Module 1: Genome Assembly

Genome assembly is the process of reconstructing a genome sequence from the short or long DNA fragments produced by sequencing machines. Because no current technology can read an entire chromosome in one pass, sequencers produce millions of overlapping reads that must be computationally stitched back together.

Short Reads vs Long Reads

Two major paradigms exist for sequencing and assembly:

Feature Short Reads (Illumina) Long Reads (PacBio / ONT)
Read length 150 – 300 bp 10 kb – 100+ kb
Error rate Very low (~0.1%) Higher (~1–5%) raw error
Repeat resolution Difficult Excellent
Cost per base Low Higher
Best for SNP calling, small genomes Structural variants, repeat-rich genomes

Learning Objectives

  • Understand the difference between short-read and long-read assembly strategies
  • Run a short-read assembly using Velvet/SPAdes in Galaxy
  • Run a long-read assembly using Flye in Galaxy
  • Assess assembly quality with QUAST and BUSCO

Hands-on Tutorials

Hands-on 1 — Short Read Assembly

Using Xanthomonas oryzae pv. oryzae Illumina reads as a model organism.

Galaxy Training Network tutorial: General Introduction to Assembly

Key tools used:

  • Velvet / SPAdes — de novo assemblers for short reads
  • QUAST — assembly quality statistics
  • BUSCO — benchmarking assembly completeness

Hands-on 2 & 3 — Long Read Assembly (PacBio)

Using a subset of rice (Oryza sativa) chromosome 9 PacBio data.

Galaxy Training Network tutorial: Genome assembly using PacBio data

Key tools used:

  • Flye — de novo assembler for single-molecule long reads
  • QUAST — assembly quality statistics
  • BUSCO — completeness assessment

Key Concepts

N50 — A summary statistic for assembly quality. Half of the total assembled bases are in contigs of N50 length or longer. A higher N50 generally indicates a more contiguous assembly.

Contig vs Scaffold — A contig is a contiguous assembled sequence with no gaps. A scaffold is a higher-order structure where contigs are ordered and oriented using additional information (e.g., paired-end reads, Hi-C), with gaps filled by Ns.

Polishing — For long-read assemblies with higher raw error rates, a polishing step uses high-accuracy short reads to correct base-level errors in the draft assembly.

Useful Resources