key: cord-0312261-n1rs3wb5 authors: Umair, M.; Ikram, A.; Rehman, Z.; Haider, S. A.; Badar, N.; Ammar, M.; Ali, Q.; Aahad, A.; Suleman, R.; Khalid, A.; Tariq, A.; Jamal, Z.; Salman, M. title: Detection and upsurge of SARS-CoV-2 Omicron variant in Islamabad Pakistan date: 2022-01-20 journal: nan DOI: 10.1101/2022.01.13.22268997 sha: 37a74e0636961a29d2b6a6d907496c7ebfb51c14 doc_id: 312261 cord_uid: n1rs3wb5 The SARS-CoV-2 omicron variant was first detected in South Africa in November, 2021 and has rapidly spread to more than 90 countries. The emergence of Omicron variant demands for enhanced genomic surveillance to track the mutation profile and spread of virus. In the current study, we have sequenced 15 whole-genome sequences of SARS-CoV-2 Omicron variant from Islamabad region of Pakistan. Among the 15 isolates, 66% were from Islamabad whereas 33% of cases had international travel history of United Kingdom, Maldives, South Africa, and Oman. The detection of Omicron in local community and in travelers highlights the need for rigorous screening at national level and at entry points in order to contain the spread of variant. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first identified in late December 2019, in Wuhan, China [1] . Subsequently, the virus has rapidly spread to several countries leading to an unprecedented pandemic that has disrupted normal life. As of January 9, . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted January 20, 2022. ; https://doi.org/10.1101/2022.01.13.22268997 doi: medRxiv preprint NOTE: This preprint reports new research that has not been certified by peer review and should not be used to guide clinical practice. During COVID-19 (coronavirus disease 2019) pandemic, SARS-CoV-2 has been evolving leading to emergence of different lineages and sub-lineages. Of note are variants of concern (VOCs) that are associated with increased transmissibility or increase in virulence or decreased effectiveness of public health and social measures or vaccines, therapeutics and diagnostics. Before November, UK the variant has spread to more than 90 countries posing a major challenge in the control of the disease. In Pakistan, the first case of Omicron was detected on December 13, 2021 and since then COVID-19 cases have started to increase however, limited data is available on the genomic surveillance of Omicron from the country. The current study aims to explore the genetic epidemiology of Omicron in Islamabad region of Pakistan. The oropharyngeal swab specimens from COVID-19 suspected subjects (n=19,404) were collected during the month of December 2021 who visited the National Institute of Health as part of routine surveillance. . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted January 20, 2022. ; https://doi.org/10.1101/2022.01.13.22268997 doi: medRxiv preprint The MagMAX Viral/Pathogen Nucleic Acid Isolation kit (ThermoFisher Scientific, USA) and the KingFisher Flex instrument (ThermoFisher Scientific, USA) were used for RNA extraction. Clinical RT-PCR testing was performed using TaqPath TM COVID-19 RT-PCR kit (ThermoFisher Scientific, USA) that targets the three genes (ORF1ab, N, and S). In case of suspected Omicron cases, there was failure in amplification of S gene termed as Spike gene target failure (SGTF). The SGTF samples with low cycle threshold values (≤30) were further confirmed for Omicron based on the positive amplification of K417N target and failure of E484K, and P681R mutation targets, using the SNPsig® SARS-CoV-2 (EscapePLEX) kit (PrimerDesign, UK). A subset of confirmed Omicron positive samples were selected for whole genome sequencing. The cDNA synthesis and amplification was performed according to the ARTIC amplicon sequencing protocol (version 2) using SuperScript™ IV VILO™ Master Mix (Invitrogen, USA) and Q5® High-Fidelity 2X Master Mix (New England BioLabs, USA), with the ARTIC nCoV-2019 Panel V3 (Integrated DNA Technologies, Inc, USA) [4] . The paired-end sequencing library (2x150 bp) was prepared from the generated amplicons using the Illumina DNA Prep Kit (Illumina, Inc, USA) by following the standard protocol. The prepared libraries were pooled and subjected to sequencing on Illumina platform, iSeq using sequencing reagent, iSeq 100 i1 Reagent v2 (300-cycle) (Illumina, Inc, USA). The Fastq files were processed for quality assessment using the FastQC tool (v0.11.9) [5] . Trimmomatic (v0.39) [6] was employed to eliminate artifacts and technical biases by removing adapter sequences and low-quality base calls (< 30). The filtered reads were aligned using the . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted January 20, 2022. ; https://doi.org/10.1101/2022.01.13.22268997 doi: medRxiv preprint Burrows-Wheeler Aligner's (BWA, v0.7.17) [7] and available reference genome (Wuhan-Hu-1, GISAID ID: EPI_ISL_402125). According to Centers for Disease Control and Prevention (CDC, USA) guidelines, variants were identified and consensus sequences for all genomes were generated [8] . Pangolin v3.1.17 and pangoLEARN model dated 06-12-2021 were used to classify the assembled genomes into PANGO lineages [9] . The phylogenetic analysis of the study isolates was performed using the Ultafast Sample Placement of Existing Trees (UShER). It rapidly places the study samples onto existing phylogenetic tree of SARS-CoV-2 genomes reported on GISAID, NCBI, COG-UK, and CNBC using maximum parsimony method. The phylogenetic tree was constructed using the updated version on December 31, 2021. The resulting tree was visualized using Auspice. The male to female ratio was 2:3 with 40% males and 60% females With reference to Omicron variant circulation within the city, 66% (n=10) of the Omicron infected samples were from the local resident of Islamabad while 33% (n=5) were identified from incoming passengers. The inbound passengers infected with omicron had travel history with two from UK (GISAID IDs: . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted January 20, 2022. All the authors declared no conflict of interest. All the sequences generated in the current study are submitted to the GISAID that are available at "https://www.gisaid.org/login/" under the accession numbers: EPI_ISL_8240498, EPI_ISL_8240500-EPI_ISL_8240513. 10. https://covid.gov.pk/. Date accessed: January 08, 2022 . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted January 20, 2022. ; https://doi.org/10.1101/2022.01.13.22268997 doi: medRxiv preprint Figure 2 : Phylogenetic tree of 15 Omicron isolates generated using USHER. The current study isolates as highlighted in red color. . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted January 20, 2022. ; https://doi.org/10.1101/2022.01.13.22268997 doi: medRxiv preprint Phylogenetic analysis of nCoV-2019 genomes nCoV-2019 sequencing protocol v2 (GunIt) V. 2. Protocols. io FastQC: a quality control tool for high throughput sequence data Trimmomatic: a flexible trimmer for Illumina sequence data Fast and accurate long-read alignment with Burrows-Wheeler transform Rapid, sensitive, full-genome sequencing of severe acute respiratory syndrome coronavirus 2 Pangolin: lineage assignment in an emerging pandemic as an epidemiological tool