Greater coverage and depth is critical for accurate genome reconstruction and subsequent phylogenetic inference. In these cases, sequences from Zika virus comprised <0.01% of the dataset, resulting in incomplete coverage.
Before sequencing these samples were depleted of human rRNA and prepared for metagenomic sequencing on the Illumina MiSeq platform as previously described 2, 16.
These samples had Ct values between 33.9 and 35.9 (equivalent to 10-48 genome copies per microlitre). During recent work on the Zika virus epidemic 15, we found that it was difficult to generate whole-genome sequences directly from clinical samples using metagenomic approaches ( Table 1). However, direct metagenomic sequencing from clinical samples poses challenges with regards to sensitivity: genome coverage may be low or absent when attempting to sequence viruses that are present at low abundance in a sample with high levels of host nucleic acid background. Whole-genome sequencing of Ebola virus directly from clinical samples without amplification was possible due to the extremely high virus copy numbers found in acute cases 13, 14. Metagenomic approaches have seen rapid adoption over the past decade, fuelled by relentless improvements in the yield of high-throughput sequencing instruments 5, 10– 12. Metagenomics, the process of sequencing the total nucleic acid content in a sample (typically cDNA or DNA), has been successfully applied to both virus discovery and diagnostics 7– 9. Sequencing directly from clinical samples is faster, less laborious, and more amenable to near-patient work than time consuming culture-based methods.
During the Ebola virus epidemic of 2013-2016, prospective viral genome sequencing was able to provide critical information on virus evolution and help inform epidemiological investigations 3– 6. Such work depends on rapid sequencing of viral material directly from clinical samples. Real-time genomic surveillance is important in managing viral outbreaks, as it can provide insights into how viruses transmit, spread, and evolve 1– 4. Genome sequencing of viruses has been used to study the spread of disease in outbreaks 1. This method has been successfully used by several groups studying Zika virus evolution and is facilitating an understanding of the spread of the virus in the Americas. Viral consensus sequences can be achieved starting with clinical samples in 1-2 days following a simple laboratory workflow. Our method relies on multiplex PCR for targeted enrichment of viral genomes from samples containing as few as 50 genome copies per reaction. The MinION protocol does not require an internet connection for analysis, making it suitable for field applications with limited connectivity.
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Here we present a protocol for generating coding-sequence complete genomes comprising an online primer design tool, a novel multiplex PCR enrichment protocol, optimised 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.
Genome sequencing has become a powerful tool for studying emerging infectious diseases however, genome sequencing directly from clinical samples without isolation remains challenging for viruses such as Zika, where metagenomic sequencing methods may generate insufficient numbers of viral reads.