Use Of High-throughput Sequencing Research Articles

Whole exome sequencing: the search for mutations associated with hereditary breast cancer in ethnic groups of Siberia

Hereditary breast cancer (HBC) is a heterogeneous disease caused by mutations in genes characterized by ethnic specifcity. The clinical heterogeneity of this disease signifcantly complicates its diagnosis. The use of high-throughput sequencing is one of the approaches that allow the search for genes and their variants associated with the development of HBC. The purpose of the study was to search for new genes associated with HBC in the understudied ethnic groups of Siberia by using whole exome sequencing (WES).Material and Methods. WES was performed on a cohort of 16 probands with BC (Tuvan, Yakut, Altai ethnos). The study material was genomic DNA isolated from peripheral blood leukocytes. Libraries were prepared using a BGI Optimal DNA Library Prep kit. An Agilent SureSelect Human All Exon V6 kit was used for hybridization. High-throughput sequencing was performed on a DNA nanoball sequencing platform (DNBSeq-G400).Results. In the overall group of patients with signs of HBC, pathogenic variants were detected in 12.5 % of cases (2/16). For the frst time, BRCA1 (rs80357635) pathogenic variant was identified in a young patient with metachronous BC (Yakut ethnic group). A pathogenic variant of the ATM gene (rs780619951 NM_000051:exon16:c.C2413T:p.R805X) was identified in a young patient with BC (Tuvinian ethnic group). A pathogenic variant of the TDP2 c.G4T:p.E2X, rs770844602 gene (DNA repair gene) was identified for the frst time in a Tuvan BC patient (metachronous) with a family history, but its contribution to HBC remains to be proven. The TDG gene variant (rs764159587 NM_001363612:exon7:c.536dupA:p.E179fs) found in the Tuvan ethnic group and affecting splicing (SpliceAI: 0.580) requires special attention.Conclusion. This report is the frst to describe the germinal variant in the BRCA1 (rs80357635) gene in the Yakut ethnic group. Further studies are required to confrm pathogenicity of germinal variants in non-well studied genes TDP2, TDG in ethnic BC patients.

Addendum – New Supporting information for PM 7/151 Considerations for the use of high throughput sequencing in plant health diagnostics

Addendum – New Supporting information for <scp>PM</scp> 7/151 Considerations for the use of high throughput sequencing in plant health diagnostics

Complete Genome Analyses of a Novel Flexivirus with Unique Genome Organization and Three Endornaviruses Hosted by the Mycorrhizal Fungus Terfezia claveryi.

The extensive use of high-throughput sequencing (HTS) has significantly advanced and transformed our comprehension of virus diversity, especially in intricate settings like soil and biological specimens. In this study, we delved into mycovirus sequence surveys within mycorrhizal fungus species Terfezia claveryi, through employing HTS with total double-stranded RNA (dsRNA) extracts. Our findings revealed the presence of four distinct members from the Alsuviricetes class, one flexivirus designated as Terfezia claveryi flexivirus 1 (TcFV1) and three endornaviruses (TcEV1, TcEV2, and TcEV3) in two different T. claveryi isolates. TcFV1, a member of the order Tymovirales, exhibits a unique genome structure and sequence features. Through in-depth analyses, we found that it shares sequence similarities with other deltaflexiviruses and challenges existing Deltaflexiviridae classification. The discovery of TcFV1 adds to the genomic plasticity of mycoviruses within the Tymovirales order, shedding light on their evolutionary adaptations. Additionally, the three newly discovered endornaviruses (TcEV1, TcEV2, and TcEV3) in T. claveryi exhibited limited sequence similarities with other endornaviruses and distinctive features, including conserved domains like DEAD-like helicase, ATPases Associated with Diverse Cellular Activities (AAA ATPase), and RNA dependent RNA polymerase (RdRp), indicating their classification as members of new species within the Alphaendornavirus genus. In conclusion, this research emphasizes the importance of exploring viral diversity in uncultivated fungi, bridging knowledge gaps in mycovirus ecology. The discoveries of a novel flexivirus with unique genome organization and endornaviruses in T. claveryi broaden our comprehension of mycovirus diversity and evolution, highlighting the need for continued investigations into viral populations in wild fungi.

