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2.2 - Assembling short-read Illumina data

Overview

time

  • Teaching: 10 minutes
  • Exercises: 10 minutes

Objectives and Key points

Objectives

  • Perform an assemble of a bacterial genome using the SPAdes assembly tool.

Keypoints

  • The SPAdes genome assembler is a powerful tool for assemnling genoms and contigs from a wide range of sampel types.

Introduction to the SPAdes assembly tool

The SPAdes assembler is a very powerful and popular assembler which utilises de Bruijn graphs to assemble short read sequence data into larger contigs.

SPAdes is particularly good at...

  • De novo assembly of short read sequences (i.e. Illumina or IonTorrent)
  • Assembling small genomes (ideally <100 Mb, such as bacterial, viral, fungal, mitochondrial genomes)

Despite this, it is possible to apply SPAdes to larger genomes and obtain very good results.

code

spades.py -h

Remember to filter your data prior to assembly!

Before beginning to work with SPAdes we need to ensure that our data is free from adapter sequences. The main reason for doing this is when we assemble sequences to form a genome, the assembler is looking for spans of nucleic acid sequence which are common to multiple reads, so that those reads can be joined together to create longer contigs. As the adapter sequence is an identical tag added to every read, these create regions of artificial homology between sequences which have no real connection to each other.

Before we attempt assembly it is critical to remove these from our data but as this was covered in the level 1 training we will not be revisiting the process here.

Performing an assembly using SPAdes

The test data is a set of Illumina MiSeq sequencing reads from an M. bovis genome. To save time, the reads have already been quality filtered with fastp and the number of reads reduced to speed up analysis.

Navigate to the assembly_illumina/ folder and run a basic SPAdes assembly.

code

cd level2/assembly_illumina/

spades.py --isolate --threads 4 \
    -1 reads/Mbovis_87900.miseq_R1.fq.gz \
    -2 reads/Mbovis_87900.miseq_R2.fq.gz \
    -o assembly/

When this job is complete, we will have a folder named assembly/ which contains a lot of different files and pieces of information. A lot of the contents are files generated internally by SPAdes and we do not need to pay attention to them. The most important files for us are:

  1. contigs.fasta - the assembled contigs, as a fasta file.
  2. scaffolds.fasta - the scaffolded contigs, as a fasta file.
  3. This is similar to the contigs file, except it will contain sequences where contigs have been joined by an indeterminate piece of sequence.
  4. This occurs when SPAdes can tell from the pairing information in our library that two contigs belong adjacent to each other, but it has no information for filling the gap between them.
  5. spades.log - the log file of the steps SPAdes performed and any warnings which occurred during assembly.
  6. assembly_graph.fastg - a map of how well the assembly is resolved.
  7. This can be useful if we are trying to obtain a complete genome, as it shows us areas which have assembled cleaning and areas which were difficult to resolve.

Different modes for running SPAdes

SPAdes was originally created as a tool for a very specific use case - the assembly of single-cell amplified genomes, which were problematic to assembly due to the highly uneven coverage of the nucleic acid content in the input libraries (Bankevich et al., 2012). It was also noted at the time that despite this fairly niche scope for use, the tool outperformed many traditional short read assemblers of the time and so over the years the team behind SPAdes have expanded the tools and added a number of alternate assembly modes into successive iterations of the tool.

There are now specific subroutines in the SPAdes assembler for working with the following data types:

  1. isolate - assembly of a single genome (what we used here)
  2. metaSPAdes - assembly of metagenomic samples (sequence libraries from multiple organisms)
  3. plasmidSPAdes - selective assembly of plasmids from genomic libraries
  4. metaplasmidSPAdes - selective assembly of plasmids from metagenomic
  5. rnaSPAdes - de novo assembly of transcriptome libraries
  6. biosyntheticSPAdes - selective assembly of biosynthetic gene clusters (BCG) from a library
  7. rnaviralSPAdes - selective de novo assembly of RNA viruses from transcriptome, metatranscriptome, and metavirome libraries