Comparison of different sequencing techniques for identification of SARS-CoV-2 variants of concern with multiplex real-time PCR
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 eva...
| Main Authors: | , , , , , , , , , , , |
|---|---|
| Format: | Journal article |
| Language: | English |
| Published: |
Public Library of Science
2022
|
| _version_ | 1797110857133457408 |
|---|---|
| author | Ranasinghe, D Jayadas, TTP Jayathilaka, D Jeewandara, C Dissanayake, O Guruge, D Ariyaratne, D Gunasinghe, D Gomes, L Wijesinghe, A Wijayamuni, R Malavige, GN |
| author_facet | Ranasinghe, D Jayadas, TTP Jayathilaka, D Jeewandara, C Dissanayake, O Guruge, D Ariyaratne, D Gunasinghe, D Gomes, L Wijesinghe, A Wijayamuni, R Malavige, GN |
| author_sort | Ranasinghe, D |
| collection | OXFORD |
| description | 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. |
| first_indexed | 2024-03-07T08:00:35Z |
| format | Journal article |
| id | oxford-uuid:7d01367e-eee5-43d4-b17e-48d81052d7fc |
| institution | University of Oxford |
| language | English |
| last_indexed | 2024-03-07T08:00:35Z |
| publishDate | 2022 |
| publisher | Public Library of Science |
| record_format | dspace |
| spelling | oxford-uuid:7d01367e-eee5-43d4-b17e-48d81052d7fc2023-09-26T15:29:35ZComparison of different sequencing techniques for identification of SARS-CoV-2 variants of concern with multiplex real-time PCRJournal articlehttp://purl.org/coar/resource_type/c_dcae04bcuuid:7d01367e-eee5-43d4-b17e-48d81052d7fcEnglishSymplectic ElementsPublic Library of Science2022Ranasinghe, DJayadas, TTPJayathilaka, DJeewandara, CDissanayake, OGuruge, DAriyaratne, DGunasinghe, DGomes, LWijesinghe, AWijayamuni, RMalavige, GNAs 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. |
| spellingShingle | Ranasinghe, D Jayadas, TTP Jayathilaka, D Jeewandara, C Dissanayake, O Guruge, D Ariyaratne, D Gunasinghe, D Gomes, L Wijesinghe, A Wijayamuni, R Malavige, GN Comparison of different sequencing techniques for identification of SARS-CoV-2 variants of concern with multiplex real-time PCR |
| title | Comparison of different sequencing techniques for identification of SARS-CoV-2 variants of concern with multiplex real-time PCR |
| title_full | Comparison of different sequencing techniques for identification of SARS-CoV-2 variants of concern with multiplex real-time PCR |
| title_fullStr | Comparison of different sequencing techniques for identification of SARS-CoV-2 variants of concern with multiplex real-time PCR |
| title_full_unstemmed | Comparison of different sequencing techniques for identification of SARS-CoV-2 variants of concern with multiplex real-time PCR |
| title_short | Comparison of different sequencing techniques for identification of SARS-CoV-2 variants of concern with multiplex real-time PCR |
| title_sort | comparison of different sequencing techniques for identification of sars cov 2 variants of concern with multiplex real time pcr |
| work_keys_str_mv | AT ranasinghed comparisonofdifferentsequencingtechniquesforidentificationofsarscov2variantsofconcernwithmultiplexrealtimepcr AT jayadasttp comparisonofdifferentsequencingtechniquesforidentificationofsarscov2variantsofconcernwithmultiplexrealtimepcr AT jayathilakad comparisonofdifferentsequencingtechniquesforidentificationofsarscov2variantsofconcernwithmultiplexrealtimepcr AT jeewandarac comparisonofdifferentsequencingtechniquesforidentificationofsarscov2variantsofconcernwithmultiplexrealtimepcr AT dissanayakeo comparisonofdifferentsequencingtechniquesforidentificationofsarscov2variantsofconcernwithmultiplexrealtimepcr AT guruged comparisonofdifferentsequencingtechniquesforidentificationofsarscov2variantsofconcernwithmultiplexrealtimepcr AT ariyaratned comparisonofdifferentsequencingtechniquesforidentificationofsarscov2variantsofconcernwithmultiplexrealtimepcr AT gunasinghed comparisonofdifferentsequencingtechniquesforidentificationofsarscov2variantsofconcernwithmultiplexrealtimepcr AT gomesl comparisonofdifferentsequencingtechniquesforidentificationofsarscov2variantsofconcernwithmultiplexrealtimepcr AT wijesinghea comparisonofdifferentsequencingtechniquesforidentificationofsarscov2variantsofconcernwithmultiplexrealtimepcr AT wijayamunir comparisonofdifferentsequencingtechniquesforidentificationofsarscov2variantsofconcernwithmultiplexrealtimepcr AT malavigegn comparisonofdifferentsequencingtechniquesforidentificationofsarscov2variantsofconcernwithmultiplexrealtimepcr |