2.5.2. Read Mapping and Differential Expression Analysis

The raw paired-end reads were clipped and quality controlled by SeqPrep https: //github.com/jstjohn/SeqPrep (accessed on 10 January 2022) and Sickle https://github. com/najoshi/sickle (accessed on 10 January 2022) with the default parameters. The clean reads of each sample were sequenced and aligned with the specified reference genome Homo\_sapien, http://asia.ensembl.org/Homo\_sapiens/Info/Index (accessed on 23 January 2022). The mapped reads of each sample were assembled by StringTie https://ccb.jhu.edu/software/stringtie/index.shtml t = example (accessed on 23 January 2022) in a reference-based approach [29].

To identify the differential expression genes (DEGs) between the two different samples, the expression level of each transcript was calculated according to the fragments per kilobase of exon per million mapped fragments (FPKM) method. RSEM http://deweylab.biostat. wisc.edu/rsem/ (accessed on 20 March 2020) [30] was used to quantify gene abundances. A differential expression analysis was performed using DESeq2 [31] with |log2FC| > 1.3, and a *Q*value ≤ 0.05 was considered to indicate significantly differentially expressed genes. Functional enrichment analyses, including gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses, were implemented to find significantly enriched DEGs in GO terms and metabolic pathways at a Bonferroni-corrected *P*value ≤0.05 compared with the whole-transcriptome background [32].
