abulenciamiguel / hyphy_tohoku

Positive selection analysis using Hyphy

Positive selection analysis using Hyphy

Install the tools needed

conda install -c bioconda minimap2
conda install -c bioconda samtools
conda install -c bioconda bcftools

1. Extraction of sequences of a specific gene

• Downloading the genome sequence fasta and annotation gff3 of the reference NC_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 genome

  samtools 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 the Spike gene into a fasta format

  samtools fasta combined.spike.sorted.bam > combined.spike.fasta
  

2. Codon-aware Multiple Sequence Alignment

• 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

3. Phylogenetic tree construction: Maximum Likelihood method

• Construct tree using maximum likelihood with GTR substitution model, +I+G invariable site plus discrete Gamma model, and 1000 bootstrapping

iqtree2 -s combined.fin.msa -m GTR+I+G -T AUTO -B 1000

4. Selection analysis

• 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