Importance about use of high-throughput sequencing in pediatric: case report of a patient with Fanconi-Bickel syndrome

BackgroundFanconi-Bickel syndrome is characterized by hepatorenal disease caused by anomalous glycogen storage. It occurs due to variants in the SLC2A2 gene. We present a male patient of 2 years 7 months old, with failure to thrive, hepatomegaly, metabolic acidosis, hypophosphatemia, hypokalemia, hyperlactatemia.ResultsExome sequencing identified the homozygous pathogenic variant NM_000340.2(SLC2A2):c.1093 C > T (p.Arg365Ter), related with Fanconi-Bickel syndrome. He received treatment with bicarbonate, amlodipine, sodium citrate and citric acid solution, enalapril, alendronate and zolendronate, and nutritional management with uncooked cornstarch, resulting in an improvement of one standard deviation in weight and height.ConclusionsThe importance of knowing the etiology in rare genetic disease is essential, not only to determine individual and familial recurrence risk, but also to establish the treatment and prognosis; in this sense, access to a new genomic technology in low- and middle-income countries is essential to shorten the diagnostic odyssey.

ANALYSIS OF ANTIBIOTIC RESISTANCE GENES OF ESCHERICHIA COLI FROM THE GUT OF THE PIGLETS WITH DIARRHEA

The study is devoted to the analysis of the prevalence of antibiotic resistance genes in cultures of E. coli isolated from the gut of the piglets with diarrhea. With the use of high-throughput sequencing, it was possible to identify 26 antibiotic resistance genes. They belonged to 7 groups of antibiotics: aminoglycosides, beta-lactam antibiotics, quinolones, sulfonamides, tetracyclines, diaminopyrimidine, phenicols. The relative abundance of the QnrD gene was maximal in the studied sample of E. coli isolates (50%). For other sequences of antibiotic resistance genes, the percentage ratio of each separate group of genes did not exceed 10%. The largest number of gene variants was characteristic of the class of genes resistant to beta-lactam antibiotics (AmpC1_Ecoli, OXA-10, OXA-14, OXA-16, Penicillin_Binding_Protein_Ecoli, TEM-143, TEM-166, TEM-215 and TEM-76, TEM- 95).

Where to draw the boundaries? Using landscape genomics to disentangle the scribbly gum species complex.

Species delimitation is an integral part of evolution and ecology and is vital in conservation science. However, in some groups, species delimitation is difficult, especially where ancestral relationships inferred from morphological or genetic characters are discordant, possibly due to a complicated demographic history (e.g., recent divergences between lineages). Modern genetic techniques can take into account complex histories to distinguish species at a reasonable cost and are increasingly used in numerous applications. We focus on the scribbly gums, a group of up to five closely related and morphologically similar "species" within the eucalypts. Multiple populations of each recognized scribbly gum species were sampled over a wide region across climates, and genomewide scans were used to resolve species boundaries. None of the taxa were completely divergent, and there were two genetically distinct entities: the inland distributed Eucalyptus rossii and a coastal conglomerate consisting of four species forming three discernible, but highly admixed groups. Divergence among taxa was likely driven by temporal vicariant processes resulting in partial separation across biogeographic barriers. High interspecific gene flow indicated separated taxa reconnected at different points in time, blurring species boundaries. Our results highlight the need for genetic screening when dealing with closely related taxonomic entities, particularly those with modest morphological differences. We show that high-throughput sequencing can be effective at identifying species groupings and processes driving divergence, even in the most taxonomically complex groups, and be used as a standard practice for disentangling species complexes.

Management and analysis of high-throughput sequence data for infectious animal diseases.

Advances in technology and decreasing costs have accelerated the use of high-throughput sequencing (HTS) for both diagnosis and characterisation of infectious animal diseases. High-throughput sequencing offers several advantages over previous techniques, including rapid turnaround times and the ability to resolve single nucleotide changes among samples, both of which are important for epidemiological investigations of outbreaks. However, due to the plethora of genetic data being routinely generated, the storage and analysis of these data are proving challenging in their own right. In this article, the authors provide insight into the aspects of data management and analysis that should be considered before adopting HTS for routine animal health diagnostics. These elements fall largely into three interrelated categories: data storage, data analysis and quality assurance. Each has numerous complexities and may need to be adapted as HTS evolves. Making appropriate strategic decisions about bioinformatic sequence analysis early on in project development will help to avert major issues in the long term.

