MOLT4 Mutation Analysis and Peptide Extraction Pipeline

Overview

This pipeline processes mutation data for the MOLT4 cell line. It performs:

1. Filtering of missense variants with valid UniProt IDs
2. Fetching canonical protein sequences via the UniProt API
3. Applying amino acid mutations to sequences
4. Generating forward and reverse protein sequences
5. Extracting peptides around mutation sites (using K/R cleavage logic)
6. Saving annotated outputs in FASTA and CSV formats
7. Verifying peptide correctness through position and boundary checks

Dependencies

Ensure the following R packages are installed:

install.packages(c("tidyverse", "httr", "stringr", "readr", "knitr"))

Input

Required File:
MOLT4 mutations.csv

Required Columns (case-insensitive):

• Uniprot ID: UniProt accession (e.g., P12345)
• Variant Info: Must include the string "missense_variant"
• Protein.Change: Mutation notation (e.g., p.P750Q)
• Gene: Gene symbol (e.g., TP53)

Output

File Name	Description
MOLT4_mutations_with_sequences.csv	Filtered entries + canonical sequences
MOLT4_analysis_summary.csv	Summary of processing/filtering stats
MOLT4_mutated_protein_output.csv	Mutated forward/reverse protein sequences + metadata
MOLT4_mutated_protein_database.fasta	FASTA-formatted protein sequences (mutated)
MOLT4_mutated_peptide_database.fasta	FASTA-formatted peptides around mutations
MOLT4_peptide_data.csv	Peptide details and positional metadata

Usage Instructions

1. Place MOLT4 mutations.csv in your working directory
2. Open the R script or R Markdown file
3. Run all code blocks from top to bottom (recommended: RStudio)
4. Output files will be saved to your working directory

How the Code Works

1. Canonical Sequence Retrieval

• Filters for rows with valid UniProt ID and missense_variant
• Fetches the canonical FASTA sequence from UniProt API
• Adds it to the dataset

2. Mutation Application

• Parses mutations (e.g., p.P750Q)
• Replaces the amino acid at the given position in the sequence
• Validates original amino acid at the target site

3. Reverse Sequence Generation

• Generates reverse of mutated sequence

4. Peptide Extraction

• Locates the second K/R residue upstream and downstream of the mutation
• Extracts peptide sequence from that window, inclusive of the K/R
• Applied to both forward and reverse sequences

5. Verification

• Checks boundary correctness of extracted peptides
• Validates mutation position
• Ensures the mutation is reflected in the peptide

FASTA Header Example (forward strand) for protein database:

Fwd_spP750Q|Q5SV97-1|PERM1_P750Q OS=Homo sapiens GN=PERM1P750Q (Sequence)

FASTA Header Example (forward strand) for peptide database:

Fwd_spP750Q|Q5SV97-1|PERM1_P750Q OS=Homo sapiens GN=PERM1 (Truncated Sequence)