The pipeline provides a quick and efficient way to obtain a matrix of read counts that can be used black with tools such as DESeq2 and edgeR for differential expression analysis. Using Nextflow as a workflow management system and Singularity for application containerisation, the nf-rnaSeqCount pipeline was developed for mapping raw RNA-seq reads to a reference genome and quantifying abundance of identified genomic features for differential gene expression analyses. The aim of this study was to develop a robust portable and reproducible bioinformatic pipeline for the automation of RNA sequencing (RNA-seq) data analyses. Even though such data promises new insights into how biological systems function and understanding disease mechanisms, computational analyses performed on such large datasets comes with its challenges and potential pitfalls. This has enabled researchers to answer many biological questions through ``multi-omics'' data analyses. The rate of raw sequence production through Next-Generation Sequencing (NGS) has been growing exponentially due to improved technology and reduced costs. nCoV-2019 sequencing protocol (RAPID barcoding, 1200bp amplicon).doi: 10.1093/biomethods/bpaa014 Our peer-reviewed paper is available here: To get information such as Primers, visit their protocol. This protocol is a modified version of Nikki Freed and Olin Silanders protocol. 11 hrs for 12 multiplexed barcoded specimens. Duration of the complete pipeline was approx. We tested the simplified and less time-consuming workflow on confirmed SARS-CoV-2-positive specimens from clinical routine and identified pre-analytical parameters, which may help to decrease the rate of sequencing failures. Subsequently, we applied the Oxford Nanopore Rapid barcoding protocol and the portable MinION Mk1C sequencer in combination with the ARTIC bioinformatics pipeline.
We adapted and simplified existing workflows using the ‘midnight’ 1,200 bp amplicon split primer sets for PCR, which produce tiled overlapping amplicons covering almost all of the SARS-CoV-2 genome. The cost per sample accumulates at around 40$, with already isolated RNA. The whole Sequencing can be done in one working day, including the bioinformatic pipeline.
Through our EPI2ME Labs environment, we hope to support laboratory scientists with simplified solutions to complex computational tasks.We established a protocol for fast, cost efficient Sars-CoV-2 sequencing with little as possible hands-on time (around 3h in total, excluding RNA extraction). He has two decades of experience in genome bioinformatics support and works with the Customer Workflows team towards demystifying bioinformatics. Stephen Rudd is an Associate Director at Oxford Nanopore and is responsible for the bioinformatic products and imagines user journeys that begin with a collection of FASTQ files. She also is a technical product manager for the Oxford Nanopore Sequencing kits. Jemma Jordan is an Associate Director at Oxford Nanopore and is responsible for the Pilot Laboratory team, who assist with the development and testing of many kits including Midnight (MRT001). Prior to joining Oxford Nanopore, he became a user of the MinION in 2014, developing methods for in situ DNA sequencing of biodiversity in the Amazon rainforest. He received his PhD from UC Berkeley, employing next generation sequencing, genome editing, and bioimaging techniques in non-model organisms. How SARS-CoV-2 sequences can be rapidly analysed to generate consensus genome sequences, and for the identification of variants of interestĪaron Pomerantz is a Segment Marketing Manager at Oxford Nanopore, helping to drive forward the development of new areas in the research life sciences market.How the Midnight Kit works, enabling fast, scalable preparation of SARS-CoV-2 genomes for nanopore sequencing.How nanopore sequencing is being utilised around the world to rapidly sequence SARS-CoV-2 genomes, providing information critical to identifying variants of interest and monitoring the spread of the virus.After an introduction to the use of nanopore sequencing for genomic epidemiology, the presenters then went through the Midnight method, from library prep, to sequencing, to data analysis. During this Knowledge Exchange, three presenters from Oxford Nanopore Technologies shared a comprehensive overview of the rapid, cost-effective sequencing of SARS-CoV-2 genomes using the Midnight Kit.
0 kommentar(er)