A Novel Rhabdovirus Associated with the Idaho Population of Potato Cyst Nematode Globodera pallida.

Globodera pallida, a potato cyst nematode (PCN), is a quarantine endoparasitic pest of potato (Solanum tuberosum) in the US due to its effects on yield and quality of potato tubers. A new rhabdovirus, named potato cyst nematode rhabdovirus (PcRV), was revealed and characterized in the G. pallida populations collected in Idaho through use of high-throughput sequencing (HTS) and RT-PCR and found to be most closely related to soybean cyst nematode rhabdovirus (ScRV). PcRV has a 13,604 bp long, single-stranded RNA genome encoding five open reading frames, including four rhabdovirus-specific genes, N, P, G, and L, and one unknown gene. PcRV was found present in eggs, invasive second-stage juveniles, and parasitic females of G. pallida, implying a vertical transmission mode. RT-PCR and partial sequencing of PcRV in laboratory-reared G. pallida populations maintained over five years suggested that the virus is highly persistent and genetically stable. Two other Globodera spp. reproducing on potato and reported in the US, G. rostochiensis and G. ellingtonae, tested negative for PcRV presence. To the best of our knowledge, PcRV is the first virus experimentally found infecting G. pallida. Based on their similar genome organizations, the phylogeny of their RNA-dependent RNA polymerase domains (L gene), and relatively high identity levels in their protein products, PcRV and ScRV are proposed to form a new genus, provisionally named "Gammanemrhavirus", within the family Rhabdoviridae.

Introduction to PM 7 Standards on Diagnostics

EPPO BulletinVolume 52, Issue 3 p. 602-603 EUROPEAN AND MEDITERRANEAN PLANT PROTECTION ORGANIZATIONFree Access Introduction to PM 7 Standards on Diagnostics The following revised Standards are included in this issue: PM 7/122 (2), PM 7/151 (1), PM 7/152 (1), PM 7/146 (2), PM 7/153 (1). First published: 20 December 2022 https://doi.org/10.1111/epp.12900AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat 1 APPROVAL EPPO Standards are approved by the EPPO Council. However, a fast-track procedure described in EPPO (2019) exists for pest-specific Diagnostic Standards. The date of approval appears in each individual Standard. In the terms of Article II of the IPPC, EPPO Standards are Regional Standards for the members of EPPO. 2 REVIEW EPPO Diagnostic Standards are subject to periodic review in the relevant Diagnostic Panels. 3 RECORD OF REVISIONS Revisions will be issued as necessary, numbered and dated. 4 SCOPE EPPO Diagnostic Protocols are intended to be used by NPPOs in their capacity as bodies responsible for the application of phytosanitary measures to detect and identify regulated pests. In 1998, EPPO started a new programme to prepare Diagnostic Protocols for the regulated pests of the EPPO region (including the EU). The work is conducted by the EPPO Panel on Diagnostics and Quality Assurance and other specialist Diagnostic Panels. The objective of the programme is to develop an internationally agreed diagnostic protocol for most of the EPPO list of pests recommended for regulation (EPPO, 2022). The protocols are based on the many years of experience of EPPO experts. The first drafts are usually prepared by a drafting team composed of experts. They are written according to a ‘common format and content of a diagnostic protocol’ agreed by the Panel on Diagnostics and Quality Assurance, modified as necessary to fit individual pests. The EPPO Standard PM 7/76 describes the purpose and use of EPPO Diagnostic Standards and includes general provisions that apply to all diagnostic protocols. 5 DEFINITIONS Definitions of the general terms used in EPPO Diagnostic Standards are described in EPPO Standard PM 7/76 (5) Use of EPPO Diagnostic Protocols, EPPO Bulletin 48, 373–377 (plus addenda). 6 OUTLINE OF REQUIREMENTS EPPO Standards of series PM 7 include general Standards on diagnostics and Diagnostic Protocols for individual pests. The Diagnostic Protocols provide all of the information necessary for a named pest to be detected and positively identified. Each Protocol begins with some short general information on the pest (its appearance, relationship with other organisms, host range, effects on host, geographical distribution and identity) and then gives details on detection and identification (including comparison with similar species, requirements for a positive diagnosis). Tests are described in the Appendices and validation data is also provided. Lists of institutes or individuals where further information on that organism can be obtained and references (on the diagnosis, detection/extraction method and test methods) are provided as well. 7 EXISTING EPPO STANDARDS IN THIS SERIES Over 150 EPPO Standards on Diagnostic Protocols, including 15 general Standards, have now been approved and published (including the new and revised Standards published here). Each Standard is numbered in the style PM 7/4 (1), meaning an EPPO Standard on Phytosanitary Measures (PM), in series no. 7 (Diagnostic Protocols), in this case Standard no. 4, first version. The full list of Diagnostic Standards (Series PM7) is available at https://www.eppo.int/RESOURCES/eppo_standards/pm7_diagnostics, with direct links to the Standards in the EPPO Bulletin. The Standards are also available in the EPPO Global Database (https://gd.eppo.int/standards/PM7/). The Standards published in the present issue of the EPPO Bulletin are listed below: PM 7/122 (2) Guidelines for the organization of interlaboratory comparisons by plant pest diagnostic laboratories, PM 7/151 (1) Considerations for the use of High Throughput Sequencing in plant health diagnostics, PM 7/152 (1) Begamoviruses, PM 7/146 (2) Tomato brown rugose fruit virus, PM 7/153 (1) Mechanical Inoculation of test plants. REFERENCES EPPO (2021) EPPO Standards PM 1/2 (30) EPPO A1 and A2 lists of quarantine pests. https://www.eppo.int/media/uploaded_images/RESOURCES/eppo_standards/pm1/pm1-002-31-en_A1A2_2022.pdf [last accessed 1 October 2022]. EPPO (2019) Procedure for preparation and approval of EPPO Standards. EPPO Bulletin 49, 159– 163. IPPC (2015) Glossary of Phytosanitary Terms. ISPM no. 5. IPPC Secretariat, FAO, Rome (IT). Available at https://www.eppo.int/media/uploaded_images/RESOURCES/eppo_standards/pm1/pm1-002-29-en.pdf [last accessed 01 November 2019]. Volume52, Issue3December 2022Pages 602-603 ReferencesRelatedInformation

