Home Pre-workhop Instructions
Lectures
1: Introduction and Key Concepts 2: Variant Detection and Phylogenetic Trees 3: Practical Applications of WGS and Phylogenetics 4: Advanced Applications of WGS
Practical 1: WGS Data Analysis
Obtaining sequencing data Viewing raw sequencing data QC of FASTQ files Cleaning and filtering FASTQs Reference mapping/alignment De novo assembly
Practical 2: Variant Calling and ML Trees
Variant calling and VCFs Building consensus sequences Aligning consensus sequences Maximum Likelihood trees
Practical 3: Timed Phylogenetic Trees
ML timed trees Bayesian trees (BactDating) Bayesian trees (BEAST2)
Practical 4: Transmission and Profiling
Identifying lineages and serotypes Transmission network reconstruction
Practical 5: Mixed Infection, Recombination and ANI
Identifying mixed infection Detecting recombination Average nucleotide identity (ANI)
Practical 6: Phylogeography and Phylodynamics
Phylogeography Inferring population dynamics
Practical 7: Fitness and Selection
Local branching index (LBI) Detecing homoplasy dN/dS GWAS


Viewing raw sequencing data and understanding the FASTQ format

We will first open a FASTQ file so you can see how the raw data looks, though you would not be expected to do any manual QC on the sequence data just by viewing it as these files can contain millions of reads.

We can view the FASTQ file in two ways, either through a text viewer or with the command-line in your terminal. If files are in the zipped format (*.fastq.gz) they will need to be unzipped first to view in this format.

Either: - Open the “Test4_R1.fastq” file in your “Data” folder in your extended text viewer.

head -10 Test4_R1.fastq

The “head’ command will output the first N lines of the file, where N is specified by -N (in our case we have asked for the first 10 lines).


The output should look like this:

Description

If we look more closely at the file, you will see that it comprised of groups of 4 lines of text for each sequenced read:

Description

These 4 lines consist of the following information:

  1. A sequence identifier with information about the sequencing run and the cluster. The exact contents of this line vary by based on the BCL to FASTQ conversion software used. If the sequence data are paired, there will be a \1 or \2 at the end of this line to denote the forward or reverse read (all reads included will have a forward and reverse entry, called a ‘mate pair’)

  2. The sequence (the base calls; A, C, T, G, and N if base cannot be confidently called).

  3. A separator (+) between the sequence and quality line.

  4. The base call quality scores. These are Phred +33 encoded, using ASCII characters to represent the numerical quality scores. Further information can be found here: https://help.basespace.illumina.com/files-used-by-basespace/quality-scores


The next step is to assess the quality of these raw sequences and perform additional steps to resolve any potential issues.


Next activity: Quality control (QC) of FASTQ files