cDNA synthesis and whole genome sequencing using the MinION
TA Talita Émile Ribeiro Adelino MG Marta Giovanetti VF Vagner Fonseca JX Joilson Xavier ÁA Álvaro Salgado de Abreu VN Valdinete Alves do Nascimento LD Luiz Henrique Ferraz Demarchi MO Marluce Aparecida Assunção Oliveira VS Vinícius Lemes da Silva AM Arabela Leal e. Silva de Mello GC Gabriel Muricy Cunha RS Roselene Hans Santos EO Elaine Cristina de Oliveira JJ Jorge Antônio Chamon Júnior FI Felipe Campos de Melo Iani AF Ana Maria Bispo de Filippis AA André Luiz de Abreu RJ Ronaldo de Jesus CA Carlos Frederico Campelo de Albuquerque JR Jairo Mendez Rico RS Rodrigo Fabiano do Carmo Said JS Joscélio Aguiar Silva NM Noely Fabiana Oliveira de Moura PL Priscila Leite LF Lívia Carla Vinhal Frutuoso SH Simone Kashima Haddad AM Alexander Martínez FB Fernanda Khouri Barreto CV Cynthia Carolina Vazquez RC Rivaldo Venâncio da Cunha EA Emerson Luiz Lima Araújo ST Stephane Fraga de Oliveira Tosta AF Allison de Araújo Fabri FC Flávia Löwen Levy Chalhoub PL Poliana da Silva Lemos FB Fernanda de Bruycker-Nogueira GL Gislene Garcia de Castro Lichs MZ Marina Castilhos Souza Umaki Zardin FS Fátima María Cardozo Segovia CG Crhistinne Cavalheiro Maymone Gonçalves ZG Zoraida Del Carmen Fernandez Grillo SS Svetoslav Nanev Slavov LP Luiz Augusto Pereira AM Ana Flávia Mendonça FP Felicidade Mota Pereira JM Jurandy Júnior Ferraz de Magalhães AJ Agenor de Castro Moreira dos Santos Júnior ML Maricélia Maia de Lima RN Rita Maria Ribeiro Nogueira AG Aristóteles Góes-Neto

Samples were selected for sequencing based on the Ct value (≤35) and availability of epidemiological metadata, such as date of symptom onset, date of sample collection, sex, age, municipality of residence, symptoms, and disease classification. For complementary DNA synthesis, the SuperScript IV Reverse Transcriptase kit (Invitrogen) was used following the manufacturer’s instructions. The cDNA generated was subjected to sequencing multiplex PCR (35-cycles) using Q5 High Fidelity Hot-Start DNA Polymerase (NEB) and a set of specific primers designed by the CADDE project (https://www.caddecentre.org/) for sequencing the complete genomes of DENV1 and DENV239 (Supplementary Table 5).

Amplicons were purified using 1x AMPure XP Beads (Beckman Coulter) and quantified on a Qubit 3.0 fluorimeter (Thermofisher Scientific) using Qubit™ dsDNA HS Assay Kit (Thermofisher Scientific). Of the 248 samples, 227 contained sufficient DNA (≥2 ng/µL) to proceed to library preparation. DNA library preparation was performed using the Ligation Sequencing Kit (Oxford Nanopore Technologies) and the Native Barcoding Kit (NBD103, Oxford Nanopore Technologies)23. Sequencing libraries were generated from the barcoded products using the Genomic DNA Sequencing Kit SQK-MAP007/SQK-LSK208 (Oxford Nanopore Technologies) and loaded into a R9.4 flow cell (Oxford Nanopore Technologies). In each sequencing run we used negative controls to prevent and check for possible contamination with less than 2% mean coverage.

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