Comparison of different sequencing techniques for identification of SARS-CoV-2 variants of concern with multiplex real-time PCR.
Comparison of different sequencing techniques for identification of SARS-CoV-2 variants of concern with multiplex real-time PCR.
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DOI：
10.1371/journal.pone.0265220
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发表时间：
2022
期刊：
影响因子：
3.7
通讯作者：
Malavige GN
中科院分区：
文献类型：
--
作者：
Ranasinghe D;Jayadas TTP;Jayathilaka D;Jeewandara C;Dissanayake O;Guruge D;Ariyaratne D;Gunasinghe D;Gomes L;Wijesinghe A;Wijayamuni R;Malavige GN
随着不同SARS-CoV-2变异体的出现以及delta变异体亚系的不断演变，至关重要的是所有国家对其至少>1%的感染进行测序，以检测具有更高传播性和逃避免疫能力的变异体的出现。然而，由于资源有限，因为许多资源贫乏的国家无法对足够数量的病毒进行测序，我们比较了两步市售多重实时PCR测定法检测与变体相关的重要单核苷酸多态性（SNP）的有用性，并比较了Illumina测序平台和Oxford Nanopore Technologies（ONT）平台的灵敏度、准确度和成本效益。由于多重真实的时间PCR确定的谱系定义SNP的存在，138/143（96.5%）鉴定为α变体，36/39（92.3%）鉴定为δ变体的样品通过两个测序平台中的任一个分配到相同的谱系。通过ONT测序的37个样品中有34个具有<5%的模糊碱基，而使用Illumina测序的37个样品中有21个产生<5%的模糊碱基。然而，Illumina读数的平均PHRED评分平均为32.35，而ONT为10.78。这种差异导致ONT的碱基错误概率为1/10，Illumina测序平台的碱基错误概率为1/1000。亚共有单核苷酸变异（SNV）在两个平台之间高度相关（R2 = 0.79），而插入缺失似乎具有较弱的相关性（R2 = 0.13）。虽然ONT与Illumina技术相比具有略高的错误率，但它以较低的读数实现了较高的覆盖率，产生较少的模糊碱基，并且比Illumina测序技术便宜得多。
As different SARS-CoV-2 variants emerge and with the continuous evolvement of sub lineages of the delta variant, it is crucial that all countries carry out sequencing of at least >1% of their infections, in order to detect emergence of variants with higher transmissibility and with ability to evade immunity. However, due to limited resources as many resource poor countries are unable to sequence adequate number of viruses, we compared to usefulness of a two-step commercially available multiplex real-time PCR assay to detect important single nucleotide polymorphisms (SNPs) associated with the variants and compared the sensitivity, accuracy and cost effectiveness of the Illumina sequencing platform and the Oxford Nanopore Technologies’ (ONT) platform. 138/143 (96.5%) identified as the alpha and 36/39 (92.3%) samples identified as the delta variants due to the presence of lineage defining SNPs by the multiplex real time PCR, were assigned to the same lineage by either of the two sequencing platforms. 34/37 of the samples sequenced by ONT had <5% ambiguous bases, while 21/37 samples sequenced using Illumina generated <5%. However, the mean PHRED scores averaged at 32.35 by Illumina reads but 10.78 in ONT. This difference results in a base error probability of 1 in 10 by the ONT and 1 in 1000 for Illumina sequencing platform. Sub-consensus single nucleotide variations (SNV) are highly correlated between both platforms (R2 = 0.79) while indels appear to have a weaker correlation (R2 = 0.13). Although the ONT had a slightly higher error rate compared to the Illumina technology, it achieved higher coverage with a lower number or reads, generated less ambiguous bases and was significantly less expensive than Illumina sequencing technology.
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影响因子：
3.7
作者：
Harper H;Burridge A;Winfield M;Finn A;Davidson A;Matthews D;Hutchings S;Vipond B;Jain N;COVID-19 Genomics UK (COG-UK) Consortium;Edwards K;Barker G
通讯作者：
Barker G
影响因子：
16.6
作者：
Dyson L;Hill EM;Moore S;Curran-Sebastian J;Tildesley MJ;Lythgoe KA;House T;Pellis L;Keeling MJ
通讯作者：
Keeling MJ
影响因子：
10.7
作者：
Nguyen LT;Schmidt HA;von Haeseler A;Minh BQ
通讯作者：
Minh BQ
影响因子：
5.8
作者：
Liu T;Chen Z;Chen W;Chen X;Hosseini M;Yang Z;Li J;Ho D;Turay D;Gheorghe CP;Jones W;Wang C
通讯作者：
Wang C
影响因子：
9.4
作者：
Tchesnokova V;Kulasekara H;Larson L;Bowers V;Rechkina E;Kisiela D;Sledneva Y;Choudhury D;Maslova I;Deng K;Kutumbaka K;Geng H;Fowler C;Greene D;Ralston J;Samadpour M;Sokurenko E
通讯作者：
Sokurenko E