Genomics Core Technology Unit, Faculty of Medicine and Health Sciences, Queen's University Belfast, Belfast, UK.
The Wellcome-Wolfson Institute for Experimental Medicine, School of Medicine, Dentistry and Biomedical Sciences, Queen's University, Belfast, UK.
J Transl Med. 2022 Mar 3;20(1):105. doi: 10.1186/s12967-022-03307-9.
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.
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.
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.
COVID-19 大流行凸显了对 SARS-CoV-2 进行全基因组测序（WGS）的重要性，以提供公共卫生政策信息。通过定义谱系，它有助于跟踪病毒在全球的传播。可以监测新变体的演变，并了解特定突变可深入了解病毒增加传染性或逃避免疫的机制。迄今为止，COVID-19 英国基因组学联盟（COG-UK）的成员已经对近 100 万个 SARS-CoV-2 基因组进行了测序。为了在未来以更具成本效益和可持续的方式实现类似的壮举，需要改进高通量病毒测序协议。因此，我们开发了一种微型化的文库制备协议，大大减少了消耗品的使用和成本。
我们提出了可在 protocols.io 上获得的“Mini-XT”微型化标签酶切文库制备协议（https://doi.org/10.17504/protocols.io.bvntn5en）。使用 ARTIC nCov-2019 多重 RT-PCR 协议扩增 SARS-CoV-2 RNA，并使用常规液体处理系统进行纯化。采用声控液体转移（Echo 525）减少反应体积和 Nextera XT 文库制备所需的吸头数量。在 Illumina MiSeq 上进行测序。最终版本的 Mini-XT 已用于对北爱尔兰的 4384 个 SARS-CoV-2 样本进行测序，COG-UK 的 QC 通过率为 97.4%。用全体积 Nextera DNA Flex（333 个样本）或使用纳米孔技术（20 个样本）处理重复样本的测序质量相当，谱系调用一致。Mini-XT 与这些技术之间的 SNP 调用一致，并且在最大似然系统发育树中配对的重复样本序列一致。
Mini-XT 协议在保持序列质量的同时，将文库制备试剂体积减少了八倍，并将样本到测序的总体吸头使用量减少了一半，与标准协议相比可实现成本节约。这将使 SARS-CoV-2 分离株和未来病原体的高通量测序更加高效。
