and amplicon-based next-generation sequencing (NGS) in samples from different mammals to identify trypanosomatid infections in several departments in Colombia. A total of 174 DNA samples (18 humans, 83 dogs, and 73 wild mammals) were analyzed by conventional PCR using a fragment of the heat shock protein 70 (Hsp70) gene and Sanger sequenced the positive samples. Twenty-seven samples were sent for amplicon-based NGS using the same gene fragment. Data obtained were used to perform diversity analyses.ResultsOne hundred and thirteen samples were positive for PCR by Hsp70 fragment; these corresponded to 22.1% Leishmania spp., 18.6% L. amazonensis, 9.7% L. braziliensis, 14.2% L. infantum, 8% L. panamensis, and 27.4% Trypanosoma cruzi. Comparison of the identified species by the two sequencing technologies used resulted in 97% concordance. Alpha and beta diversity indices were significant, mainly for dogs; there was an interesting index of coinfection events in the analyzed samples: different Leishmania species and the simultaneous presence of T. cruzi and even T. rangeli in one of the samples analyzed. Moreover, a low presence of L. braziliensis was observed in samples from wild mammals. Interestingly, to our knowledge, this is the first report of Leishmania detection in Hydrochaeris hydrochaeris (capybara) in Colombia.ConclusionsThe Hsp70 fragment used in this study is an optimal molecular marker for trypanosomatid identification in many hosts and allows the identification of different species in the same sample when amplicon-based sequencing is used. However, the use of this fragment for molecular diagnosis through conventional PCR should be carefully interpreted because of this same capacity to identify several parasites. This point is of pivotal importance in highly endemic countries across South America because of the co-circulation of different genera from the Trypanosomatidae family. The findings show an interesting starting point for One Health approaches in which coevolution and vector-host interactions can be studied.Graphical

We report the whole-genome sequence of monkeypox virus obtained using MinION technology (Oxford Nanopore Technologies) from a French clinical specimen during the 2022 epidemic. Amplicon-based sequencing and shotgun metagenomic approaches were directly applied to the sample.

Oxford Nanopore Technologies’ MinION has proven to be a valuable tool within human and microbial genetics. Its capacity to produce long reads in real time has opened up unique applications for portable sequencing. Examples include tracking the recent African swine fever outbreak in China and providing a diagnostic tool for disease in the cassava plant in Eastern Africa. Here we review the current applications of Oxford Nanopore sequencing in livestock, then focus on proposed applications in livestock agriculture for rapid diagnostics, base modification detection, reference genome assembly and genomic prediction. In particular, we propose a future application: ‘crush-side genotyping’ for real-time on-farm genotyping for extensive industries such as northern Australian beef production. An initial in silico experiment to assess the feasibility of crush-side genotyping demonstrated promising results. SNPs were called from simulated Nanopore data, that included the relatively high base call error rate that is characteristic of the data, and calling parameters were varied to understand the feasibility of SNP calling at low coverages in a heterozygous population. With optimised genotype calling parameters, over 85% of the 10,000 simulated SNPs were able to be correctly called with coverages as low as 6×. These results provide preliminary evidence that Oxford Nanopore sequencing has potential to be used for real-time SNP genotyping in extensive livestock operations.

Phylogenetic analysis based on core genome single nucleotide polymorphisms (cgSNPs) using whole-genome sequencing (WGS) is increasingly used in epidemiological investigations of bacteria. The approach, however, is both resource intensive and time-consuming. Oxford Nanopore Technologies (ONT) sequencing is capable of real-time data analysis but the high error rate hampers its application in cgSNP-based phylogenetic analysis. Here, we developed a cgSNP-independent phylogenetic analysis method using ONT read assemblies by focusing on open reading frame (ORF) content patterns. WGS data of 66 Enterobacter hormaechei strains acquired by both ONT and Illumina sequencing and 162 strains obtained from NCBI database were converted to binary sequences based on the presence or absence of ORFs using BLASTn. Phylogenetic trees calculated from binary sequences (ORF trees) were compared with cgSNP trees derived from Illumina sequences. Clusters of closely related strains in the cgSNP trees formed comparable clusters in the ORF trees built with binary sequences, and the tree topologies between them were similar based on Fowlkes-Mallows index. The ORF-based phylogenetic analysis using ONT sequencing may be useful in epidemiological investigations and offer advantages over the cgSNP-based approach. Conversion of assembled WGS data to binary sequences based on the presence or absence of ORFs circumvents read error concerns with ONT sequencing. Since ONT sequencing generates data in real time and does not require major investment, this ORF-based phylogenetic analysis method has the potential to enable phylogenetic and epidemiological analysis at the point of care.

