Viral-Amplicon-Pipeline

Introduction

This repository details the viral amplicon sequencing pipeline developed by the DAMP Lab for the Connor Lab, both at Boston University. The experimental goal is to extract RNA from viral samples, generate cDNA and amplicons, and prepare the amplicons for sequencing. This is a fully automated process that includes eight automated scripts that were created and validated in the DAMP Lab utilizing Hamilton Microlab STAR liquid handlers. The full Standard Operating Procedures can be found in the Full Pipeline SOPs and Intermediary Steps Document.

Pipeline Steps with Approximate Times

Automated Scripts are linked.

1. Sample Receipt & Aliquot ~20 minutes
2. RNA Extraction ~2 hours and 15 minutes
3. RT and cDNA Generation ~1 hour and 20 minutes
4. Target Amplicon Generation ~5 hours and 10 minutes
5. QC Checkpoint: Gel Electrophoresis ~2 hours and 20 minutes
6. Ampure XP Bead Clean-Up ~2 hours
7. Sample Dilution ~25 minutes
8. Library Preparation ~4 hours and 15 minutes
9. Quantification ~1 hour and 10 minutes
10. Sample Dilution (Optional) ~25 minutes
11. Pooling ~30 minutes
12. QC Checkpoint: TapeStation/Fragment Analysis ~1 hour
13. Sequencing Preparation ~30 minutes
14. Sequencing ~17 hours

Software and Equipment Requirements

1. VENUS 4 (or above)
2. Hamilton Liquid Handler and Appropriate Modules (HHS, CPAC, Magnetic Stands)
3. Thermocycler
4. Plate Reader
5. Agilent TapeStation or Fragment Analyzer

Authors

• Kristen Sheldon
• Courtney Tretheway