Sequencing of mRNA from Whole Blood using Nanopore Sequencing.
J Vis Exp
; (148)2019 06 03.
Article
em En
| MEDLINE
| ID: mdl-31205309
ABSTRACT
Sequencing in remote locations and resource-poor settings presents unique challenges. Nanopore sequencing can be successfully used under such conditions, and was deployed to West Africa during the recent Ebola virus epidemic, highlighting this possibility. In addition to its practical advantages (low cost, ease of equipment transport and use), this technology also provides fundamental advantages over second-generation sequencing approaches, particularly the very long read length, ability to directly sequence RNA, and real-time availability of data. Raw read accuracy is lower than with other sequencing platforms, which represents the main limitation of this technology; however, this can be partially mitigated by the high read depth generated. Here, we present a field-compatible protocol for sequencing of the mRNAs encoding for Niemann-Pick C1, which is the cellular receptor for ebolaviruses. This protocol encompasses extraction of RNA from animal blood samples, followed by RT-PCR for target enrichment, barcoding, library preparation, and the sequencing run itself, and can be easily adapted for use with other DNA or RNA targets.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
RNA Mensageiro
/
Análise de Sequência de RNA
/
Sequenciamento por Nanoporos
Tipo de estudo:
Guideline
Limite:
Animals
Idioma:
En
Revista:
J Vis Exp
Ano de publicação:
2019
Tipo de documento:
Article