7  Clean Raw Read Data

As you have seen in Chapter 6 sequence read data can be of variable quality. The usual approach to improving the overall quality of a dataset is to discard low quality reads, and low-quality sections of reads. This is referred to as cleaning raw read data.

Trimmomatic is a commonly-used software tool for cleaning raw read data that

  1. identifies and excludes (drops) low-quality reads
  2. trims (deletes) any remaining Illumina adapter sequences
  3. trims (deletes) any low-quality read ends
Good practice

It is good practice always to use a tool like trimmomatic to clean your raw sequencing data.

When reporting how you cleaned your sequence data in a manuscript or dissertation, you should always state:

  1. The software tool you used, with its version number and a citation of the paper describing it (if available; provide a URL to the software if there is no paper)
  2. The parameters used when running the tool (if default parameters were used, state this)
  3. The number of reads that remained after cleaning
Tip

Tools like trimmomatic return two kinds of cleaned read data:

  1. R1-paired, R2-paired: these are high quality paired-end reads
  2. R1-unpaired, R2-unpaired: these are single reads, where one member of the read pair was low quality

This part of the workshop will guide you through using trimmomatic to clear your sequence read data.

7.1 Using trimmomatic

  1. Navigate to the trimmomatic tool using the Tools sidebar in Galaxy
  • You can use the search tools field to find trimmomatic, or look under the FASTA/FASTQ section in the sidebar
  1. Select the trimmomatic tool
  2. Make sure that Paired-end (two separate input files) is chosen
  3. Select the forward and reverse raw read sets (trimmed_pe_aln.qsorted.mapped.fixed.1.fastq.gz trimmed_pe_aln.qsorted.mapped.fixed.2.fastq.gz) as the Input FASTQ files
  4. Set Perform initial ILLUMINACLIP to Yes; this will trim any remaining Illumina adapters from the read sequences
  5. Under Adapter sequences to use select TruSeq3 (paired-ended for MiSeq and HiSeq)
  6. Click Run Tool
Video: Using trimmomatic to clean raw sequencing reads

7.2 trimmomatic Output

Cleaning your data with trimmomatic generates four output files (Figure 7.1).

Figure 7.1: FastQC output files in Galaxy. From the top: unpaired reverse (R2) reads, unpaired forward (R1) reads, paired reverse (R2) reads, and paired forward (R1) reads.

You can inspect the contents of these files by clicking on the filename in the History sidebar, and also by clicking on the corresponding eye icon to view the trimmed reads in the workspace.

Video: Inspecting trimmomatic output files

7.3 Next steps

Now that we have clean, high-quality sequencing reads, we can move on to assembling the SARS-CoV-2 genome, in Chapter 8.