Trimming reads and removing adapter sequences
Sometimes Illumina adapter sequences are still present in some reads because adapters can form adapter dimers and then one of them gets sequenced or if a DNA fragment is shorter than the read length, the sequencer continues to “read-through” into the adapter at the end of the DNA fragment. In the latter case the forward and the reverse read will contain adapter sequences, which is called a “palindrome”. While a full adapter sequence can be identified relatively easily, reliably identifying a short partial adapter sequence is inherently difficult. However, if there is a short partial adapter present at the end of the forward read and the beginning of the reverse read, that is a good sign for a “palindrome” sequence.
Reads that start or end with very low quality can be aligned better if the bad quality parts are trimmed off. We will use Trimmomatic to trim reads and remove adapter sequences. As we have paired reads, we will run it in Paired-end (PE) mode which requires 2 input files (for forward and reverse reads) and 4 output files (for forward paired, forward unpaired, reverse paired and reverse unpaired reads).
The current processing steps are:
- ILLUMINACLIP: Cut adapter and other illumina-specific sequences from the read.
- SLIDINGWINDOW: Perform a sliding window trimming, cutting once the average quality within the window falls below a threshold.
- LEADING: Cut bases off the start of a read, if below a threshold quality
- TRAILING: Cut bases off the end of a read, if below a threshold quality
- CROP: Cut the read to a specified length
- HEADCROP: Cut the specified number of bases from the start of the read
- MINLEN: Drop the read if it is below a specified length
In Trimmomatic, different processing steps take one or more settings, delimited by ‘:’ (a colon): ILLUMINACLIP:<fastaWithAdapters>:<seed mismatches>:<palindrome clip threshold>:<simple clip threshold>:<minAdapterLength>
See the Trimmomatic manual for all other options.
cd # Make a directory for the trimmed reads mkdir trimmed cd trimmed # Run Trimmomatic java -jar /home/scripts/trimmomatic.jar PE \ /home/data/wgs_100k/10558.PunPundMak.R1.100k.fastq.gz \ /home/data/wgs_100k/10558.PunPundMak.R2.100k.fastq.gz \ 10558.PunPundMak.R1.100k.paired.fastq.gz 10558.PunPundMak.R1.100k.unpaired.fastq.gz \ 10558.PunPundMak.R2.100k.paired.fastq.gz 10558.PunPundMak.R2.100k.unpaired.fastq.gz \ ILLUMINACLIP:/home/scripts/Trimmomatic-0.39/adapters/TruSeq3-PE.fa/:2:30:10 \ LEADING:5 TRAILING:5 SLIDINGWINDOW:5:10 MINLEN:50