Summary and Setup

Welcome to the Genome Annotation Workshop

This workshop provides a hands-on introduction to genome annotation, covering structural and functional annotation techniques optimized for the RCAC cluster. You’ll learn best practices for gene prediction using BRAKER3, Helixer, and Easel, as well as functional annotation with EnTAP.

Designed for researchers and bioinformaticians, this workshop will equip you with the skills to generate high-quality, reproducible genome annotations, leveraging transcriptomic, proteomic, and homology-based evidence. You’ll also explore strategies for quality assessment and troubleshooting common challenges in annotation workflows.

Learning Objectives

By the end of this workshop, you will be able to:

  • Set up and run multiple genome annotation pipelines (BRAKER3, Helixer, EASEL) on an HPC cluster
  • Perform functional annotation using EnTAP to assign gene functions, GO terms, and pathway associations
  • Assess annotation quality using BUSCO, OMArk, and structural metrics
  • Compare gene predictions across tools and select the most appropriate approach for your organism

Prerequisites

  • Basic command-line skills (navigating directories, editing files, running commands)
  • An account on the Purdue RCAC cluster (Negishi and Gilbreth)
  • Familiarity with basic genomics concepts

See the Setup page for detailed instructions on data access and software configuration.

Instructors

  1. Arun Seetharam, Ph.D.: Arun is a lead bioinformatics scientist at Purdue University’s Rosen Center for Advanced Computing. With extensive expertise in comparative genomics, genome assembly, annotation, single-cell genomics, NGS data analysis, metagenomics, proteomics, and metabolomics. Arun supports a diverse range of bioinformatics projects across various organisms, including human model systems.

  2. Charles Christoffer, Ph.D.: Charles is a Senior Computational Scientist at Purdue University’s Rosen Center for Advanced Computing. He has a Ph.D. in Computer Science in the area of structural bioinformatics and has extensive experience in protein structure prediction.

Schedule

Time Session
8:30 AM Arrival & Setup
9:00 AM Introduction & Annotation Strategies – Overview of genome annotation, structural vs. functional annotation, key challenges, and selecting the right pipeline
10:30 AM Break
10:40 AM Gene Annotation with BRAKER – Running BRAKER for ab initio and RNA-seq-supported annotation, gene model evaluation
12:00 PM Lunch Break
1:00 PM Interpreting BRAKER Results & Gene Annotation with Helixer – Reviewing BRAKER outputs, refining predictions, and using Helixer for deep-learning-based annotation
2:30 PM Gene Annotation with EASEL – Running the EASEL Nextflow pipeline for automated genome annotation
2:50 PM Break
3:10 PM Functional Annotation with EnTAP & Annotation Quality Assessment – Assigning gene functions, GO term mapping, evaluating completeness with BUSCO, and benchmarking gene models
4:30 PM Wrap-Up & Discussion – Troubleshooting, Q&A, and next steps

What is not covered

  1. Gene prediction using MAKER
  2. Evidence based gene prediction (EviAnn, EVidenceModeler)
  3. Genome assembly
  4. Comparative analyses

Pre-requisites

  1. Basic knowledge of genomics
  2. Basic knowledge of command line interface
  3. Basic knowledge of bioinformatics tools

Data Sets

To copy only data:

BASH 

rsync -avP /depot/workshop/data/annotation_workshop ${RCAC_SCRATCH}/

The worked out folder is available at /depot/workshop/data/annotation_workshop-results on the training cluster. You can copy the data to your scratch space using the following command:

BASH 

rsync -avP /depot/workshop/data/annotation_workshop-results ${RCAC_SCRATCH}/

Only use this if you are unable to finish the exercises in the workshop.

You only need one directory on Gilbreth cluster. See below for details.

BASH 

rsync -avP /depot/workshop/data/annotation_workshop/05_helixer ${RCAC_SCRATCH}/

Software Setup

Discussion

Details

SSH key setup for different systems is detailed in the expandable sections below.

Open a terminal and run:

SH 

ssh-keygen -b 4096 -t rsa
type .ssh\id_rsa.pub | ssh trainXX@negishi.rcac.purdue.edu "mkdir -p ~/.ssh; cat >> ~/.ssh/authorized_keys"

Open Terminal and run

SH 

ssh-keygen -b 4096 -t rsa
cat .ssh/id_rsa.pub | ssh trainXX@negishi.rcac.purdue.edu "mkdir -p ~/.ssh; cat >> ~/.ssh/authorized_keys"

Open a terminal and run:

SH 

ssh-keygen -b 4096 -t rsa
cat .ssh/id_rsa.pub | ssh trainXX@negishi.rcac.purdue.edu "mkdir -p ~/.ssh; cat >> ~/.ssh/authorized_keys"