Since December 2019, the coronavirus disease 2019 (COVID-19) caused by a novel coronavirus SARS-CoV-2 has rapidly spread to almost every nation in the world. Soon after the pandemic was recognized by epidemiologists, a group of biologists comprising the ARTIC Network, has devised a multiplexed polymerase chain reaction (PCR) protocol and primer set for targeted whole-genome amplification of SARS-CoV-2. The ARTIC primer set amplifies 98 amplicons, which are separated only in two PCRs, across a nearly entire viral genome. The original primer set and protocol showed a fairly small amplification bias when clinical samples with relatively high viral loads were used. However, when sample's viral load was low, several amplicons, especially amplicons 18 and 76, exhibited low coverage or complete dropout. We have determined that these dropouts were due to a dimer formation between the forward primer for amplicon 18, 18_LEFT, and the reverse primer for amplicon 76, 76_RIGHT. Replacement of 76_RIGHT with an alternatively designed primer was sufficient to produce a drastic improvement in coverage of both amplicons. Based on this result, we replaced 12 primers in total in the ARTIC primer set that were predicted to be involved in 14 primer interactions. The resulting primer set, version N1 (NIID-1), exhibits improved overall coverage compared to the ARTIC Network's original (V1) and modified (V3) primer set.

中文翻译：

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分离引物相互作用可通过ARTIC Network的多重PCR改善SARS-CoV-2基因组测序

自2019年12月以来，由新型冠状病毒SARS-CoV-2引起的2019年冠状病毒疾病（COVID-19）已迅速传播到世界几乎每个国家。在大流行病被流行病学家认可后不久，由ARTIC Network组成的一组生物学家就设计出了一种多重聚合酶链反应（PCR）方案和引物组，用于SARS-CoV-2的靶向全基因组扩增。ARTIC引物组可在几乎整个病毒基因组中扩增98个扩增子，仅在两个PCR中分离。当使用具有较高病毒载量的临床样品时，原始引物组和方案显示出相当小的扩增偏差。但是，当样品的病毒载量低时，几个扩增子，尤其是扩增子18和76表现出低覆盖率或完全脱落。我们已经确定这些缺失是由于在扩增子18的正向引物18_LEFT和扩增子76的反向引物76_RIGHT之间形成了二聚体。用另一种设计的引物替换76_RIGHT足以显着提高两个扩增子的覆盖率。基于此结果，我们替换了ARTIC引物中的12个引物，这些引物预计将参与14个引物相互作用。与ARTIC Network的原始（V1）和改良（V3）引物组相比，所得的引物组版本N1（NIID-1）具有更好的总体覆盖率。用另一种设计的引物替换76_RIGHT足以显着提高两个扩增子的覆盖率。基于此结果，我们替换了ARTIC引物中的12个引物，这些引物预计将参与14个引物相互作用。与ARTIC Network的原始（V1）和改良（V3）引物组相比，所得的引物组版本N1（NIID-1）具有更好的总体覆盖率。用另一种设计的引物替换76_RIGHT足以显着提高两个扩增子的覆盖率。基于此结果，我们替换了ARTIC引物中的12个引物，这些引物预计将参与14个引物相互作用。与ARTIC Network的原始（V1）和改良（V3）引物组相比，所得的引物组版本N1（NIID-1）具有更好的总体覆盖率。