The use of new sequencing technologies in forensic biology has been explored recently, particularly using Illumina and Ion Torrent platforms. The MinION™, a single‐molecule nanopore sequencer from Oxford Nanopore Technologies™ could advance the field further due to the portability, lower overall instrument cost, and ease of use. This review focuses on exploring applications and the associated benefits and limitations of this technology relative to those currently used in forensic science. The MinION sequences single molecules directly, without amplification, and has the ability to generate long reads in real time. Due to the large amount of sequencing data that the MinION generates, having systems in place for storage and sophisticated analysis tools needs to be considered. The user is able to stop sequencing when the sought after information has been gathered, decreasing the time from sampling to sequence output. Possible forensic applications for the MinION where long sequence reads would be helpful include sequencing whole mitochondrial and bacterial genomes. Sequencing of short tandem repeats and single nucleotide polymorphisms may prove useful for rapid identification at the crime scene. Limitations of the current technology include accuracy of the base calling that has still not reached the standard required for forensic DNA analysis but can be counteracted by read depth and consensus. In addition, the optimum input sample size is currently greater than most real‐world forensic samples. Given the rapid pace of change, future developments by Oxford Nanopore Technologies are likely to overcome these barriers in the coming years.This article is categorized under: Forensic Biology > Forensic DNA Technologies

ABSTRACTShort‐read metabarcoding analysis is the gold standard for accessing partial 16S and ITS genes with high read quality. With the advent of long‐read sequencing, the amplification of full‐length target genes is possible, but with low read accuracy. Moreover, 16S rRNA gene amplification in seaweed results in a large proportion of plastid reads, which are directly or indirectly derived from cyanobacteria. Primers designed not to amplify plastid sequences are available for short‐read sequencing, while Oxford Nanopore Technology (ONT) offers adaptive sampling, a unique way to remove reads in real time. In this study, we compare three options to address the issue of plastid reads: deleting plastid reads with adaptive sampling, using optimised primers with Illumina MiSeq technology, and sequencing large numbers of reads with Illumina NovaSeq technology with universal primers. We show that adaptive sampling using the default settings of the MinKNOW software was ineffective for plastid depletion. NovaSeq sequencing with universal primers stood out with its deep coverage, low error rate, and ability to include both eukaryotes and bacteria in the same sequencing run, but it had limitations regarding the identification of fungi. The ONT sequencing helped us explore the fungal diversity and allowed for the retrieval of taxonomic information for genera poorly represented in the sequence databases. We also demonstrated with a mock community that the SAMBA workflow provided more accurate taxonomic assignment at the bacterial genus level than the IDTAXA and KRAKEN2 pipelines, but many false positives were generated at the species level.

AB1037 Interobserver reliability of knee osteoarthritis lesions using musculoskeletal ultrasound: differences between static versus real time reading

