Studying the impact of NTD mutations on SARS-CoV-2 spike expression using deep mutational scanning

Dependencies

• Python (version 3.9)
• Biopython
• R (version 4.1)
• PEAR
• ProDy

Input files

• ./Fasta/SARS2-NTD.fa: NTD sequences with 21 nt upstream (5' flank)
• ./Fasta/Amplicon.fa: Amplicon sequences for NGS (including recovery primer regions)
• ./Fasta/NTD_ref.fa: Reference (i.e. wild type) amino acid sequences (primer regions not included)
• ./PDB/6zge.pdb: Spike cryoEM structure from Wrobel et al. (2020)
• ./PDB/6zge_RBD.pdb: RBD only from the Spike cryoEM structure from Wrobel et al. (2020)
• ./PDB/7b62.pdb: NTD crystal structure from Rosa et al. (2021)
• ./PDB/spike_with_complete_NTD.pdb: 6zge with NTD in chain A being replaced by 7b62 (chain X)
• ./data/ASA.table: Maximum accessible surface area for individual amino acids from Tien et al. (2013)
• ./data/RBD_DMS_data.csv: DMS data of RBD from Chan et al. (2021)
• Raw read files in fastq format from NIH SRA database BioProject PRJNA792013

Primer design for DMS library construction

1. Generating foward (NNK + internal barcode) and reverse primers (constant)
   python3 script/lib_primer_design.py

   • Input file:
     • ./Fasta/SARS2-NTD.fa
   • Output files:
     • ./primer/SARS2-NTD_lib_Fprimer_bc.fa
     • ./primer/SARS2-NTD_lib_Rprimer_bc.fa

2. Generating barcode file
   python3 script/check_barcode.py

   • Input files:
     • ./primer/SARS2-NTD_lib_Fprimer_bc.fa
     • ./Fasta/Amplicon.fa
   • Output file:
     • ./data/barcodes.tsv

Calculating experssion score from DMS data

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_merged/ folder and named as described in ./doc/filename_merged_fastq.txt

2. Counting variants based on nucleotide sequences
   python3 script/NTD_fastq2count.py

   • Input files:
     • Merged read files in fastq_merged/ folder
   • Output files:
     • result/NTD_DMS_count_nuc_A.tsv
     • result/NTD_DMS_count_nuc_B.tsv

3. Convert nucleotide sequences to amino acid mutations
   python3 script/NTD_count_nuc2aa.py

   • Input files:
     • ./data/barcodes.tsv
     • ./Fasta/NTD_ref.fa
     • result/NTD_DMS_count_nuc_A.tsv
     • result/NTD_DMS_count_nuc_B.tsv
   • Output files:
     • result/NTD_DMS_count_aa_A.tsv
     • result/NTD_DMS_count_aa_B.tsv

4. Compute expression score
   python3 script/NTD_count2score.py

   • Input files:
     • result/NTD_DMS_count_aa_A.tsv
     • result/NTD_DMS_count_aa_B.tsv
   • Output file:
     • ./result/NTD_DMS_scores.tsv
     • ./result/NTD_DMS_scores_by_resi.tsv

5. Plot correlation between replicates as quality control
   Rscript script/plot_QC.R

   • Input file:
     • ./result/NTD_DMS_scores.tsv
   • Output files:
     • ./graph/QC_replicate_exp.png
     • ./graph/Exp_by_class.png
     • ./result/NTD_DMS_expression_score.tsv Rscript script/plot_QC_mean_exp_by_replicates.R
   • Input file:
     • ./result/NTD_DMS_scores_by_resi.tsv
   • Output files:
     • ./graph/QC_replicate_mean_exp.png Rscript script/QC_bin0_vs_bin3_counts.R
   • Input file:
     • ./result/NTD_DMS_scores.tsv
   • Output files:
     • ./graph/bin0_bin_3_ratio_rep_1_all.png
     • ./graph/bin0_bin_3_ratio_rep_2_all.png Rscript script/QC_bin0_vs_bin3_biomodality.R
   • Input file:
     • ./result/NTD_DMS_scores.tsv
   • Output files:
     • ./graph/bin0_by_replicates.png
     • ./graph/bin3_by_replicates.png

6. Plot heatmap for input frequencies of individual mutations Rscript script/plot_input_freq_heatmap.R

   • Input file:
     • ./result/NTD_DMS_scores.tsv
   • Ouput file:
     • ./graph/NTD_input_freq_heatmap.png

7. Plot heatmap for the expression scores of individual mutations
   Rscript script/plot_score_heatmap.R

   • Input file:
     • ./result/NTD_DMS_scores.tsv
   • Ouput file:
     • ./graph/NTD_exp_heatmap.png

8. Plot mutational tolerability in loops vs others
   Rscript script/NTD_loop_vs_other_residues.R

   • Input file:
     • ./result/NTD_DMS_scores_by_resi.tsv
   • Output file:
     • ./graph/ntd_loop_vs_other_residues.png

9. Plot the mutational tolerability in selected hotspot regions Rscript script/Hot_spots_vs_other_residues.R

   • Input file:
     • ./result/NTD_DMS_scores_by_resi.tsv
   • Output file:
     • ./graph/hot_spots_vs_other_residues.png

