Nanopore sequencing and full genome de novo assembly of human cytomegalovirus TB40/E reveals clonal diversity and structural variations.
van Wilgenburg, Bonnie
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Karamitros, T., van Wilgenburg, B., Wills, M., Klenerman, P., & Magiorkinis, G. (2018). Nanopore sequencing and full genome de novo assembly of human cytomegalovirus TB40/E reveals clonal diversity and structural variations.. BMC genomics, 19 (1), 577. https://doi.org/10.1186/s12864-018-4949-6
Background: Human cytomegalovirus (HCMV) has a double-stranded DNA genome of approximately 235 Kbp that is structurally complex including extended GC-rich repeated regions. Genomic recombination events are frequent in HCMV cultures but have also been observed in vivo. Thus, the assembly of HCMV whole genomes from technologies producing shorter than 500 bp sequences is technically challenging. Here we improved the reconstruction of HCMV full genomes by means of a hybrid, de novo genome-assembly bioinformatics pipeline upon data generated from the recently released MinION MkI B sequencer from Oxford Nanopore Technologies. Methods: Total DNA was extracted from concentrated preparations of HCMV (strain TB40/E). The MinION libraries were constructed using the Transposase-based “Rapid Sequencing Kit”. We developed a novel, self-correcting bioinformatics algorithm to assemble the pooled HCMV genomes in three stages. In the first stage, long contigs (N50= 21,892) of lower accuracy were reconstructed. In the second stage, short contigs (N50= 5,686) of higher accuracy were assembled, while in the final stage the high quality contigs served as template for the correction of the longer contigs resulting in a high-accuracy, full genome assembly (N50= 41,056). Results: The MinION run resulted in ~47,000 reads from a single R9 flowcell and in ~100x average read depth across the virus genome. We were able to reconstruct a single representative haplotype without employing any scaffolding steps. The majority (98.8%) of the genomic features from the reference strain were accurately annotated on this full genome construct. Our method also allowed the detection of multiple alternative sub-genomic fragments and non-canonical structures suggesting rearrangement events between the unique (UL /US) and the repeated (T/IRL/S) genomic regions. Conclusions: Third generation high-throughput sequencing technologies can accurately reconstruct full-length HCMV genomes including their low-complexity and highly repetitive regions. Full-length HCMV genomes could prove crucial in understanding the genetic determinants and viral evolution underpinning drug resistance, virulence and pathogenesis.
Cell Line, Humans, Cytomegalovirus, Sequence Analysis, DNA, Evolution, Molecular, Genome, Viral, Algorithms, Nanopores, High-Throughput Nucleotide Sequencing, Genome Size
External DOI: https://doi.org/10.1186/s12864-018-4949-6
This record's URL: https://www.repository.cam.ac.uk/handle/1810/284714
Attribution 4.0 International
Licence URL: https://creativecommons.org/licenses/by/4.0/