Pectinids constitute one of the most conspicuous groups of marine bivalves, and include some of the most important species from the point of view of fisheries and aquaculture. In spite of this, their systematics and evolution are not well understood. Only two molecular phylogenetic analyses based on relatively wide taxonomic samplings have been published. These studies largely neglected American species, some of which are central for testing current models of pectinid evolution and diversification, or are commercially valuable. We have sequenced 820 nucleotide base pairs of the 12S and 16S ribosomal RNA genes in nine species of pectinids belonging to six genera living along American coasts. Sequences from homologous regions of 19 other species were gathered from public databases. We constructed phylogenetic maximum-parsimony and maximum-likelihood trees of this set of 28 taxa. Our phylogenetic analysis indicates that Crassadoma is polyphyletic, and cementation to the substrate as a life habit could have appeared independently in two geographic chlamydinid lineages. Nodipecten is placed in the subfamily Pectininae, and the suspected close relationship of Amusium, Euvola and Pecten within this subfamily is also supported. Zygochlamys patagonica appears in the Chlamydinae subfamily, as expected. The existence of a separate subfamily Palliolinae is suggested but not supported statistically. The position of Argopecten, Aequipecten and Flexopecten within the subfamily Pectinidae, suggested by a recent study, could not be confirmed, and we argue that it could be due to a combination of long branch attraction and incomplete sequencing.

Structure of 16S and 23S Ribosomal RNA Genes in Campylobacter Species: Phylogenetic Analysis of the Genus Campylobacter and Presence of Internal Transcribed Spacers

We read with interest the letter ‘Pathogenic causes and outcomes of endophthalmitis after vitreoretinal surgeries in Finland from 2009 to 2018’ by Loukovaara et al. (2020). They discuss the problems concerning identification of microorganisms in patients with endophtalmitis (EO) and also mention the future perspective of ‘…more sensitive modalities for causative pathogen detection including Next Generation Sequencing technique…’ (NGS). In this letter, we want to highlight the advantages of using NGS (16S/18S microbiome sequencing) as a supplement to culture in the identification of microorganisms. With this technique, DNA from bacteria (16S), fungi, and parasites (18S) can be used for identification of living and non-living microorganisms (Ring et al. 2017). The technique is already available as a standard method from Statens Serum Institute, Copenhagen, Denmark, for hospitals in Denmark. Even though infectious EO is a rare condition, it is a serious ocular complication with potentially devastating visual outcomes. Normally, the microbiological diagnosis of EO is based on culture of bacteria or fungi. As others (Ramakrishnan et al. 2009; Melo et al. 2011), Loukovaara et al. (2020) only obtained a positive culture in a limited number of cases (52.9%). The low rate of positive cultures is probably due to the small sample volume, prior antibiotic treatment, insufficient culture techniques or a sterile inflammation. We performed a quality assurance project regarding the identification of causative microorganisms with culture, supplemented with Sanger sequencing (Hartmeyer & Justesen 2010) in selected cases, compared to 16S/18S microbiome sequencing in all patients with EO. The project included two university hospitals in Denmark for a period of 1 year. Vitreous samples were sent to Statens Serum Institute for 16S/18S amplicon-based microbiome sequencing (Ring et al. 2017). Fifteen patients were included, and concordance between methods was evaluated to be observed in 13 cases (seven cases were without any microorganisms). In one of the discrepant cases, culture was negative, but Staphylococcus aureus was diagnosed with 16S/18S microbiome sequencing after anti-VEGF therapy. The other discrepant case is described below. An immunocompromised patient with liver failure and a transjugular intrahepatic portosystemic shunt (TIPS) was admitted to hospital with a history of 3 weeks of blurred vision and a suspicion of retinal detachment. The examination at the hospital showed diffuse unilateral anterior and posterior segment inflammation with posterior synechiae, dense vitritis and blurry white retinal infiltrates (Fig. 1), and fungal endophthalmitis was suspected. Acute vitrectomy was performed with injection of vancomycin, ceftazidime and amphotericin B. Culture and 16S Sanger sequencing of the vitreous sample were negative, but 16S/18S microbiome sequencing identified Candida albicans. The patient received intravitreal amphotericin B again and started oral treatment with fluconazole. After 1 month and during fluconazole treatment, the patient developed candidemia. A positron emission tomography-computed tomography demonstrated a possible site of infection in the thoracic spine (T12) and lumbar spine (L1). A biopsy showed C. albicans. We cannot for sure determine whether the spine or possibly the TIPS was the main site of infection. The patient died 2 months later from several complications, but without 16S/18S microbiome sequencing, treatment would have been delayed significantly. The case demonstrates the value of 16S/18S microbiome sequencing and superiority to Sanger sequencing and should be considered in culture-negative cases where infection with bacteria or fungi is suspected.

