Whole Genome Sequencing of SARS-CoV-2: Adapting Illumina Protocols for Quick and Accurate Outbreak Investigation During a Pandemic

Abstract

The COVID-19 pandemic spread very fast around the world. A few days after the first detected case in South Africa, an infection started a large hospital outbreak in Durban, KwaZulu-Natal. Phylogenetic analysis of SARS-CoV-2 genomes can be used to trace the path of transmission within a hospital. It can also identify the source of the outbreak and provide lessons to improve infection prevention and control strategies. In this manuscript, we outline the obstacles we encountered in order to genotype SARS-CoV-2 in real-time during an urgent outbreak investigation. In this process, we encountered problems with the length of the original genotyping protocol, reagent stockout and sample degradation and storage. However, we managed to set up three different library preparation methods for sequencing in Illumina. We also managed to decrease the hands on library preparation time from twelve to three hours, which allowed us to complete the outbreak investigation in just a few weeks. We also fine-tuned a simple bioinformatics workflow for the assembly of high-quality genomes in real-time. In order to allow other laboratories to learn from our experience, we released all of the library preparation and bioinformatics protocols publicly and distributed them to other laboratories of the South African Network for Genomics Surveillance (SANGS) consortium.

Novel SARS-CoV-2 Whole-genome sequencing technique using Reverse Complement PCR enables easy, fast and accurate outbreak analysis in hospital and community settings

“…The technique has subsequently been optimized and analysis, albeit in small sample numbers, concluded that it delivers sufficient quality to perform phylogenetic analysis. [18][19][20] It had been used as targeted and random RT-PCR screening with subsequent sequencing of the population in order to study the spread through the community. was early on in the pandemic, it seems more likely that a common source of infection, in-or outside the hospital is the cause.…”

A genomics network established to respond rapidly to public health threats in South Africa

“…We have set up protocols for Illumina, Oxford Nanopore Technologies, PacBio, and Ion Torrent sequencers largely based on open methods developed by the ARTICnetwork. We have also modified Illumina protocols to use an alternative library, which has reduced the hands-on sample processing time by 9 h. 3 As some of the laboratories in the network were prepared when the first cases of COVID-19 were reported in South Africa, we were able to use genomics early in the epidemic to understand the initial spread of the virus, which was characterised by clustered outbreaks. For example, we did a detailed investigation into a nosocomial outbreak in KwaZulu-Natal that began just 4 days after the first reported case in the country.…”

“…2,5,6 Short read data are deposited in the Sequence Read Archive every week. Assembled genomes are rapidly shared via the Global Initiative on Sharing All Influenza Data, 3 which is working in collaboration with the NGS-SA to ensure that data generated in South Africa can also be quality controlled and analysed in-country. We believe that open access to data is crucial to facilitate a rapid response to the SARS-CoV-2 pandemic.…”

Real Time Viral Sub-Strains Discovery in Emerging Infectious Disease Situation – The African Perspective

“…Furthermore, DUET web server prognosticated the destabilizing and stabilizing effect of D614G variant of the spike protein and P322L mutation of the nsp12 respectively. As a supplemental study to the obstacles encountered during near-real time SARS-CoV-2 genotyping during pandemic, Pillay et al [45] established 3 lineages (B, B.1 and B.2) from a phylogenetic analysis of 54 near-full-length genome, using Phylogenetic Assignment of Named Global Outbreak LINeages (PANGOLIN) software suite. The 54 genome sequences and 10,959 GISAID reference genomes were aligned using MAFFT v7.313 and a maximum likelihood tree topology constructed in IQ-TREE.…”