Illumina iSeq 100 and MiSeq exhibit similar performance in freshwater fish environmental DNA metabarcoding.

Environmental DNA (eDNA) analysis is a method of detecting DNA from environmental samples and is used as a biomonitoring tool. In recent studies, Illumina MiSeq has been the most extensively used tool for eDNA metabarcoding. The Illumina iSeq 100 (hereafter, iSeq), one of the high-throughput sequencers (HTS), has a relatively simple workflow and is potentially more affordable than other HTS. However, its utility in eDNA metabarcoding has still not been investigated. In the present study, we applied fish eDNA metabarcoding to 40 water samples from river and lake ecosystems to assess the difference in species detectability and composition between iSeq and MiSeq. To check differences in sequence quality and errors, we also assessed differences in read changes between the two HTS. There were similar sequence qualities between iSeq and MiSeq. Significant difference was observed in the number of species between two HTS, but no difference was observed in species composition between the two HTS. Additionally, the species compositions in common with the conventional method were the same between the two HTS. According to the results, using the same amplicon library for sequencing, two HTS would exhibit a similar performance of fish species detection using eDNA metabarcoding.

Targeted next generation sequencing (NGS) to classify melanocytic neoplasms.

This study piloted a pan-solid-tumor next generation sequence (NGS)-based laboratory developed test as a diagnostic aid in melanocytic tumors. 31 cases (4 "epithelioid" nevi, 5 blue nevi variants, 7 Spitz tumors [3 benign and 4 malignant] and 15 melanomas) were evaluated. All tumors [median diameter 7 mm (range 4-15 mm); median thickness 2.25 mm (range 0.25-12 mm)] yielded satisfactory results. The number of small nucleotide variants/tumor was significantly different between melanoma (median 18/tumor, range 4-71) and all other lesions (median 8/tumor, range 3-17) (P < 0.004) and malignant (median 16/tumor, range 4-71) vs benign lesions (median 7/tumor, range 3-14) (P = 0.01). BRAF, MET, NTRK1, and ROS fusions only occurred in benign Spitz tumors; EML4 fusion, BRAF, MAP2K1 and TERT mutations occurred in malignant Spitz tumors and/or melanoma. Amplifications and NRAS and NF1 mutations only occurred in melanoma. Most melanomas contained >1 pathogenic alteration. Developed NGS-based criteria correctly classified all malignant lesions in this series. 10/12 cases showed concordance with FISH; consensus diagnosis agreed with NGS classification in FISH-non-concordant cases. This pilot study suggests that NGS may be an effective diagnostic adjunct comparable to FISH, but further studies with larger numbers of cases are needed.

Standard finishing categories for high-throughput sequencing of viral genomes.

Viral genome sequencing has become the cornerstone of almost all aspects of virology. In particular, high-throughput, next-generation viral genome sequencing has become an integral part of molecular epidemiological investigations into outbreaks of viral disease, such as the recent outbreaks of Middle Eastern respiratory syndrome, Ebola virus disease and Zika virus infection. Multiple institutes have acquired the expertise and necessary infrastructure to perform such investigations, as evidenced by the accumulation of thousands of novel viral sequences over progressively shorter time periods. The authors recently proposed a nomenclature comprised of five high-throughput sequencing standard categories to describe the quality of determined viral genome sequences. These five categories (standard draft, high quality, coding complete, complete and finished) cover all levels of viral genome finishing and can be applied to sequences determined by any technology platform or assembly technique.

Le séquençage des génomes viraux est devenu la pierre angulaire de pratiquement toutes les facettes de la virologie. En particulier, le séquençage à haut débit de nouvelle génération est désormais une partie intégrante des enquêtes d'épidémiologie moléculaire relatives aux foyers de maladies virales, par exemple les récentes épidémies du syndrome respiratoire du Moyen-Orient, la maladie due au virus Ebola ou l'infection par le virus Zika. Nombre d'institutions ont acquis les compétences techniques et les infrastructures nécessaires pour réaliser ce type d'enquêtes, comme en témoigne l'accumulation de milliers de séquences virales nouvelles obtenues en un laps de temps de plus en plus court. Les auteurs ont récemment élaboré une nomenclature constituée de cinq catégories de référence décrivant la qualité des séquences d'un génome viral obtenues par séquençage à haut débit. Ces cinq catégories (ébauche de référence, séquence de haute qualité, séquence codante complète, séquence complète et séquence finie) couvrent toutes les étapes de la finition du génome viral et s'appliquent quelle que soit la plateforme technologique ou la technique d'assemblage utilisée pour déterminer la séquence.

La secuenciación del genoma vírico se ha erigido a día de hoy en la piedra angular de casi todos los aspectos de la virología. La secuenciación de alto rendimiento de próxima generación, en particular, es ahora un componente integral de las investigaciones de epidemiología molecular sobre brotes de enfermedades víricas como los registrados últimamente de síndrome respiratorio de Oriente Medio, enfermedad por el virus del Ebola o infección por el virus Zika. Numerosas instituciones se han dotado de las competencias técnicas y la infraestructura necesaria para llevar a cabo tales investigaciones, como deja patente la acumulación de miles de nuevas secuencias víricas en periodos de tiempo cada vez más cortos. En fechas recientes los autores han propuesto una nomenclatura compuesta de cinco categorías de referencia que sirven para describir la calidad de las secuencias de genoma vírico determinadas por secuenciación de alto rendimiento. Estas cinco categorías (borrador normal, gran calidad, codificación completa, completa y acabada) cubren toda la gradación de acabados en la secuenciación de genoma vírico y pueden ser aplicadas a las secuencias obtenidas por cualquier dispositivo técnico o cualquier técnica de ensamblaje.

Aplicación de la secuenciación masiva de nueva generación al diagnóstico molecular de la hipercolesterolemia familiar / Applicability of next generation sequencing to the molecular diagnosis of familial hypercholesterolemia

Introducción: La hipercolesterolemia familiar es una enfermedad autosómica dominante que cursa con niveles plasmáticos elevados de colesterol unido a lipoproteínas de baja densidad. La presencia de esta alteración en el metabolismo lipídico se asocia a un aumento del riesgo cardiovascular en los pacientes que la padecen, siendo de gran importancia la realización de un diagnóstico genético para ofrecer un tratamiento adecuado y disminuir la morbimortalidad. El objetivo de este trabajo es describir la aplicación de la secuenciación de nueva generación al diagnóstico genético de la hipercolesterolemia familiar, en comparación con la secuenciación Sanger, la técnica convencional usada hasta el momento. Material y métodos: Se analizaron 110 muestras de sangre venosa periférica procedente de pacientes que presentaban cuadro clínico de hipercolesterolemia familiar mediante secuenciación de nueva generación, utilizando un panel comercial que permite la identificación de mutaciones en los genes LDLR, APOB, PCSK9 yLDLRAP1 (SEQPRO Lipo, Progenika) con la tecnología GS JUNIOR 454 (Roche). Resultados: Aplicando esta tecnología fue posible secuenciar los genes asociados a la hipercolesterolemia familiar descritos hasta el momento en grupos de hasta 20 pacientes simultáneamente. Se detectaron un total de 35 mutaciones en las 110 muestras analizadas, localizándose el 94,29% en el gen LDLR. Todas las mutaciones identificadas fueron confirmadas mediante el método de secuenciación Sanger. Conclusiones: La utilización de la secuenciación masiva de nueva generación permite la realización de un diagnóstico genético más rápido y un análisis molecular más eficiente de los genes implicados en la hipercolesterolemia familiar con una fiabilidad similar a la técnica convencional Sanger (AU)

Introduction: Familial hypercholesterolemia is an autosomal dominant disorder that causes increased levels of cholesterol associated with low density lipoproteins in plasma. The presence of altered lipid metabolism in these patients increases their level of risk of suffering from a cardiovascular disease. The certainty of having this genetic disorder by making a timely and precise molecular diagnostic is crucial for the appropriate treatment and the reduction of the disease morbidity-mortality. The aim of this work is to describe the applicability of next generation sequencing technology to the genetic diagnosis of familial hypercholesterolemia and compare this novel method with the conventional Sanger sequencing method. Material and methods: A next generation sequencing commercial panel (SEQPRO Lipo, Progenika) was used to analyze 110 peripheral venous blood samples from patients with familial hypercholesterolemia. This enables the assessment of mutations in genes associated with the disease (e.g. LDLR, APOB, PCSK9 and LDLRAP1)with the GS JUNIOR 454 technology (Roche). Results: Application of next generation sequencing enables the sequencing of the genes involved in the familial hypercholesterolemias, described so far, in groups of 20 patients simultaneously. Using this novel technology, a total of 35 mutations were detected in the 110 analysed samples, with 94.29% being located in the LDLR gene. Mutations were confirmed by Sanger sequencing. Conclusion: Next generation sequencing enables a quick genetic diagnosis and a more efficient molecular analysis of all genes described so far to be involved in familial hypercholesterolemia, with similar reliability to that of conventional Sanger sequencing (AU)

Comparison of custom capture for targeted next-generation DNA sequencing.

Targeted, capture-based DNA sequencing is a cost-effective method to focus sequencing on a coding region or other customized region of the genome. There are multiple targeted sequencing methods available, but none has been systematically investigated and compared. We evaluated four commercially available custom-targeted DNA technologies for next-generation sequencing with respect to on-target sequencing, uniformity, and ability to detect single-nucleotide variations (SNVs) and copy number variations. The technologies that used sonication for DNA fragmentation displayed impressive uniformity of capture, whereas the others had shorter preparation times, but sacrificed uniformity. One of those technologies, which uses transposase for DNA fragmentation, has a drawback requiring sample pooling, and the last one, which uses restriction enzymes, has a limitation depending on restriction enzyme digest sites. Although all technologies displayed some level of concordance for calling SNVs, the technologies that require restriction enzymes or transposase missed several SNVs largely because of the lack of coverage. All technologies performed well for copy number variation calling when compared to single-nucleotide polymorphism arrays. These results enable laboratories to compare these methods to make informed decisions for their intended applications.