The long-read sequencers from Pacific Bioscience (PacBio) and Oxford Nanopore Technologies (ONT) offer the opportunity to phase mutations multiple kilobases apart directly from sequencing reads. In this study, we used long-range PCR with ONT and PacBio sequencing to phase two variants 9 kb apart in the RET gene. We also re-analysed data from a recent paper which had apparently successfully used ONT to phase clinically important haplotypes at the CYP2D6 and HLA loci. From these analyses, we demonstrate PCR-chimera formation during PCR amplification and reference alignment bias are pitfalls that need to be considered when attempting to phase variants using amplicon-based long-read sequencing technologies. These methodological pitfalls need to be avoided if the opportunities provided by long-read sequencers are to be fully exploited.

Ineffective hole cleaning leads to a complicated drilling hole problems such as stuck pipe incidents, and difficult tripping operations. The objective of this paper to introduce a unique Hole Cleaning Advisory System which is showing six real time hole cleaning indicators that can provide an obvious vision on hole cleaning performance during drilling operations and thereby improves drilling efficiency. They are real time drilling fluid density (MWeff), real time equivalent circulating density (ECDeff), real time developed carrying capacity index (HCI), and real time developed cuttings concentration in annulus (CCA), real time developed transport ratio (CTRm) and real time transport index (TIm). Artificial intelligence tool such as artificial neural network (ANN) was applied for validation and confirmation of models parameters and obtaining similar real time profiles of hole cleaning indices. The system was utilizing rig sensors’ real time values, surface and field operations readings by collecting historical and real time readings , selecting the most relevant and important factors. Performing data analytics, preparation and interpretation. Finding the relationships between parameters. Modifying critical parameters. Validating models with referenced tools those are applicable and utilized in drilling operations. Integrating developed models in real time drilling operations to evaluate and monitor drilling performance. The conducted process, validations of real time models were done and compared with other commercial software and advanced drilling tool such as Pressure while drilling (PWD) tool and other sensors tools provided by Services Company for drilling operations. The system were applied in the fields and provided a firm and solid validations of the developed system that empowered the new technology to be applied in drilling rigs fleet. The results showed that the MWeff and ECDeff were validated with pressure while drilling (PWD) and resulted in 2 % errors. MWeff was validated with other commercial software shows real time mud as output, the developed real time model of mud weight while drilling showed errors 2 %. That means the developed models for mud weight and equivalent circulating density were robust and can be utilize while drilling operation to optimize well drilling performance. regarding other hole cleaning indices such as HCI, CCA, CTRm & TIm were applied in several types of well, it was able to identify the hole cleaning issues and improves the drilling efficiency specifically rate of penetration (ROP) by 65 %. The drilling engineering and operation team was performing immediate intervention to avoid drilling troubles due to hole cleaning. The final results contributed for optimizing drilling efficiency significantly in safety and environmental manners with cost and time effectiveness. In addition, participating in drilling automation and digitalization in compatibility with fourth industrial revolution (4-IR).

