Mini-XT, a miniaturized tagmentation-based protocol for efficient sequencing of SARS-CoV-2

Abstract

Background: The 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 1 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.

Results: We present the 'Mini-XT' miniaturized tagmentation-based library preparation protocol available on protocols.io ( https://doi.org/10.17504/protocols.io.bvntn5en ). SARS-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. The final version of Mini-XT has been used to sequence 4384 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.

Conclusions: The Mini-XT protocol maintains sequence quality while reducing library preparation reagent volumes eightfold 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.

Fig. 1

Overview of the Mini-XT protocol. The workflow can be paused at any stage and the plates stored at the indicated temperature

Fig. 2

Mini-XT sequencing metrics for individual runs. All runs over a 3-month period are indicated by date (YYMMDD)

Fig. 3

Sequencing results from Mini-XT and Illumina DNA Flex library preparation are comparable. A The percentage coverage was very similar between each of 333 samples prepared by both library preparation protocols. B A subset of the consensus sequences (named according to protocol and sample number) were placed in a phylogenetic tree with branch lengths shown in nucleotide substitutions per site and the lineages comprising the main branches indicated. The same samples sequenced using both protocols cluster together on the tree. C The same SNVs were identified in each sample by both platforms. This is illustrated in a SnipIt plot of 15 pairs of sequences representing each branch of the tree and aligned to a reference Wuhan sequence modified to contain all invariant SNVs identified in these samples

Fig. 4

Mini-XT and Illumina sequencing results are comparable with those from Nanopore technology. Twenty samples with B.1.1.7 lineage were sequenced on both platforms. A The percentage coverage was very similar between platforms. B The same SNVs were identified by both platforms, as exemplified in a SnipIt plot of 10 pairs of sequences aligned to a reference Wuhan sequence modified to contain all invariant SNVs identified in these samples. C In a phylogenetic tree of all 20 samples the same samples (named according to protocol and sample number) clustered together, reflecting consistent SNV calling (branch lengths shown in nucleotide substitutions per site)

Fig. 5

Comparison of samples amplified with ARTIC v3 and v4 primer sets. The percent coverage (top) and longest run with no N’s (bottom) were consistently greater with the v4 primers