PM 7/151 (1) Considerations for the use of high throughput sequencing in plant health diagnostics1

<scp>PM</scp> 7/151 (1) Considerations for the use of high throughput sequencing in plant health diagnostics¹

Drastic reduction of false positive species in samples of insects by intersecting the default output of two popular metagenomic classifiers.

The use of high-throughput sequencing to recover short DNA reads of many species has been widely applied on biodiversity studies, either as amplicon metabarcoding or shotgun metagenomics. These reads are assigned to taxa using classifiers. However, for different reasons, the results often contain many false positives. Here we focus on the reduction of false positive species attributable to the classifiers. We benchmarked two popular classifiers, BLASTn followed by MEGAN6 (BM) and Kraken2 (K2), to analyse shotgun sequenced artificial single-species samples of insects. To reduce the number of misclassified reads, we combined the output of the two classifiers in two different ways: (1) by keeping only the reads that were attributed to the same species by both classifiers (intersection approach); and (2) by keeping the reads assigned to some species by any classifier (union approach). In addition, we applied an analytical detection limit to further reduce the number of false positives species. As expected, both metagenomic classifiers used with default parameters generated an unacceptably high number of misidentified species (tens with BM, hundreds with K2). The false positive species were not necessarily phylogenetically close, as some of them belonged to different orders of insects. The union approach failed to reduce the number of false positives, but the intersection approach got rid of most of them. The addition of an analytic detection limit of 0.001 further reduced the number to ca. 0.5 false positive species per sample. The misidentification of species by most classifiers hampers the confidence of the DNA-based methods for assessing the biodiversity of biological samples. Our approach to alleviate the problem is straightforward and significantly reduced the number of reported false positive species.

The Quest to Identify a New Virus DiseaseofSunflower from Nebraska.

Between 2010 and 2018, sunflower plants exhibiting virus-like symptoms, including stunting, mottling, and chlorotic ringspots on leaves, were observed from commercial fields and research plots from four sites within three distinct counties of western Nebraska (Box Butte, Kimball, and Scotts Bluff). Near identical symptoms from field samples were reproduced on seedlings mechanically in the greenhouse on multiple occasions, confirming the presence of a sap-transmissible virus from each site. Symptomatic greenhouse-inoculated plants from the 2010 and 2011 Box Butte samples tested negative for sunflower mosaic virus (SuMV), sunflower chlorotic mottle virus (SuCMoV), and all potyviruses in general by ELISA and RT-PCR. Similar viral-like symptoms were later observed on plants in a commercial sunflower field in Kimball County in 2014, and again from volunteers in research plots in Scotts Bluff County in 2018. Samples from both of these years were again successfully reproduced on seedlings in the greenhouse as before following mechanical transmissions. Symptom expression for all years began 12 to 14 days after inoculation as mild yellow spots followed by the formation of chlorotic ringspots from the mottled pattern. The culture from 2014 tested negatively for three groups of nepoviruses via RT-PCR, ruling this group out. However, transmission electron microscopy assays of greenhouse-infected plants from both 2014 and 2018 revealed the presence of distinct, polyhedral virus particles. With the use of high throughput sequencing and RT-PCR, it was confirmed that the infections from both years were caused by a new virus in the tombusvirus genus and was proposed to be called Sunflower ring spot mottle virus (SuRSMV). Although the major objective of this project was to identify the causal agent of the disease, it became evident that the diagnostic journey itself, with all the barriers encountered on the 10-year trek, was actually more important and impactful than identification.

Analysis results of 169 cases of chorionic villus samples of missed abortion using high throughput sequencing.

This study aimed to evaluate the use of high-throughput sequencing (HTS) technology to detect chromosomes in chorionic villus samples of missed abortion embryos and investigate its utility in the genetic diagnosis of missed abortion. HTS was used to assess chorionic villus samples obtained from 169 patients with missed abortions from August 2020 to March 2022, at the Second Affiliated Hospital of Guangxi Medical University. The test results were statistically analyzed. To investigate the impact of advanced age on the incidence of chromosomal abnormalities, the patients were divided into two groups: elderly (≥35 years) and nonelderly pregnant women (<35 years). (1) Among the samples of 169 patients, 100 (59.17%) cases of chromosomal abnormalities were detected. Among these 100, 90 (90%) had chromosomal numerical abnormalities and 10 (10%) had chromosomal structural abnormalities. (2) Chromosomal numerical abnormality was abnormalities mainly included aneuploidy (92.22%, 83/90), with trisomy (62.22%, 56/90) and monosomy (22.22%, 20/90) accounting for the majority. The top three numerical abnormalities included 18 cases of Turner syndrome (monosomy X; 20%, 18/90), 10 cases of trisomy 16 (11.11%, 10/90), and 10 cases of trisomy 22 (11.11%, 10/90). (3) Villous chromosomal abnormalities were found in 48 (70.59%) elderly pregnant women, and 52 (51.48%) nonelderly pregnant women, with statistically significant differences (p < 0.05). (1) Chromosomal abnormality is an important cause of missed abortion, it majorly includes chromosomal numerical abnormality, of which most cases are of aneuploidy. (2) Advanced age may increase the risk of embryonic chromosomal abnormalities. (3) Villus chromosome detection using HTS has a positive value and can be used for analyzing and determining the causes of missed abortion.

Reproducibility and Sensitivity of High-Throughput Sequencing (HTS)-Based Detection of Citrus Tristeza Virus and Three Citrus Viroids.

The credibility of a pathogen detection assay is measured using specific parameters including repeatability, specificity, sensitivity, and reproducibility. The use of high-throughput sequencing (HTS) as a routine detection assay for viruses and viroids in citrus was previously evaluated and, in this study, the reproducibility and sensitivity of the HTS assay were assessed. To evaluate the reproducibility of HTS, the same plants assayed in a previous study were sampled again, one year later, and assessed in triplicate using the same analyses to construct the virome profile. The sensitivity of the HTS assay was compared to routinely used RT-PCR assays in a time course experiment, to compensate for natural pathogen accumulation in plants over time. The HTS pipeline applied in this study produced reproducible and comparable results to standard RT-PCR assays for the detection of CTV and three viroid species in citrus. Even though the limit of detection of HTS can be influenced by pathogen concentration, sample processing method and sequencing depth, detection with HTS was found to be either equivalent or more sensitive than RT-PCR in this study.

