Contamination Screening

Screen FASTQ files against multiple genomes to identify contamination sources using FastQ Screen.

FastQ Screen Overview

FastQ Screen aligns a subset of reads against multiple reference genomes to identify:

• Cross-species contamination
• Bacterial/viral contamination
• Adapter sequences
• PhiX spike-in
• Sample swaps

Basic Usage

# Screen against configured genomes
fastq_screen sample.fastq.gz

# Multiple files
fastq_screen *.fastq.gz

# Specify output directory
fastq_screen --outdir qc_results/ sample.fastq.gz

# Custom config file
fastq_screen --conf my_screen.conf sample.fastq.gz

Configuration File

Create fastq_screen.conf:

# Database locations
DATABASE	Human	/path/to/human/genome
DATABASE	Mouse	/path/to/mouse/genome
DATABASE	Ecoli	/path/to/ecoli/genome
DATABASE	PhiX	/path/to/phix/genome
DATABASE	Adapters	/path/to/adapters
DATABASE	rRNA	/path/to/rrna

# Aligner (bowtie2 recommended)
BOWTIE2	/path/to/bowtie2

# Or use BWA
# BWA	/path/to/bwa

# Threads
THREADS	8

Pre-built Databases

# Download common screening databases
fastq_screen --get_genomes

# Downloads to ~/fastq_screen_databases/
# Includes: Human, Mouse, Rat, E.coli, PhiX, Adapters, etc.

Screening Options

# Number of reads to sample (default 100000)
fastq_screen --subset 200000 sample.fastq.gz

# Use all reads (slow)
fastq_screen --subset 0 sample.fastq.gz

# Set threads
fastq_screen --threads 8 sample.fastq.gz

# Paired-end (screen R1 only by default)
fastq_screen sample_R1.fastq.gz

# Force screening both pairs
fastq_screen --paired sample_R1.fastq.gz sample_R2.fastq.gz

Output Options

# Generate PNG plot (default)
fastq_screen sample.fastq.gz

# No plot (text only)
fastq_screen --nograph sample.fastq.gz

# Generate additional mapping statistics
fastq_screen --tag sample.fastq.gz

# Filter reads by mapping (keep unmapped to all genomes)
fastq_screen --filter 0000 sample.fastq.gz

# Keep only reads mapping to first genome (e.g., Human)
fastq_screen --filter 1--- sample.fastq.gz

Filter Codes

Use --filter to select reads based on mapping status:

Code	Meaning
0	Did not map to genome
1	Mapped uniquely
2	Mapped more than once
3	Mapped (unique or multi)
-	Ignore this genome

# Example: Keep reads mapping only to Human (first genome)
# Human:1, all others:0
fastq_screen --filter 10000 sample.fastq.gz

# Keep reads NOT mapping to anything (clean reads)
fastq_screen --filter 00000 sample.fastq.gz

Output Files

File	Description
*_screen.txt	Tab-delimited results
*_screen.png	Visualization
*_screen.html	HTML report

Results Format

#Fastq_screen version: 0.15.3
Genome	#Reads_processed	#Unmapped	%Unmapped	#One_hit_one_genome	%One_hit_one_genome	#Multiple_hits_one_genome	%Multiple_hits_one_genome	#One_hit_multiple_genomes	%One_hit_multiple_genomes	Multiple_hits_multiple_genomes	%Multiple_hits_multiple_genomes
Human	100000	2000	2.00	95000	95.00	1000	1.00	1500	1.50	500	0.50
Mouse	100000	98000	98.00	100	0.10	50	0.05	1500	1.50	350	0.35

Interpreting Results

Expected Results by Sample Type

Sample Type	Expected Pattern
Human sample	>90% Human, <1% others
Mouse sample	>90% Mouse, <1% others
Human + PhiX	>80% Human, ~10% PhiX
Contaminated	Significant % to unexpected genome

Common Issues

Pattern	Likely Cause
High adapter %	Library prep issue
High PhiX %	Spike-in not removed
High E.coli %	Bacterial contamination
High rRNA %	rRNA depletion failed
Multiple species	Sample swap or contamination

MultiQC Integration

FastQ Screen results are automatically detected by MultiQC:

# Screen all samples
for f in *.fastq.gz; do
    fastq_screen --outdir screen_results/ "$f"
done

# Aggregate with MultiQC
multiqc screen_results/

Custom Database Setup

Create Bowtie2 Index

# Index a FASTA file
bowtie2-build reference.fa reference

# Add to config
# DATABASE	MyGenome	/path/to/reference

Common Databases to Include

Genome	Purpose
Human (GRCh38)	Human samples
Mouse (GRCm39)	Mouse samples
E. coli	Bacterial contamination
PhiX	Illumina spike-in
Adapters	Library prep
rRNA	Ribosomal RNA
Vectors	Cloning vectors
Mycoplasma	Cell culture contamination

Example Workflows

Standard Screening

# Download databases
fastq_screen --get_genomes

# Screen samples
fastq_screen --outdir screen_results/ --threads 8 *.fastq.gz

# Check results
multiqc screen_results/

Remove Contamination

# Screen and tag reads
fastq_screen --tag sample.fastq.gz

# Filter to keep only Human reads (assuming Human is first database)
fastq_screen --filter 3----- --tag sample.fastq.gz

# Or use BBDuk for removal
bbduk.sh in=sample.fastq.gz out=clean.fastq.gz \
    ref=contaminants.fa k=31 hdist=1

Related Skills

• quality-reports - FastQC shows overrepresented sequences
• adapter-trimming - Remove adapter contamination
• metagenomics - Deeper taxonomic analysis