conda install -c bioconda minimap2
conda install -c bioconda samtools
conda install -c bioconda bcftools
-
Downloading the genome sequence
fasta
and annotationgff3
of the referenceNC_045512.2
wget -O - "https://eutils.ncbi.nlm.nih.gov/entrez/eutils/efetch.fcgi?db=nuccore&id=NC_045512.2&rettype=fasta" > NC_045512.2.fasta wget -O - "https://ftp.ncbi.nlm.nih.gov/genomes/all/GCF/009/858/895/GCF_009858895.2_ASM985889v3/GCF_009858895.2_ASM985889v3_genomic.gff.gz" | gunzip > NC_045512.2.gff
-
Extract the
Spike gene
sequence from the reference genomesamtools faidx NC_045512.2.fasta NC_045512.2:21563-25384 > NC_045512.2.spike.fasta
-
Map the sequences to the Spike gene sequence then sort and index
minimap2 -ax asm5 NC_045512.2.spike.fasta combined.fasta | samtools sort -o combined.spike.sorted.bam samtools index combined.spike.sorted.bam
-
Convert the
bam
file which contains the sequences aligned to theSpike gene
into afasta
formatsamtools fasta combined.spike.sorted.bam > combined.spike.fasta
- Correct for frame-shift mutations
hyphy pre-msa.bf --reference NC_045512.2.spike.fasta --input combined.spike.fasta CPU=10
- Generate multiple sequence alignment of the spike protein sequence
muscle -align combined.spike.fasta_protein.fas -output combined.spike.fasta_protein.msa -threads 10
- Generate corrected aligned nucleotide sequence of the spike gene
hyphy post-msa.bf --protein-msa combined.spike.fasta_protein.msa --nucleotide-sequences combined.spike.fasta_nuc.fas --output combined.fin.msa CPU=10
- Construct tree using maximum likelihood with
GTR
substitution model,+I+G
invariable site plus discrete Gamma model, and1000 bootstrapping
iqtree2 -s combined.fin.msa -m GTR+I+G -T AUTO -B 1000
- Test using
FUBAR
(Fast, Unconstrained Bayesian AppRoximation)
hyphy fubar --alignment combined.fin.msa --tree combined.fin.msa.treefile CPU=10
5. Viewing of results using Hyphy vision
- Upload/load the
combined.fin.msa.FUBAR.json
file