*2.4. Read Alignment and Mapping Reads to the Reference Genome*

To analyze the sequencing results effectively and accurately, low-quality raw data or connectors in the sequencing data were filtered. Cutadapt was used to remove the 3 sequencing adapter (https://cutadapt.readthedocs.io/en/stable/) (accessed on 10 March 2021), and reads with an average mass fraction lower than Q20 were removed [38]. The high-quality clean reads from each library were mapped to the pepper reference genome CM334 (https: //ftp.solgenomics.net/genomes/Capsicum\_annuum/C.annuum\_cvCM334/) (accessed on 10 March 2021) using HISAT2 software (http://ccb.jhu.edu/software/hisat2/index.shtml) (accessed on 10 March 2021). The read count value of each gene was mapped using HTSeq as the original expression of the gene [39]. Fragments per kilobase of transcript per million mapped reads (FPKM) was used to standardize the gene expression levels based on Cufflinks software [40].
