Introduction

This workshop aims to provide a comprehensive understanding of microbial genome assembly using short-read sequencing data. It showcases the different stages involved in assembling bacterial genomes from raw sequencing data of bacterial isolates, including quality control, genome assembly using a de novo approach, as well as genome annotation and visualisation.

This workshop assumes that attendees have basic knowledge in commandline and R scripting.

Sequencing and genome assembly

The majority of microbial genome assemblies today are based on short-read sequencing platforms such as Illumina, which offer the advantages of high throughput, low cost, and relatively low error rates. However, short reads present challenges in genome assembly due to their limited length and the presence of repetitive regions in genomes. While longer reads (e.g., from PacBio or Oxford Nanopore) can provide better coverage of these regions, short-read sequencing remains the most common and accessible method for routine microbial genome assembly.

De novo vs. reference-guided assembly

De novo genome assembly refers to the process of constructing a genome from scratch without using a reference genome. This approach is particularly useful when working with organisms for which no high quality, closed reference genome is available (which also is quite the norm for microorganisms). The process of de novo assembly relies on the identification of overlap regions on the sequence reads themselves to reconstruct genomes. It is often preferred for microbial genomes when high-quality, non-contaminated sequencing data is available, as it avoids biases that may arise from comparison to reference genomes that would be, for example, phylogenetically too distant.

In contrast, reference-guided assembly involves mapping short-read sequences to an existing reference genome of a closely related species. This method relies on the reference genome to guide the assembly process, making it faster and potentially more accurate, particularly for well-studied organisms.

Genome assembly workflow

Key considerations

There are a few general points that are worth considering when setting up a study involving sequencing and genome assembly from microbial isolates, with emphasis on each point likely influenced by the research question(s):

  • Sequencing read length: “short” read length usually sits between 50 and 300 bp, longer reads can improve assembly contiguity and genome quality.
  • Sequencing depth: number of times each base is read during sequencing. Higher depth reduces sequencing errors and noise, leading to more accurate base calls.
  • Contamination: non-target DNA (e.g., from host or environment) can interfere with the assembly process.

A bit of background on the data

Data used in this workshop was retrieved from Tee et al., 2021: “Genome streamlining, plasticity, and metabolic versatility distinguish co-occurring toxic and nontoxic cyanobacterial strains of Microcoleus” (https://doi.org/10.1128/mBio.02235-21).

This workshop works through sequencing data from 10 isolates of Microcoleus. Species from this benthic cyanobacterial are ubiquitous and form thick mats in freshwater systems, such as rivers, that are sometimes toxic due to the production of potent neurotoxins (anatoxins).

Attribution notice

This workshop was created using materials from BIOSCI701 by Dr. Kim Handley at the University of Auckland.