For these 11 genes, we further investigated whether they still produce spliced transcripts using available RNA sequencing (RNA-seq) data of cetaceans. We downloaded RNA-seq data of the bottlenose dolphin skin and blood (PRJNA385781, SRA study: SRP106690) and the minke whale brain, heart, kidney, liver, lung, and muscle (PRJNA72723, SRA study: SRP025154) (6) from the National Center for Biotechnology Information (NCBI) SRA. We processed all SRA read files with fastq-dump using parameters for removing technical reads (skip technical), filtering (read-filter = pass) and removing tags (clip), splitting paired-end reads into according files (split files), keeping read identifiers (readids), and formatting data into base space (dumpbase). Reads were then mapped to the genome assembly of the bottlenose dolphin (turTru3) and the minke whale (balAcu1) using STAR [Spliced Transcripts Alignment to a Reference (version 2.4.2a); https://github.com/alexdobin/STAR/releases/tag/STAR_2.4.2a). For bottlenose dolphin, genome indexes were generated with default parameters. For minke whale, we adjusted the number of bins for the indexes according to the number of total bases and scaffolds in the assembly (genomeChrBinNbits = 18). The minke whale RNA-seq data consist of paired-end reads, whereas the dolphin RNA-seq data consist of single-end reads. During mapping, we specified input files according to paired- or single-end read data. All runs were mapped separately using parameters for removing reads that map to many different locations (outFilterMultimapNmax = 20) and limiting the number of allowed mismatches for mapped reads (outFilterMismatchNoverLmax = 0.04). We used bedtools (https://bedtools.readthedocs.io/en/latest/) and bedGraphToBigWig (UCSC genome browser source code) to visualize the read coverage in the UCSC genome browser. The dolphin RNA-seq data contain 10 runs from blood samples and 25 runs from skin samples. We combined all runs from blood samples and all runs from skin samples using bigWigMerge. The read coverage across the seven candidate gene losses was then inspected to assess whether remnants of the lost genes are still expressed and properly spliced in dolphin and minke whale.

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