MOLECULAR PROFILING IN LOW-GRADE EC 69 3 eMethod. Detailed information on DNA analysis, smMIP design and library preparation, sequencing and immunochemistry analysis DNA analysis Representative areas of EC in the surgical specimen were marked and selected for formalin-fixed paraffin-embedded (FFPE) 20 μm thick sections. Slides were cut from these FFPE section and stained with hematoxylin and eosin (H&E). Tumor areas were marked on these slides and the tumor cell percentage was estimated. These specimens were digested overnight at 56°C in TET-lysis buffer (10mmol/L Tris/HCL pH 8.5, 1 mmol/L EDTA pH 8.0, 0.01% Tween-20) with 5% Chelex-100 (BioRad, Hercules, CA) and 0.2% proteinase K, with subsequent inactivation at 95°C for 10 min. After this was centrifugated, the supernatant was transferred into a clean tube. DNA concentration was determined using the Qubit Broad Range Kit (Thermo Fisher Scientific, Waltham, MA). smMIP design and library preparation The panel consisted of 10 genes important for EC oncogenesis (ARID1A, CTNNB1, ERBB2, KRAS, MTOR, NRAS, PIK3CA, PTEN, POLE, TP53). The smMIPs were designed in a tilling manner for hotspots in oncogenes and all coding as well as splice site consensus sequences of tumor suppressor genes (TSGs), with preferential targeting of both strand by two independent smMIPs. All the smMIP probes are constructed by an extension and ligation probe arm (40 bp long) with a 112 bp gap and a common backbone sequence for PCR-based library amplification. The backbone and ligation probe arm are connected by means of an 8 bp degenerate sequence (8xN) serving as a Unique Molecular Identifier (UMI, “single-molecule tag”). Following, the smMIP probes were mixed and phosphorylated with 1 µl of T4 polynucleaotide kinase (M0201; New England Biolabs). The molecular ratio between gDNA and smMIPs was set at 1:3,200 for each individual smMIP and the standard genomic DNA input was set at 100ng. A capture mix was made (volume 25 µl) with the phosphorylated smMIP pool, 1 unit of Ampligase DNA ligase (A0110K; EpiBio, Madison, WI) and Ampligase Buffer (A1905B, DNA ligase buffer), 3.2 units of Hemo Klentaq (M0332; New England Biolabs), 8 mmol of dNTPs (28-406520/-12/-22/-32; GE Healthcare, Little Chalfont, UK) and 100 ng of genomic DNA in a 20 µl volume. This capture mix was denatured at 95°C for 10 min and subsequently incubated for probe hybridization, extension and ligation for 18hr at 60°C. To perform the exonuclease treatment, Exonuclease 1 (10 units; M0293; New England Biolabs) and III (50 units; M0206; New England Biolabs) and Ampligase Buffer was added to the capture mix after cooling (total of 27 µl). This mix was incubated at 37°C for 45 min, with subsequent inactivation at 95°C for 2 min. From the 27 µl, 20 µl was used for PCR in at total volume of 50 µl including a common forward primer, bar-coded reverse primers, and iProof high fidelity master mix (1725310, Bio-Rad, Veenendaal, the Netherlands). The resulting PCR products were then pooled and purified with 0.8x volume of Agencourt Ampure XP Beads (a63881, Beckman Coulter, Woerden, the Netherlands). Sequencing The purified libraries were denatured and diluted to 1.2pmol/l, and then sequenced on a NexSeq500 device (Illumina, San Diego, CA) using the manufacturer’s instructions (300 cycles High Output sequencing kit, v2), resulting in 2x150bp paired-end reads. All Bcl files were converted to fastq files and bar-coded reads were then demultiplexed. Single-molecule-directed assembly of the duplicate reads was conducted generating consensus (‘unique’) reads with the software Sequence Pilot (version 4.4.0; JSI medical system, Ettenheim, Germany). Variants were annotated as ‘malignant’, ‘likely malignant’, ‘unknown significance’, ‘likely benign’ and ‘benign’ using amongst others publicly available databases such as ClinVar (https://www.ncbi.nlm.
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