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RNA-seq is a powerful tool for studying gene expression and identifying novel transcripts. However, before the data can be analyzed, it must first be cleaned and processed to remove low-quality reads and adapter sequences. This process is known as trimming.

Adapter sequences should be removed from reads because they interfere with downstream analyses, such as alignment of reads to a reference. The adapters contain the sequencing primer binding sites, the index sequences, and the sites that allow library fragments to attach to the flow cell lawn.

fastq ), then you have paired end (PE) reads. If you have only one FASTQ file per sample (e.g., something_R1. fastq only), then you have single end (SE) reads. You call Trimmmomatic on whatever data you have (SE or PE).

To trim the adapter we need to provide Trimmomatic with a fasta file containing the adapters we want to remove. For common Illumina adapters, these are provided in the Trimmomatic directory under adapters . Here we have single end data, so we will use the fasta Trimmomatic-0.39/adapters/TruSeq3-SE.

Should I trim adapters from my Illumina reads? This depends on the objective of your experiments. In case you are sequencing for counting applications like differential gene expression (DGE) RNA-seq analysis, ChIP-seq, ATAC-seq, read trimming is generally not required anymore when using modern aligners.

Adapter trimming is a prerequisite step for analyzing next-generation sequencing (NGS) data when the reads are longer than the target DNA/RNA fragments.

Trimmomatic supports sequence quality data in both standard (phred+33) and Illumina 'legacy' formats (phred+64), and can also convert between these formats if required. The quality format is determined automatically if not specified by the user. The trimming status of each read can optionally be written to a log file.

To configure your environment for use of Trimmomatic, run the following command: module load trimmomatic . The default version will be loaded. To select a particular Trimmomatic version, use module load trimmomatic/version . For example, use module load trimmomatic/0.38 to load Trimmomatic 0.38.