Extracted DNA was sent to CIBIO (Centro de Investigação em Biodiversidade e Recursos Genéticos) in Vairão, Portugal, to primer optimization, library preparation and amplicon sequencing. The primer-pairs specifically designed for this study are available on Table S2 . All obtained sequences are available in Text S1 . Detailed protocols on library preparation and amplicon sequencing are available in Appendix A.1 .
Demultiplexing, barcode removal and QC control were performed in Illumina’s Basespace online platform. Only reads with Phred quality score (Q) > 30 were utilized in this study. Demultiplexed reads were aligned against a reference, i.e., sardine’s mitogenome (reference NCBI: NC_009592.1) with bwa-mem algorithm [30 ] implemented in bwa [31 (link)]. Only reads that were properly paired were kept, mapping qualities above 10 and coverage above 2 were kept for variant calling. Variant calling was performed with Stacks v 1.48 [32 (link)] through imported .bam files. Specifically, we ran ref_map.pl pipeline with the sardine mitogenome as reference and default parameters.
Demultiplexing, barcode removal and QC control were performed in Illumina’s Basespace online platform. Only reads with Phred quality score (Q) > 30 were utilized in this study. Demultiplexed reads were aligned against a reference, i.e., sardine’s mitogenome (reference NCBI: NC_009592.1) with bwa-mem algorithm [30 ] implemented in bwa [31 (link)]. Only reads that were properly paired were kept, mapping qualities above 10 and coverage above 2 were kept for variant calling. Variant calling was performed with Stacks v 1.48 [32 (link)] through imported .bam files. Specifically, we ran ref_map.pl pipeline with the sardine mitogenome as reference and default parameters.