Structural analysis

1. Computing relative solvent accessibility (RSA) for individual residues
   python3 script/RSA_analysis.py

   • Input files:
     • ./PDB/7b62.pdb
     • ./PDB/6zge.pdb
     • ./data/ASA.table
   • Ouput file:
     • ./result/NTD_RSA.tsv

2. Compute expression score for RBD DMS data Rscript script/compute_exp_score_RBD.R

   • Input file:
     • ./data/RBD_DMS.csv
   • Output file:
     • ./data/RBD_DMS_exp.tsv

3. Compute RSA for RBD residues python3 script/RBD_analysis.py

   • Input files:
     • ./data/RBD_DMS_exp.tsv
     • ./PDB/6zge_RBD.pdb
     • ./data/ASA.table
   • Output file:
     • ./result/RBD_exp_RSA.tsv

4. Computing the distance of individual NTD residues to RBD or S2
   python3 script/Dist_analysis.py

   • Input file:
     • ./PDB/spike_with_complete_NTD.pdb
   • Output file:
     • ./result/Dist_NTD_to_RBD_S2.tsv

5. Replace the B-factor by expression score in the PDB file
   python3 script/Bfactor_to_score.py

   • Input file:
     • ./PDB/7b62.pdb
     • ./result/NTD_DMS_scores_by_resi.tsv
   • Output file:
     • ./PDB/7b62_exp.pdb

6. Plot mean expression score vs RSA for individual residues
   Rscript script/plot_RSA.R

   • Input file:
     • ./result/NTD_DMS_scores_by_resi.tsv
     • ./result/NTD_RSA.tsv
   • Output file:
     • ./graph/Exp_vs_RSA.png

7. Plot mutational tolerability vs RSA for RBD DMS data
   Rscript script/Mean_expression_score_in_mammalian_system.R

   • Input file:
     • ./result/RBD_DMS_data.csv
   • Output file:
     • ./result/RBD_exp_RSA.tsv Rscript script/plot_RSA_RBD.R
   • Input file:
     • ./result/RBD_exp_RSA.tsv
   • Output file:
     • ./graph/Exp_vs_RSA_RBD.png

8. Plot mutational tolerability vs distance to RBD/S2 for individual residues and categorized by antibody epitopes
   Rscript script/Dist_to_RBD_S2_exp_by_Ab.R

   • Input file:
     • ./result/NTD_DMS_scores_by_resi.tsv
     • ./result/Dist_NTD_to_RBD_S2.tsv
   • Output file:
     • ./graph/Exp_vs_dist.png
     • ./graph/antibody_epi_vs_mean_exp.png Rscript script/antibody_epitopes_vs_distances.R
   • Input file:
     • ./result/NTD_DMS_scores_by_resi.tsv
     • ./result/Dist_NTD_to_RBD_S2.tsv
   • Output file:
     • ./graph/Exp_vs_dist.png
     • ./graph/antibody_epitopes_vs_distance.png

9. Plot mutational tolerability vs sequence conservation for individual residues
   Rscript script/align_freq_vs_score.R

   • Input files:
     • ./result/NTD_DMS_scores_by_resi.tsv
     • ./result/residue_freq.csv
   • Output file:
     • ./graph/dms_expression_vs_seq_conservation.png

10. Plot mutational tolerability vs sequence conservation for individual residues
    Rscript script/RSA_vs_alignment_frequency.R

    • Input files:
      • ./result/residue_freq.csv
      • ./result/NTD_RSA.tsv
    • Output files:
      • ./graph/RSA_vs_seq_con.png

11. Visualizing the mutational tolerability on the S protein structure Pymol script/plot_Bfactor_as_exp.pml

    • Input files:
      • ./PDB/7b62_exp.pdb
      • ./PDB/6zge.pdb
    • Output files:
      • ./PDB/overlay.png
      • ./PDB/Overlay_closeup_0.png
      • ./PDB/Overlay_closeup_180.png
      • ./PDB/Overlay_closeup_topdown.png
      • ./PDB/circulating_mutation_1.png
      • ./PDB/circulating_mutation_2.png
      • ./PDB/circulating_mutation_3.png
      • ./PDB/circulating_indel_1.png
      • ./PDB/circulating_indel_2.png
      • ./PDB/circulating_indel_3.png

12. Analysis of the ciculating NTD mutations/indels among 17 major variants Rscript script/NTD_circulating_mutation_vs_other_residues.R

    • Input files:
      • ./result/NTD_DMS_scores.tsv
      • ./result/NTD_DMS_scores_by_resi.tsv
    • Output files:
      • ./graph/ntd_circulating_mutations_vs_other_mutations.png
      • ./graph/ntd_indel_sites_vs_other_sites.png

13. Mutational tolerability of selected regions compared to other residues Rscript script/NTD_loop_vs_other_residues.R

    • Input files:
      • ./result/NTD_DMS_scores_by_resi.tsv
    • Output files:
      • ./graph/NTD_loop_vs_other_residues.png Rscript script/Hot_spots_vs_other_residues.R
    • Input files:
    • ./result/NTD_DMS_scores_by_resi.tsv
    • Output files:
    • ./graph/hot_spots_vs_other_residues.png