4.3.3. Reference Genome Preparation and Mapping of Clean Reads

To enable the mapping of clean reads, we first prepared a reference genome of *P. mume* [1]. Then, we performed reverse complementation process (C to T, and G to A) using Bismarkv 0.16.3 software [88]

and Bowtie2 [89]. In addition, a gene annotation file in gene transfer format, a Gene Ontology (GO) annotation file, a description file, and a gene region file in browser extensible data format were generated for subsequent annotation and function analyses.

The quality-checked clean reads generated by BS-seq were aligned against the two strands of the converted reference genome (X700-dovetail). The best unique alignment of these sequence reads was selected from the two sets of pairwise comparisons. To infer cytosine methylation states and positions, the sequences were then compared against the normal genomic sequence. Identical sequences aligned to a unique genomic region were regarded as duplicates and used to estimate sequencing depth and coverage. To allow their visualization in the IGV browser, sequences were transformed into bigWig format (non-overlap) [88,90]. The bisulfite non-conversion rate was defined as the number of sequenced cytosines at all of the cytosine reference positions divided by the number in the lambda genome.
