Evolutionary contingency in human H3N2 influenza neuraminidase

This README describes the deep sequencing analysis in:
Prevalence and mechanisms of evolutionary contingency in human influenza H3N2 neuraminidase

Contents

• Identifying permissive mutations for N387K
• Identifying permissive mutations for N336H

Dependencies

• Python3
• PEAR
• BioPython
• Distance
• R

Input files

• ./Fasta/SD93_mutlib_ref.fa: Reference sequence for SD93 mutant library
• ./Fasta/Bil69_mutlib_ref.fa: Reference sequence for Bil69 mutant library
• Raw read files in fastq format from NIH SRA database BioProject PRJNA790468

Identifying permissive mutations for N387K

1. Merge overlapping paired-end reads using PEAR
   pear -f [FASTQ FILE FOR FORWARD READ] -r [FASTQ FILE FOR FORWARD READ] -o [OUTPUT FASTQ FILE]

   • Output files should be placed in the fastq/ folder and named as:
     • SD93_ipt.assembled.fastq for input library
     • SD93_rep1.assembled.fastq for post-passaged library (replicate 1)
     • SD93_rep2.assembled.fastq for post-passaged library (replicate 2)

2. Count variants from fastq files
   python3 script/SD93_NA_fastq2count.py

   • Input files:
     • ./Fasta/SD93_mutlib_ref.fa
     • fastq/SD93_ipt.assembled.fastq
     • fastq/SD93_rep1.assembled.fastq
     • fastq/SD93_rep2.assembled.fastq
   • Output file:
     • ./result/SD93_MultiMutLib.tsv

3. Filtering low count variants
   python3 script/SD93_NA_filter.py

   • Input file:
     • ./result/SD93_MultiMutLib.tsv
   • Output file:
     • ./result/SD93_MultiMutLib_filtered.tsv

4. Plot post-passaged frequency of each variant
   Rscript script/SD93_NA_plot_freq.R

   • Input file:
     • ./result/SD93_MultiMutLib_filtered.tsv
   • Output file:
     • ./graph/SD93_mutlib_rep_compare.png

Identifying permissive mutations for N336H

1. Merge overlapping paired-end reads
   pear -f [FASTQ FILE FOR FORWARD READ] -r [FASTQ FILE FOR FORWARD READ] -o [OUTPUT FASTQ FILE]

   • Output files should be placed in the fastq/ folder and named as:
     • Bil69_ipt.assembled.fastq for input library
     • Bil69_rep1.assembled.fastq for post-passaged library (replicate 1)
     • Bil69_rep2.assembled.fastq for post-passaged library (replicate 2)

2. Compute enrichment value for each variant from fastq files
   python3 script/Bil69_NA_fastq2enrich.py

   • Input files:
     • ./Fasta/Bil69_mutlib_ref.fa
     • fastq/Bil69_ipt.assembled.fastq
     • fastq/Bil69_rep1.assembled.fastq
     • fastq/Bil69_rep2.assembled.fastq
   • Output files:
     • ./result/Bil69_MultiMutLib.tsv

3. Filter irrelevant variants
   python3 script/Bil69_NA_filter.py

   • Input file:
     • ./result/Bil69_MultiMutLib.tsv
   • Output file:
     • ./result/Bil69_MultiMutLib_filtered.tsv

4. Plot enrichment of each variant
   Rscript script/Bil69_NA_plot_enrich.R

   • Input file:
     • ./result/Bil69_MultiMutLib.tsv
   • Output file:
     • ./graph/Bil69_mutlib_rep_compare.png