Distribution and Genetic Diversity of Coguvirus eburi in South African Citrus and the Development of a Real-Time RT-PCR Assay for Citrus-Infecting Coguviruses

Citrus virus A (CiVA), a novel negative-sense single-stranded RNA virus assigned to the species Coguvirus eburi in the genus Coguvirus, was detected in South Africa with the use of high-throughput sequencing after its initial discovery in Italy. CiVA is closely related to citrus concave gum-associated virus (CCGaV), recently assigned to the species Citrus coguvirus. Disease association with CiVA is, however, incomplete. CiVA was detected in grapefruit (C. paradisi Macf.), sweet orange [C. sinensis (L.) Osb.], and clementine (C. reticulata Blanco) in South Africa, and a survey to determine the distribution, symptom association, and genetic diversity was conducted in three provinces and seven citrus production regions. The virus was detected in 'Delta' Valencia trees in six citrus production regions, and a fruit rind symptom was often observed on CiVA-positive trees. Additionally, grapefruit showing symptoms of citrus impietratura disease were positive for CiVA. This virus was primarily detected in older orchards that were established prior to the application of shoot tip grafting for virus elimination in the South African Citrus Improvement Scheme. The three viral-encoded genes of CiVA isolates from each cultivar and region were sequenced to investigate sequence diversity. Genetic differences were detected between the Delta Valencia, grapefruit, and clementine samples, with greater sequence variation observed with the nucleocapsid protein (NP) compared with the RNA-dependent RNA polymerase (RdRp) and the movement protein (MP). A real-time detection assay, targeting the RdRp, was developed to simultaneously detect citrus-infecting coguviruses, CiVA and CCGaV, using a dual priming reverse primer to improve PCR specificity.

Exploring the tymovirales landscape through metatranscriptomics data.

Tymovirales is an order of viruses with positive-sense RNA genomes that mostly infect plants, but also fungi and insects. The number of genome sequences of viruses that could fit this taxon has been growing in the last few years with the extensive use of high-throughput sequencing. Here, we report the discovery of 31 novel viral genome sequences associated with 27 different host plant species, which were hidden in public databases. These viral sequences were identified through homology searches in more than 3,000 plant transcriptomes from the NCBI Sequence Read Archive (SRA) using known tymovirales sequences as queries. Identification, assembly, and curation of raw SRA reads resulted in 29 viral genome sequences with complete coding regions, and two representing partial genomes. Some of the obtained sequences highlight novel genome organizations for members of the order. Phylogenetic analysis showed that six of the novel viruses are related to alphaflexiviruses, 17 to betaflexiviruses, two to deltaflexiviruses, and six to tymovirids. These findings shed new light on the phylogenetic relationships and evolutionary landscape of this group of viruses. Furthermore, this study illustrates the complexity and genome diversity among members of the order and demonstrates that analyzing public SRA data provides an invaluable tool to accelerate virus discovery and refine virus taxonomy.

A Robust and Fast/Multiplex Pharmacogenetics Assay to Simultaneously Analyze 17 Clinically Relevant Genetic Polymorphisms in CYP3A4, CYP3A5, CYP1A2, CYP2C9, CYP2C19, CYP2D6, ABCB1, and VKORC1 Genes.

In the field of pharmacogenetics, the trend is to analyze a panel of several actionable genetic polymorphisms. It may require the use of high-throughput sequencing which demands expensive reagents/instruments and specific skills to interpret results. As an alternative, the aim of this work was to validate an easy, fast, and inexpensive multiplex pharmacogenetics assay to simultaneously genotype a panel of 17 clinically actionable variants involved in drug pharmacokinetics/pharmacodynamics. We designed primers to perform a multiplex PCR assay using a single mix. Primers were labeled by two fluorescent dye markers to discriminate alleles, while the size of the PCR fragments analyzed by electrophoresis allowed identifying amplicon. Polymorphisms of interest were CYP3A4*22, CYP3A5*3, CYP1A2*1F, CYP2C9*2-*3, CYP2C19*2-*3-*17, VKORC1-1639G > A, ABCB1 rs1045642-rs1128503-rs2229109-rs2032582, and CYP2D6*3-*4-*6-*9. The assay was repeatable and a minimum quantity of 10 ng of DNA/ sample was needed to obtain accurate results. The method was applied to a validation cohort of 121 samples and genotyping results were consistent with those obtained with reference methods. The assay was fast and cost-effective with results being available within one working-day. This robust assay can easily be implemented in laboratories as an alternative to cumbersome simplex assays or expensive multiplex approaches. Together it should widespread access to pharmacogenetics in clinical routine practice.

