Illumina MiSeq sequencing; 220313_M05607_0212_000000000-DDM4Y
Zebrafish genomic DNA was extracted by the zebrafish embryo genotyper or by whole embryo lysis at 3 days postfertilization. The DNA was used for a PCR-based targeted amplification locus-specific primers with overhangs containing unique molecular identifiers and binding sites for paired-end sequencing NGS primers. Overhang primers containing unique sample identifiers for each embryo and flow cell binding sites were added in an additional PCR run. The concentration of the resulting PCR products was determined with the Qubit dsDNA HS (High Sensitivity) Assay Kit (ThermoFisher Scientific), with simultaneous quantification for all the samples by use of the VICTOR Multilabel Plate Reader (PerkinElmer). Equal amounts of all samples were pooled, purified with magnetic beads, diluted to a concentration of 10pM and spiked with 30%PhiX Control v3 Library (Illumina). Paired-end, dual-indexed sequencing with 2×150 bp cycles was performed on a MiSeq instrument (Illumina).