title: DNA sequencing of Diplostomidae-specific mtDNA CO1 fragment: Perca fluviatilis eye
design: We screened eye samples from 212 perch using modified diplostomid-specific primers (Moszczynska et al. 2009) that included linkers for Illumina-compatible adapters at their 5 ends (Clarke et al. 2014; Kaunisto et al. 2017). In total, 142 PCR-positive samples were chosen for sequencing. For library preparation PCR-products were purified and used for the second PCR to attach Illumina adapters and unique individual bar-codes following the modified PCR protocol described in Kaunisto et al. (2017): for a reaction volume of 12.5 l, 6.25 l KAPA HiFi HotStart MasterMix (product nr KK2602, KAPA Biosystems, Wilmington, Massachusetts, USA), 3.75 pmol of each primer, 1.25 l of sterile deionized water and 2 l of purified locusspecific PCR product were mixed. The PCR cycling conditions were similar to those described in Kaunisto et al. (2017), except that the extension step was 20 s. After the second PCR, 2 l of each indexed sample was pooled together and purified using SPRI beads. The quality of library pool was ensured using Agilent Bioanalyzer 2100 and the library pool concentration was measured with Qubit DNA HS kit. The samples were sequenced in a single run with Illumina MiSeq instrument (Illumina Inc., San Diego, California, USA) by the Turku Centre for Biotechnology, Turku, Finland, using v3 sequencing chemistry. Paired-end sequencing with 2 x 300 bp read length was used with 8 bp dual index run.
identifiers.namespace: SUB6534379
identifiers.id: Sa38_S262