Miniseq sequencing system

Tumors were fixed in 10% buffered formalin, embedded in paraffin, and stained with hematoxylin–eosin and PAS. Immunostaining was performed with primary antibodies against GH, Prolactin, ACTH, TSH, FSH, LH, alpha-subunit, PIT1, TPIT, SF1, synaptophysin, chromogranin, S100 protein, Keratins (AE1/AE3, Cam5.2), p53, and Ki67 (MiB1).
DNA panel sequencing was performed using a self-customized targeted panel manufactured by Qiagen (CDHS-21330Z-424). This panel targets the complete coding regions and splice-sites of six genes (ATRX, EGFR, NF1, NF2, PTEN, TP53), as well as mutation hotspots of 14 further genes (AKT, BRAF, CTNNB1, FGFR1, FGFR2, H3F3A, HIST1H3B, HIST1H3C, IDH1, IDH2, KRAS, PI3CA, PIK3R1, TERT-promoter). The library was constructed according the manufacturer’s instructions. Sequencing was performed on an Illumina MiniSeq sequencing system (paired-end, 2 × 151 bp, average coverage 500 ×). Data was analyzed with the Qiagen CLC Genomics workbench, using a self-customized workflow. Variants were annotated with information from the 1000 genome project, dbSNP, ClinVar, and COSMIC. Only variants with an allele frequency ≥ 5% and a total target coverage of ≥ 40 × were analyzed further. Variants not annotated by ClinVar were additionally analyzed with VarSome (www.varsome.com).

Genomic DNA, extracted from each zebrafish embryo at 3 dpf, was used as template DNA for primary PCR amplification. Deep sequencing was carried out as previously described^{16 (link)}. The primers used for deep sequencing are listed in Suppl. Table 3. After nested PCR, index sequences were added to the amplicons. The index sequences were matched to samples as described in Suppl. Table 4. Sequencing reactions were performed with a MiniSeq sequencing system (Illumina, CA, USA) to obtain paired 151 bp read lengths. The obtained reads were mapped to each reference sequence from the zebrafish genome database (DanRer10) by the following setting: Masking mode = no masking; Mismatch cost = 2; Insertion cost = 3; Deletion cost = 3; Length fraction = 0.5; Similarity fraction = 0.8; Global alignment = No; Auto detect paired distances = Yes; Nonspecific match handling = Map randomly. The variant calling was performed with the following settings: Ignore positions with coverage = 150,000; Ignore broken pairs = Yes; Ignore Nonspecific matches = Reads; Minimum coverage = 10; Minimum count = 2; Minimum frequency = 0.5%; Base quality filter = No; Read detection filter = No; Relative read direction filter = 0.5%; Read position filter = No; Remove pyro-error variants = No. Rearrangement of the output file was made using Excel (Microsoft, WA, USA).

Hundred nanogram of genomic DNA or 2–3 μL of Proteinase K extraction solution was amplified by three-round PCR using SUN-PCR blend (SUN GENETICS). The following programs were used for the first round PCR: 3 min at 95 °C; 25 cycles of 30 s at 95 °C, 30 s at 60 °C, and 60 s at 72 °C; and, finally, 3 min at 72 °C. 1 µL of the first round PCR product was used for the second round PCR. The following programs were used: 3 min at 95 °C; 25 cycles of 30 s at 95 °C, 30 s at 60 °C, and 20 s at 72 °C; and, finally, 3 min at 72 °C. 1 µL of the second round PCR product was used for the third round PCR. The following programs were used: 3 min at 95 °C; 30 cycles of 30 s at 95 °C, 30 s at 62 °C, and 20 s at 72 °C; and, finally, 3 min at 72 °C. The amplified genomic DNA regions containing a Cas9-targeted site were purified using Expin™ PCR SV mini (GeneAll) according to the manufacturer’s protocol. The PCR amplicons were sequenced using a MiniSeq Sequencing System (Illumina), and the results were analyzed using Cas-Analyzer (http://www.rgenome.net/cas-analyzer/). A list of primers for PCR is provided in Supplementary Fig. 6.