Ineffective hole cleaning leads to a complicated drilling hole problems such as stuck pipe incidents, and difficult tripping operations. The objective of this paper to introduce a unique Hole Cleaning Advisory System which is showing six real time hole cleaning indicators that can provide an obvious vision on hole cleaning performance during drilling operations and thereby improves drilling efficiency. They are real time drilling fluid density (MWeff), real time equivalent circulating density (ECDeff), real time developed carrying capacity index (HCI), and real time developed cuttings concentration in annulus (CCA), real time developed transport ratio (CTRm) and real time transport index (TIm). Artificial intelligence tool such as artificial neural network (ANN) was applied for validation and confirmation of models parameters and obtaining similar real time profiles of hole cleaning indices. The system was utilizing rig sensors’ real time values, surface and field operations readings by collecting historical and real time readings, selecting the most relevant and important factors. Performing data analytics, preparation and interpretation. Finding the relationships between parameters. Modifying critical parameters. Validating models with referenced tools those are applicable and utilized in drilling operations. Integrating developed models in real time drilling operations to evaluate and monitor drilling performance. The conducted process, validations of real time models were done and compared with other commercial software and advanced drilling tool such as Pressure while drilling (PWD) tool and other sensors tools provided by Services Company for drilling operations. The system were applied in the fields and provided a firm and solid validations of the developed system that empowered the new technology to be applied in drilling rigs fleet. Results, Observations, Conclusions: the results showed that the MWeff and ECDeff were validated with pressure while drilling (PWD) and resulted in 2 % errors .MWeff was validated with other commercial software shows real time mud as output, the developed real time model of mud weight while drilling showed errors 2 %. That means the developed models for mud weight and equivalent circulating density were robust and can be utilize while drilling operation to optimize well drilling performance. regarding other hole cleaning indices such as HCI, CCA, CTRm & TIm were applied in several types of well, it was able to identify the hole cleaning issues and improves the drilling efficiency specifically rate of penetration (ROP) by 65 %. The drilling engineering and operation team was performing immediate intervention to avoid drilling troubles due to hole cleaning. The final results contributed for optimizing drilling efficiency significantly in safety and environmental manners with cost and time effectiveness. In addition, participating in drilling automation and digitalization in compatibility with fourth industrial revolution (4-IR).

In matrix treatments, placement of the injected fluids is essential for success. Over the years, several diversion and placement techniques have been applied to obtain a desired fluid distribution. Real-time evaluation of a treatment was limited to observing injection pressures or bottomhole pressures. These measured pressures provided some information on the diversion process. The application of distributed temperature sensing (DTS) during matrix treatments to monitor the temperature profiles along the wellbore in real time is a recent method to obtain a qualitative indication of the fluid distribution. In this paper, we will discuss whether DTS can also be used to quantify the fluid distribution during a matrix treatment. For the real-time quantification of the fluid distribution during a matrix treatment from temperature surveys, both real-time read outs of the temperature surveys and an accurate real-time model are needed. With DTS, the real-time read out is a feasible technique that has been developed to present and evaluate the temperature surveys in real time. Further, a coupled wellbore and near-wellbore thermal model is available that runs in real time. This paper will describe these techniques and models and validations using several case histories. In addition, an analysis of matrix treatments using DTS temperature surveys, where available, will be presented. The models will be used in the analysis to obtain calculated fluid flow distribution. We will discuss how this methodology can be applied in real time and what benefits quantification of fluid flow distribution offers. Further, we will describe what other benefits can be obtained from real-time temperature profiles during stimulation treatments.

ABSTRACTSequencing of the 16S ribosomal RNA (rRNA) gene is an important tool in addition to conventional methods for the identification of bacterial pathogens in human infections. In polymicrobial samples, Sanger sequencing can produce uninterpretable chromatograms. This limitation can be overcome by Next Generation Sequencing (NGS) of the 16S rRNA gene. We investigated the applicability of Oxford Nanopore Technologies (ONT) sequencing of the partial 16S rRNA gene as a diagnostic routine method for pathogen detection in clinical samples. From June 2021 to August 2022, 101 clinical samples positive in PCR for partial 16S rRNA gene analysis were subjected to both Sanger and ONT sequencing. Sanger sequences were edited and compared with deposited sequences in the NCBI database using BLAST, while ONT data were processed using EPI2ME Fastq 16S. The positivity rate (clinically relevant pathogen) was higher for ONT vs. Sanger sequencing: 72% and 59%, respectively. Concordance between Sanger and ONT sequencing was 80%. Furthermore, ONT detected more samples with polymicrobial presence compared to Sanger (13 vs. 5) sequencing. Interestingly, in one joint fluid sample, Borrelia bissettiiae was identified by ONT but not by Sanger. The results show that the detection of both monobacterial and multiple bacterial species is improved using ONT.