README.md

• Introduction
• Reference
• Software Requirements
• Analysis Pipeline
• Sample Preparation
• Library Preparation
• Read Preparation and Analysis

Introduction

For a detailed view of the code breakdown and analysis, please visit the GitHub documentation here.

This repository hosts the analysis pipeline and software inputs used to analyze sequencing data generated in the following manuscript (currently available as a preprint):

Reference

M. Stumpf M, Brunetti T, J. Davenport B, K. McCarthy M, E. Morrison T. 2025 (in press). Deep mutationally scanned (DMS) CHIKV E3/E2 virus library maps viral amino acid preferences and predicts viral escape mutants of neutralizing CHIKV antibodies. J. Virol.

Software Requirements

For this analysis:

• fastqc
• multiqc
• cutadapt
• seqtk
• virvarseq
• dms_tools2
• megalogo
• dmslogo
• dms-viz
• R >= v4.2.1

Analysis Pipeline

Diagram 1. Analysis Pipeline Workflow

%%{init: {"theme": "neutral"}}%%

flowchart TB
  A{"Raw FASTQ<br>Files"}
  A --> B["Trim Reads<br><code>CutAdapt</code>"]
  A -.-> C("QC Check<br><code>FastQC</code><br><code>MultiQC</code>")
  B -.-> D("QC Check<br><code>FastQC</code><br><code>MultiQC</code>")
  B --> E["Subsample<br>Reads<br><code>seqtk</code>"]
  E -.-> F("QC Check<br><code>FastQC</code><br><code>MultiQC</code>")
  E --> G["Alignment/<br>Codon Inference"<br><code>VirVarSeq</code>]
  G --> H["Formatting for<br><code>batchdiffsel</code>"]
  G --> I["Calculate<br><code>ndet</code>"]
  G -.-> J("QC Check")
  I --> K["Visualization<br><code>dms-viz</code>"]
  H --> L["Differential<br>Selection<br><code>batchdiffsel</code>"]
  L --> K
  L --> M["Visualization<br><code>dmslogo</code>"]
  I --> N["Visualization<br><code>megaLogo</code>"]

Loading

Sample Preparation

Brief Methods: (For full detailed methods, see Manuscript)

• Viral supernatants were treated with RNase ONE to remove non-virion associated RNAs.
• RNA was isolated from treated supernatants using Trizol and treated with DNase H on-column and aliquoted into 3 x 10 uL aliquots and frozen at -80°C.
• RNA aliquots were thawed and reverse transcribed using SuperScript IV following manufacturer recommendations.
• Full cDNA reaction volume served as a template for an amplicon PCR reaction to amplify the mutagenized CHIKV p62 region for sequencing.
• PCR products were PCR purified and eluted in molecular grade water.

Library Preparation

Completed at the CU Anschutz Genomics Shared Resource

• Libraries were mechanically sheared via Covaris
• Fragments were barcoded using Ovation Ultralow System V2 (Tecan)
• Barcoded libraries were batched and loaded onto an Illumina NovaSeq 6000
• Each sample was sequenced with 2x150bp reads at a depth of 25M paired-end reads (50M pairs)
• Samples were demultiplexed and RAW fastq files delivered

Read Preparation and Analysis

Overview of code breakdown and purpose(s) for execution of each code block.

For a detailed view of the code breakdown and analysis, please visit the GitHub documentation here.

All raw FASTQ files can be found at the following Zenodo DOIs:

https://doi.org/10.5281/zenodo.14269994

https://doi.org/10.5281/zenodo.14510616

https://doi.org/10.5281/zenodo.14510626