Unveiling Prolonged COVID Variants: A Protocol for Clustering and Phylogenetic Analysis

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.

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

#Indexing the reference genome 
bwa index Reference_genome.fasta

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.

#Aligning the reference genome with sample genome 
bwa mem Reference_genome.fasta sample_ID.fasta > sample_ID.sam

#SAM to BAM conversion using SAM tools 
samtools view -@ 4 -Sb -o sample_ID.bam sample_ID.sam

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.

#VCF generation  
samtools mpileup -uf Reference_genome.fasta sample_ID.bam | bcftools call -O v -mv -o 1_output.vcf

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.

#MAFFT version 7 web server  
https://mafft.cbrc.jp/alignment/server/

Citation

As depicted in the image, the acquired FASTA files can be uploaded by opting for the designated "choose File" button, and then selecting the alignment parameter located in the upper-left corner of the web server interface.

Clustering and Phylogenetic Tree Analysis

Citation