Genome sequencing has become a powerful tool for studying emerging infectious diseases; however, genome sequencing directly from clinical samples (i.e., without isolation and culture) remains challenging for viruses such as Zika, for which metagenomic sequencing methods may generate insufficient numbers of viral reads. Here we present a protocol for generating coding-sequence-complete genomes, comprising an online primer design tool, a novel multiplex PCR enrichment protocol, optimized library preparation methods for the portable MinION sequencer (Oxford Nanopore Technologies) and the Illumina range of instruments, and a bioinformatics pipeline for generating consensus sequences. The MinION protocol does not require an Internet connection for analysis, making it suitable for field applications with limited connectivity. Our method relies on multiplex PCR for targeted enrichment of viral genomes from samples containing as few as 50 genome copies per reaction. Viral consensus sequences can be achieved in 1-2 d by starting with clinical samples and following a simple laboratory workflow. This method has been successfully used by several groups studying Zika virus evolution and is facilitating an understanding of the spread of the ...Continue Reading
Effects of primer-template mismatches on the polymerase chain reaction: human immunodeficiency virus type 1 model studies
Unusual distribution of mutations associated with serial bottleneck passages of human immunodeficiency virus type 1
Genetic and phenotypic variation of foot-and-mouth disease virus during serial passages in a natural host
Direct metagenomic detection of viral pathogens in nasal and fecal specimens using an unbiased high-throughput sequencing approach
Skip the alignment: degenerate, multiplex primer and probe design using K-mer matching instead of alignments
Automated degenerate PCR primer design for high-throughput sequencing improves efficiency of viral sequencing
A modified RNA-Seq approach for whole genome sequencing of RNA viruses from faecal and blood samples
Bacterial and viral identification and differentiation by amplicon sequencing on the MinION nanopore sequencer
Rapid metagenomic identification of viral pathogens in clinical samples by real-time nanopore sequencing analysis
Isolation of a Novel Insect-Specific Flavivirus from Culiseta melanura in the Northeastern United States
Use of Unamplified RNA/cDNA-Hybrid Nanopore Sequencing for Rapid Detection and Characterization of RNA Viruses
Comparison of Next-Generation Sequencing Technologies for Comprehensive Assessment of Full-Length Hepatitis C Viral Genomes.
Outbreak of Zika Virus Infection, Chiapas State, Mexico, 2015, and First Confirmed Transmission by Aedes aegypti Mosquitoes in the Americas
Genome Sequence of a Candidate World Health Organization Reference Strain of Zika Virus for Nucleic Acid Testing
Harmonization of nucleic acid testing for Zika virus: development of the 1st World Health Organization International Standard
Rapid outbreak sequencing of Ebola virus in Sierra Leone identifies transmission chains linked to sporadic cases.
Reassembling haplotypes in a mixture of pooled amplicons when the relative concentrations are known: A proof-of-concept study on the efficient design of next-generation sequencing strategies
ALT-803 Transiently Reduces Simian Immunodeficiency Virus Replication in the Absence of Antiretroviral Treatment
Ticks and Tick-Borne Pathogens of the Caribbean: Current Understanding and Future Directions for More Comprehensive Surveillance
Infection via mosquito bite alters Zika virus tissue tropism and replication kinetics in rhesus macaques
Phylogenetic analysis of West Nile Virus in Maricopa County, Arizona: Evidence for dynamic behavior of strains in two major lineages in the American Southwest
Genome-enhanced detection and identification of fungal pathogens responsible for pine and poplar rust diseases
Systematic and stochastic influences on the performance of the MinION nanopore sequencer across a range of nucleotide bias
A framework and an algorithm to detect low-abundance DNA by a handy sequencer and a palm-sized computer
Zika viruses of African and Asian lineages cause fetal harm in a mouse model of vertical transmission
Recent establishment of tick-borne encephalitis foci with distinct viral lineages in the Helsinki area, Finland
Detection of atypical porcine pestivirus genome in newborn piglets affected by congenital tremor and high preweaning mortality1
Return of the founder Chikungunya virus to its place of introduction into Brazil is revealed by genomic characterization of exanthematic disease cases.
Oropouche virus cases identified in Ecuador using an optimised qRT-PCR informed by metagenomic sequencing
Community-acquired in name only: A cluster of carbapenem-resistant Acinetobacter baumannii in a burn intensive care unit and beyond.
Real-time, MinION-based, amplicon sequencing for lineage typing of infectious bronchitis virus from upper respiratory samples.
Genomic epidemiology supports multiple introductions and cryptic transmission of Zika virus in Colombia
The Robust Restriction of Zika Virus by Type-I Interferon in A549 Cells Varies by Viral Lineage and Is Not Determined by IFITM3.
CREs: Gene & Cell Therapy
Gene and cell therapy advances have shown promising outcomes for several diseases. The role of cis-regulatory elements (CREs) is crucial in the design of gene therapy vectors. Here is the latest research on CREs in gene and cell therapy.