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