ABSTRACT
IntroductionThe COVID-19 pandemic has highlighted the importance of whole genome sequencing (WGS) of SARS-CoV-2 to inform public health policy. By enabling definition of lineages it facilitates tracking of the global spread of the virus. The evolution of new variants can be monitored and knowledge of specific mutations provides insights into the mechanisms through which the virus increases transmissibility or evades immunity. To date almost one million SARS-CoV-2 genomes have been sequenced by members of the COVID-19 Genomics UK (COG-UK) Consortium. To achieve similar feats in a more cost-effective and sustainable manner in future, improved high throughput virus sequencing protocols are required. We have therefore developed a miniaturized library preparation protocol with drastically reduced consumable use and costs. MethodsSARS-CoV-2 RNA was amplified using the ARTIC nCov-2019 multiplex RT-PCR protocol and purified using a conventional liquid handling system. Acoustic liquid transfer (Echo 525) was employed to reduce reaction volumes and the number of tips required for a Nextera XT library preparation. Sequencing was performed on an Illumina MiSeq. ResultsWe present the Mini-XT miniaturized tagmentation-based library preparation protocol available on protocols.io (https://dx.doi.org/10.17504/protocols.io.bvntn5en). The final version of Mini-XT has been used to sequence 4,384 SARS-CoV-2 samples from N. Ireland with a COG-UK QC pass rate of 97.4%. Sequencing quality was comparable and lineage calling consistent for replicate samples processed with full volume Nextera DNA Flex (333 samples) or using nanopore technology (20 samples). SNP calling between Mini-XT and these technologies was consistent and sequences from replicate samples paired together in maximum likelihood phylogenetic trees. ConclusionThe Mini-XT protocol maintains sequence quality while reducing library preparation reagent volumes 8-fold and halving overall tip usage from sample to sequence to provide concomitant cost savings relative to standard protocols. This will enable more efficient high-throughput sequencing of SARS-CoV-2 isolates and future pathogen WGS.
Subject(s)
COVID-19ABSTRACT
CD8+ T cell immunity to SARS-CoV-2 has been implicated in COVID-19 severity and virus control, though direct evidence has been lacking so far. Here, we identified non-synonymous mutations in MHC-I restricted CD8+ T cell epitopes after deep sequencing of 747 SARS-CoV- 2 virus isolates. Mutant peptides exhibited diminished or abrogated MHC-I binding, which was associated with a loss of recognition and functional responses by CD8+ T cells isolated from HLA-matched COVID-19 patients. Our findings highlight the capacity of SARS-CoV-2 to subvert CD8+ T cell surveillance through escape mutations in MHCI-restricted viral epitopes. This provides evolutionary evidence for CD8+ T cell immunity controlling SARS-CoV-2 with consequences for COVID-19 vaccine design.
Subject(s)
COVID-19ABSTRACT
Superspreading events shape the COVID-19 pandemic. Here we provide a national-scale analysis of SARS-CoV-2 outbreaks in Austria, a country that played a major role for virus transmission across Europe and beyond. Capitalizing on a national epidemiological surveillance system, we performed deep whole-genome sequencing of virus isolates from 576 samples to cover major Austrian SARS-CoV-2 clusters. Our data chart a map of early viral spreading in Europe, including the path from low-frequency mutations to fixation. Detailed epidemiological surveys enabled us to calculate the effective SARS-CoV-2 population bottlenecks during transmission and unveil time-resolved intra-patient viral quasispecies dynamics. This study demonstrates the power of integrating deep viral genome sequencing and epidemiological data to better understand how SARS-CoV-2 spreads through populations. Graphical Abstract O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY