Quality Control and Preprocessing Exercise

Motivation

NGS can be affected by a range of artefacts that arise during the library preparation and sequencing processes including:

• low base quality
• contamination with adapter sequences
• biases in base composition

Aims

In this part you will learn to:

• assess the intrinsic quality of raw reads using metrics generated by the sequencing platform (e.g. quality scores)
• pre-process data, i.e. trimming the poor quality bases and adapters from raw reads

You will use the following tools, which are available through the module load/unload system:

• FastQC: http://www.bioinformatics.babraham.ac.uk/projects/fastqc/

module load FASTQC

• FastqMcf: https://code.google.com/p/ea-utils/wiki/FastqMcf

module load ea-utils

• The data set you'll be using is downloaded from ENA (http://www.ebi.ac.uk/ena/data/view/SRP019027).
• The reads belong to sample SRR769316, though this has been modified for course timing reasons.

View data set

cd $HOME/i2rda_data/Quality_control_and_preprocessing
zcat Read_1.fastq.gz |head
zcat Read_2.fastq.gz |head

Assessment of data quality

Run FastQC on the raw data:

fastqc --nogroup Read_1.fastq.gz Read_2.fastq.gz
firefox Read_*_fastqc.html &

where:

• --nogroup disables grouping of bases for reads >50bp. All reports will show data for every base in the read.

Look at the FastQC results and answer the following questions:

• What is the quality encoding?
• How many reads are present in each fastq file?
• What is the length of the reads?
• Are there any adapter sequences observed?
• Which parameters you think should be used for trimming the reads?

Pre-processing of data

Trim reads using Fastq-Mcf:

fastq-mcf -o Read_1_q30l50.fastq -o Read_2_q30l50.fastq \
-q 30 -l 50 \
--qual-mean 30 adapters.fasta Read_1.fastq Read_2.fastq

where:

• -o output file
• -q quality threshold causing base removal
• -l Minimum remaining sequence length
• --qual-mean - Minimum mean quality score

Reassessment of data quality

Run FastQC on the trimmed reads:

fastqc --nogroup Read_1_q30l50.fastq Read_2_q30l50.fastq
firefox Read*q30l50*fastqc.html

Look at the FastQC results and answer the following questions:

• How many reads are present in each fastq file?
• What is the length of the reads?
• Did qualities improve?