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name: bio-genome-assembly-short-read-assembly description: De novo genome assembly from Illumina short reads using SPAdes. Covers bacterial, fungal, and small eukaryotic genome assembly, as well as metagenome and transcriptome assembly modes. Use when assembling genomes from Illumina reads. tool_type: cli primary_tool: SPAdes

Short-Read Assembly

Assemble genomes from Illumina paired-end or single-end reads using SPAdes.

SPAdes Overview

SPAdes (St. Petersburg genome Assembler) uses de Bruijn graph approach with multiple k-mer sizes for robust assembly.

Installation

Copyconda install -c bioconda spades

Basic Usage

Paired-End Assembly

Copyspades.py -1 R1.fastq.gz -2 R2.fastq.gz -o output_dir

Single-End Assembly

Copyspades.py -s reads.fastq.gz -o output_dir

With Unpaired Reads

Copyspades.py -1 R1.fastq.gz -2 R2.fastq.gz -s unpaired.fastq.gz -o output_dir

Assembly Modes

Isolate Mode (Default for Bacteria)

Copyspades.py --isolate -1 R1.fq.gz -2 R2.fq.gz -o isolate_assembly

Best for single-organism isolates with uniform coverage.

Careful Mode

Copyspades.py --careful -1 R1.fq.gz -2 R2.fq.gz -o careful_assembly

Reduces misassemblies at cost of speed. Recommended for small genomes.

Meta Mode (Metagenomes)

Copyspades.py --meta -1 R1.fq.gz -2 R2.fq.gz -o meta_assembly

For mixed microbial communities with varying coverage.

RNA Mode (Transcriptomes)

Copyspades.py --rna -1 R1.fq.gz -2 R2.fq.gz -o rna_assembly

Assembles transcripts from RNA-seq data.

Plasmid Mode

Copyspades.py --plasmid -1 R1.fq.gz -2 R2.fq.gz -o plasmid_assembly

Extracts plasmid sequences from bacterial isolates.

Key Options

Multiple Libraries

Paired Libraries with Different Insert Sizes

Copyspades.py \
    --pe1-1 short_R1.fq.gz --pe1-2 short_R2.fq.gz \
    --pe2-1 long_R1.fq.gz --pe2-2 long_R2.fq.gz \
    -o output_dir

With Mate Pairs

Copyspades.py \
    --pe1-1 paired_R1.fq.gz --pe1-2 paired_R2.fq.gz \
    --mp1-1 mate_R1.fq.gz --mp1-2 mate_R2.fq.gz \
    -o output_dir

With PacBio/Nanopore (Hybrid)

Copyspades.py \
    -1 illumina_R1.fq.gz -2 illumina_R2.fq.gz \
    --pacbio pacbio.fq.gz \
    -o hybrid_assembly

# Or with Nanopore
spades.py \
    -1 illumina_R1.fq.gz -2 illumina_R2.fq.gz \
    --nanopore nanopore.fq.gz \
    -o hybrid_assembly

K-mer Selection

Auto Selection (Recommended)

SPAdes automatically selects appropriate k-mers based on read length.

Manual K-mer Specification

Copy# For 150bp reads
spades.py -k 21,33,55,77 -1 R1.fq.gz -2 R2.fq.gz -o output

# For 250bp reads
spades.py -k 21,33,55,77,99,127 -1 R1.fq.gz -2 R2.fq.gz -o output

Output Files

Copyoutput_dir/
├── scaffolds.fasta     # Final scaffolds (use this)
├── contigs.fasta       # Contigs before scaffolding
├── assembly_graph.gfa  # Assembly graph
├── spades.log          # Log file
├── params.txt          # Parameters used
└── K*/                 # Intermediate k-mer assemblies

Scaffold FASTA Headers

Copy>NODE_1_length_500000_cov_50.5

• NODE_1 - Contig/scaffold ID
• length_500000 - Sequence length
• cov_50.5 - Average k-mer coverage

Memory and Performance

Reduce Memory Usage

Copy# Limit memory to 32GB
spades.py -m 32 -1 R1.fq.gz -2 R2.fq.gz -o output

# Use fewer threads
spades.py -t 8 -1 R1.fq.gz -2 R2.fq.gz -o output

Resume Interrupted Assembly

Copyspades.py --continue -o output_dir

Skip Error Correction

Copy# If reads already corrected
spades.py --only-assembler -1 R1.fq.gz -2 R2.fq.gz -o output

Complete Workflows

Bacterial Genome Assembly

Copy#!/bin/bash
set -euo pipefail

R1=$1
R2=$2
OUTDIR=$3
THREADS=${4:-16}

echo "=== Bacterial Genome Assembly ==="

# Run SPAdes in isolate mode
spades.py \
    --isolate \
    --careful \
    -t $THREADS \
    -1 $R1 -2 $R2 \
    -o $OUTDIR

# Basic stats
echo "Assembly statistics:"
grep -c "^>" ${OUTDIR}/scaffolds.fasta
seqkit stats ${OUTDIR}/scaffolds.fasta

Metagenome Assembly

Copy#!/bin/bash
set -euo pipefail

R1=$1
R2=$2
OUTDIR=$3

spades.py \
    --meta \
    -t 32 \
    -m 200 \
    -1 $R1 -2 $R2 \
    -o $OUTDIR

echo "Metagenome assembly complete: ${OUTDIR}/scaffolds.fasta"

Transcriptome Assembly

Copyspades.py \
    --rna \
    -t 16 \
    -1 rnaseq_R1.fq.gz -2 rnaseq_R2.fq.gz \
    -o transcriptome_assembly

Alternative Assemblers

MEGAHIT (Alternative for Metagenomes)

Copymegahit -1 R1.fq.gz -2 R2.fq.gz -o megahit_output -t 16

Troubleshooting

Out of Memory

• Reduce -m limit
• Use --meta mode (more memory efficient)
• Try MEGAHIT instead

Poor Assembly

• Check read quality with FastQC
• Trim adapters and low-quality bases
• Increase coverage if possible
• Try --careful mode

Long Runtime

• Reduce k-mer values
• Use --only-assembler if reads pre-corrected
• Increase threads

Related Skills

• read-qc - Preprocess reads before assembly
• assembly-polishing - Polish assembly with Pilon
• assembly-qc - Assess with QUAST/BUSCO
• long-read-assembly - Long-read alternatives