Downregulation of HULC Induces Ferroptosis in Hepatocellular Carcinoma via Targeting of the miR-3200-5p/ATF4 Axis.

Hepatocellular carcinoma is a malignant tumor that poses a serious threat to human health. Ferroptosis, which represents an identified type of regulated iron-dependent cell death, may play an important role in hepatocellular carcinoma. However, it is unclear as to whether ferroptosis is involved with the mechanisms of lncRNA HULC in liver cancer cells. Here, we show that knockdown of HULC increases ferroptosis and oxidative stress in liver cancer cells. We also found changes in some related miRNAs in cells treated with HULC siRNA. Differential miRNA expression levels were determined with the use of high-throughput sequencing and prediction target genes identified using bioinformatics analysis. HULC was found to function as a ceRNA of miR-3200-5p, and miR-3200-5p regulates ferroptosis by targeting ATF4, resulting in the inhibition of proliferation and metastasis within HCC cells. In summary, these findings illuminate some of the molecular mechanisms through which downregulation of HULC induces liver cancer cell ferroptosis by targeting the miR-3200-5p/ATF4 axis to modulate the development of hepatocellular carcinoma.

Efficient Screening of Long Oligonucleotides Against Hundred Thousands of SARS-CoV-2 Genome Sequences

An unprecedented use of high-throughput sequencing for routine monitoring of SARS-CoV-2 viruses in patient samples has created a dataset of over 6 million SARS-CoV-2 genomes. To monitor genomes, deposited in the GISAID database, and to track the continuous sequence evolution of molecular assay oligonucleotide target sequences. A simple pipeline tool for non-experts was developed to mine this database for nucleotide changes in oligonucleotides and tested with the long oligonucleotides of a Recombinase polymerase amplification (RPA) assay targeting the RNA-dependent RNA polymerase (RdRP) gene of the SARS-CoV-2. Results indicate the emergence of a single nucleotide change in the reverse oligonucleotide from 0.03 to 26.23% (January to May 2021) in Alpha variant genomes, which however reduced to 17.64% by September after which the Alpha variant was completely displaced by the Delta variant. For all other variants, no relevant nucleotide changes were observed. The oligonucleotide screening pipeline allows efficient screening of nucleotide changes in oligonucleotides of all sizes in minutes.

Development of a multiprimer metabarcoding approach to understanding trophic interactions in agroecosystems.

To understand trophic interactions and the precise ecological role of each predatory species, it is important to know which arthropod and plant resources are used by generalist predators in agroecosystems. Molecular approaches, such as the use of high-throughput sequencing (HTS), play a key role in identifying these resources. This study develops a multiprimer metabarcoding approach for screening the most common trophic interactions of two predatory arthropods with contrasting morphologies, Rhagonycha fulva (Coleoptera: Cantharidae) and Anthocoris nemoralis (Hemiptera: Anthocoridae) collected from a peach crop. To reduce the time and cost of this metabarcoding approach, we first evaluated the effect of using two different predator-pools of different size (10 and 23 individuals of the same species). We also used our system to analyze the performance of one and two primer pairs in the same library. Our results show that the analysis of 23 individuals together with the use of two primer pairs in the same library optimize the HTS analysis. Using these best-performing conditions, we then analyzed the entire bodies of field-collected predators as well as the washing solutions used to clean the insect bodies. We were able to identify both gut content (i.e., diet) and external pollen load (i.e., on the insects' bodies). This study also demonstrates the importance of washing predatory insects' bodies prior to HTS analysis when the target species have a considerable size (>10 mm) and hairy structures. This metabarcoding approach has significant potential for the study of trophic links in agriculture, revealing expected and unexpected trophic relationships.