European Journal of Cardiovascular Medicine

SARS-COV 2 responsible for global pandemic, there has been a continuous evolution in the SARS-CoV-2 genome since it was first reported, particularly in the S-gene. It is necessary to monitor the shifts in the SARS-CoV-2 variants. Genome sequencing has emerged as one of the widely used approaches to understand the genetic epidemiology of SARS-CoV-2. The variant Omicron (B.1.1.529) first case was reported on 11 NOV 2021 at Bostwana and it spreads worldwide. WHO designated it as a Variant of concern (VOC) on 26 NOV 2021. In, INDIA first case was reported in Kerala on 12^th DEC 2021. SARS COV-2 is a RNA virus of 30 kb nucleotides that encodes 29 proteins, 23 of them localized in spike protein which bound to mutate and the efforts should be aimed for prevention. In Omicron, 30 mutations occurred in the Spike protein that leading to reduced affinity of antibody binding to S- protein and is 10 times more contagious and has higher chance of reinfection in those who had COVID -19 disease. The mutations on the Omicron variant are widely distributed on multiple proteins of SARS-CoV-2 such as NSP3, NSP4, NSP5, NSP6, NSP12, NSP14, S protein, envelope protein, membrane protein, and nucleocapsid protein. The focus is the mutations on the S protein receptor-binding domain (RBD) for the potential impact on infectivity and antibody resistance caused by this new variant. This is due to the fact that the RBD located on the S protein facilitates the binding between the S protein and the host angiotensin-converting enzyme 2 (ACE2). Though previous VoCs emerged in which natural immunity from COVID-19 infections was common but in this, VoC which has emerged at a time when vaccine immunity is increasing in the world. Omicron has reduced vaccine effectiveness against infection to 33% from 80% for Delta. By PCR, “S” genes dropout or “S” genes failure was observed, hence Sequencing was introduced.

Retrospective study was conducted at State level Viral Research Diagnostic Laboratory  (VRDL) in Department of Microbiology, Guntur Medical College, Guntur. Institutional Ethical committee approval was taken before the commencement of the study.

Study period: 8 months

Sample size & type: 519, RT PCR positive COVID 19 samples

STUDY PROCEDURE: A total of 519 covid-19 Positive samples were selected for sequencing with cycle threshold, CT<30.

In short, viral nucleic acid was extracted using the QIAamp viral RNA mini kit – Manual extraction procedure column based procedure.

• After extraction of RNA, Extracted RNA reaches the Sample preparation room. First strand cDNA synthesis was done using the Go Script reverse transcriptase system by using Elution Prime Fragment 3HC Mix, First stand mix and Reverse Transcriptase.
• Enrichment was done using COVIDSeq illumina PCR Mix, COVIDSeq primer pool 1 and 2 (CPP1 and CPP2, respectively).
• The library was prepared using Illumina COVIDSeq assay kit according to guidelines by using Tagmentation buffer1, Enrichment BLT and Nuclease free water.
• After that post tagmentation clean up was done by using Tagmentation wash buffer and stop Tagment buffer 2
• Then done pool up & clean up the libraries from Tag1 Plate.
• Prepared libraries were purified and pooled and then quantified with a Qubit double-stranded DNA (dsDNA) high-sensitivity (HS) assay kit.
• Libraries were normalized to a 4 nM concentration.
• Pooled normalized libraries were denatured and finally diluted to a 1.2 nM concentration according to the Illumina Miniseq system denature and dilute libraries.
• The libraries were then loaded and run on an Illumina MiniSeq instrument following the standard protocol for 150-bp paired-end reads.
• DRAGEN COVID Lineage app v3.5.8 was used to process and assemble the raw reads as well as for variant calling and lineage determination.
• Finally confirmed results again with ICMR NIV Pune - bioinformatics team

Out of 519 samples , Females were 52% and Males were 48 %. Hospitalized were 62% and quarantined were 38%. Mean age for females were 28 yrs and males were 31 yrs. CT value ranged from 10 to 30 processed of which CT value of 16-20 were 36.8% followed by 21-25 were 31.2% . All 519 samples were omicron positive and  by Next Clade analyzes it showed in order of prevalence 21L omicron -40.26%, 22 B omicron-24%, 22 D omicron – 20.2%, lowest prevalence is 21B Kappa, 21J Delta, 21K Omicron 22C omicron - 0.19%.

Fig 1.Gender Distribution

Table 1 : Age group and gender wise distribution of samples

Table 2 : ORF1ab gene CT value wise distribution of samples

Table 3 : NEXT CLADE analysis

Above table represents the Variants of Concern (VOCs) that have been identified from different samples. The majority of the Variants of Concern (VOCs) detected in this study were found to be mutants of OMICRON-21L Omicron (40.26%) and its subvariants, as well as BA2 and BA.5.2.1, BA5.2.

In order to decrease the spread of pandemic it is very important to identify which VOC is prevalent and responsible for highest mortality and morbidity, and to take health care preventive measures. In VRDL laboratory of Guntur Medical College, Guntur, the whole genomes are sequenced in Covid positive samples with CT value of < 30, to identify different mutational variants.

As per materials and methods mentioned above the Covid positive samples of CT value <30 was processed for extraction of RNA by QIA ampviral RNA minikit.

These are sequenced using an Illumina MiSeq instrument and detected different VOC, pipelines were used to process files, using Next Clade pipelines. total 519 samples were sequenced; Females were 52% and Males were 48 %. Hospitalized were 62% and quarantined were 38%. Mean age for females were 28 yrs and males were 31 yrs. CT value ranged from 10 to 30 processed of which CT value of 16-20 were 36.8% followed by 21-25 were 31.2% . All 519 samples were omicron positive and by Next Clade analyzes it showed in order of prevalence 21L omicron -40.26%, 22 B omicron-24%, 22 D omicron – 20.2%, lowest prevalence is 21B Kappa, 21J Delta, 21K Omicron 22C omicron - 0.19%. The majority of the Variants of Concern (VOCs) detected in this study were found to be mutants of OMICRON-21L Omicron (40.26%) and its subvariants, as well as BA2 and BA.5.2.1, BA5.2.