TITLE: Unveiling Prolonged COVID Variants: A Protocol for Clustering and Phylogenetic Analysis
AUTHORS: Spoorthi R Kulkarni, Lavanya C, Baishali Garai, Vidya Niranjan

[DESCRIPTION]
Prolonged COVID-19 has emerged as a significant concern globally, with a subset of individuals experiencing persistent symptoms long after the acute phase of the infection. Understanding the genomic basis of prolonged COVID-19 can provide crucial insights into its pathophysiology and aid in the development of targeted therapeutic strategies. In this study, we retrieved genome sequences of COVID-19 from the National Center for Biotechnology Information (NCBI) database and employed a comprehensive pipeline to identify single nucleotide polymorphisms (SNPs). The primary objective was to investigate the clustering similarity of different variants of COVID-19 prevalent in the Indian population. By focusing solely on Indian population genome sequences, we aimed to capture the unique genetic landscape of COVID-19 variants circulating in this demographic. Our analysis revealed distinct SNP patterns across the Indian population, indicative of genetic diversity within the viral strains. Furthermore, we plan to map these identified SNPs to relevant pathways to elucidate their potential functional significance in the context of prolonged COVID-19. 

[STEPS]
SECTION: SAMPLE COLLECTION AND REFERENCE GENOME
1. The genomic sequences utilized in this protocol were sourced from the NCBI Virus Database, which encompasses region-specific genome sequences. Focused on the Indian population, the protocol primarily analyzes sample sequences provided in FASTA format, alongside reference genomes obtained from the NCBI Virus Database.

 





SECTION: REFERENCE GENOME INDEXING
2. Index the reference genome of COVID-19 by executing the 'bwa index' command followed by the name of the FASTA file containing the reference genome. This step facilitates efficient alignment of sequencing reads to the reference genome during subsequent analyses

 

SECTION: ALIGNMENT AND MAPPING
3. The bwa command is used to align the sequencing reads to the indexed reference genome. This command efficiently aligns the reads, taking into account possible mismatches, insertions, deletions, and sequencing errors. SAMtools is a suite of programs for interacting with high-throughput sequencing data in SAM/BAM format.


 

SECTION: ALIGNMENT AND MAPPING
4. 
 

SECTION: VARIANT CALLING AND SNP GENERATION USING SAMtools MPILEUP AND BCFTOOLS CALL
5. This protocol step involves variant calling and single nucleotide polymorphism (SNP) generation using SAMtools mpileup and BCFtools call. The SAMtools mpileup command is used to generate a pileup format file from multiple BAM files aligned to a reference genome. This file contains information about the alignment of sequencing reads to the reference genome at each genomic position. The BCFtools call command then analyzes the pileup data to identify variants, including SNPs, insertions, deletions, and complex variants. The output is generated in variant call format (VCF), providing detailed information about the detected variants, including their genomic coordinates, allele frequencies, and quality scores. This protocol step is crucial for identifying genetic variations and understanding the genomic landscape of the samples under investigation.

 

SECTION: VARIANT GENERATION USING MULTIPLE SEQUENCE ALIGNMENT
6. The Multiple Sequence Alignment (MSA) was conducted using the MAFFT version 7 tool, leveraging the alignment parameters specifically chosen for the analysis. The FASTA files, obtained from earlier downloads, served as input for the MSA procedure. The primary objective of this step was to align the genomic sequences to visualize shared single nucleotide polymorphisms (SNPs) and subsequently generate a phylogenetic tree and similarity score clusters. 

 


 

 


SECTION: VARIANT GENERATION USING MULTIPLE SEQUENCE ALIGNMENT
7. Clustering and Phylogenetic Tree Analysis 

 



SECTION: VARIANT GENERATION USING MULTIPLE SEQUENCE ALIGNMENT
8.