Genomic surveillance of the SARS-CoV-2 pandemic is crucial and mainly achieved by amplicon sequencing protocols. Overlapping tiled-amplicons are generated to establish contiguous SARS-CoV-2 genome sequences, which enable the precise resolution of infection chains and outbreaks. We investigated a SARS-CoV-2 outbreak in a local hospital and used nanopore sequencing with a modified ARTIC protocol employing 1200 bp long amplicons. We detected a long deletion of 168 nucleotides in the ORF8 gene in 76 samples from the hospital outbreak. This deletion is difficult to identify with the classical amplicon sequencing procedures since it removes two amplicon primer-binding sites. We analyzed public SARS-CoV-2 sequences and sequencing read data from ENA and identified the same deletion in over 100 genomes belonging to different lineages of SARS-CoV-2, pointing to a mutation hotspot or to positive selection. In almost all cases, the deletion was not represented in the virus genome sequence after consensus building. Additionally, further database searches point to other deletions in the ORF8 coding region that have never been reported by the standard data analysis pipelines. These findings and the fact that ORF8 is especially prone to deletions, make a clear case for the urgent necessity of public availability of the raw data for this and other large deletions that might change the physiology of the virus towards endemism.

中文翻译：

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SARS-CoV-2 ORF8 中多次出现 168 个核苷酸缺失，但标准扩增子测序和变体调用流程未注意到

SARS-CoV-2 大流行的基因组监测至关重要，主要通过扩增子测序方案来实现。生成重叠的平铺扩增子以建立连续的 SARS-CoV-2 基因组序列，从而能够精确解决感染链和爆发。我们调查了当地一家医院的 SARS-CoV-2 疫情，并使用纳米孔测序和修改后的 ARTIC 方案，采用 1200 bp 长的扩增子。我们在医院疫情爆发的 76 个样本中检测到 ORF8 基因中存在 168 个核苷酸的长缺失。这种删除很难用经典的扩增子测序程序来识别，因为它删除了两个扩增子引物结合位点。我们分析了公开的 SARS-CoV-2 序列和来自 ENA 的测序读取数据，并在属于 SARS-CoV-2 不同谱系的 100 多个基因组中发现了相同的缺失，表明存在突变热点或正选择。在几乎所有情况下，在建立共识后，病毒基因组序列中都没有体现出删除。此外，进一步的数据库搜索指向标准数据分析管道从未报告过的 ORF8 编码区中的其他缺失。这些发现以及 ORF8 特别容易发生删除的事实清楚地表明，迫切需要公开获得该删除和其他可能将病毒的生理学改变为特有性的大型删除的原始数据。