Sureselect enzymatic fragmentation kit

Exome libraries were prepared according to the Agilent SureSelect XT HS Human All exon v7 instructions. Briefly, 200 ng of DNA was enzymatically fragmented with Agilent SureSelect Enzymatic Fragmentation Kit. Fragmented DNA was end-repaired and dA-tail was added at DNA ends; then, molecular barcode adaptors were added, followed by AMPure XP bead purification. The adaptor-ligated library was amplified by PCR and purified by AMPure XP beads. DNA libraries were hybridized with targeting exon probes and purified with streptavidin-coated magnetic beads. The retrieved libraries were amplified by PCR and purified by AMPure XP beads and pooled for sequencing in NextSeq 500 using Illumina flow cell High Output 300 cycles chemistry. All quality controls of the libraries were carried out using Screen tape assays and quantified by Qubit fluorometer. Quality parameters included a DNA integrity number above 8 and a 100X sequencing depth aimed with at least 85% of coverage.

About 150–200 ng of dsDNA, according to Qubit dsDNA HS assay kits fluorometric quantification, were sheared by the Sure Select Enzymatic Fragmentation kit (Agilent Technologies Inc., Santa Clara, CA, USA). The NGS library was created using Sure Select XT2 Low input Custom library probes (Agilent Technologies Inc.), and sequencing was performed on MiSeq (Illumina Inc., San Diego, CA, USA) using 2 × 150 bp paired-end sequencing. Data collection was performed with the MiSeq Reporter (MSR) software v.2.6.2.3, using the ‘FastQ only’ workflow. The run quality was evaluated by Illumina Sequencing Analysis Viewer v.1.9.1, while the Bioinformatics pipeline for the annotation of the vcf files was developed in-house, in collaboration with enGenome Software Company (Pavia, Italy). Paired-end reads were mapped on the Human hg19 genome. Full coverage of the coding regions, mean read depth of 650× and read coverage of >50× were obtained.
The NGS custom panel, named Oncopan, can detect Single Nucleotide Variants (SNV) and small ins/del of the following genes: APC, ATM, BARD1, BMPR1A, BRCA1, BRCA2, BRIP1, CDH1, CDKN2A (α e β), CDK4 (exon 2), CHEK2, CTNNA1, EPCAM, FANCM, MLH1, MSH2, MSH3, MSH6, MUTYH, NBN, NHTL1, PALB2, PMS2, POLD1, POLE, PTEN, RAD51C, RAD51D, SMAD4, STK11, TP53, KRAS, NRAS, BRAF, EGFR, HER2(ERBB2), PIK3CA. The analysis was carried out on tumor samples from Patient 2.

Total gDNA was extracted from hiPSCs and hiPSC-CMs at 0, 2, and 4 weeks after differentiation using a QIAamp DNA Mini Kit (QIAGEN). After the quantification of the gDNA concentration, 200 ng of each gDNA sample was digested using a SureSelect Enzymatic Fragmentation Kit (Agilent Technologies) to create a library of gDNA restriction fragments. The enzymatically fragmented gDNA samples were enriched using ONCO AccuPanel (NGeneBio, Seoul, Republic of Korea) according to the manufacturer’s recommendations. At each step of library enrichment, the gDNA was purified using AMPure XP beads (Beckman Coulter) to selectively bind nucleic acids based on their size. Prior to sample pooling, the quantity and quality of the library were measured using a Quibit dsDNA BR Assay Kit (Invitrogen) and 1% agarose gel electrophoresis. DNA libraries were pooled, hybridized with biotin-labeled RNA probes, and eluted using Dynabeads^TM MyOne^TM Streptavidin T1 (Invitrogen). The captured library was amplified by Veriti^TM 96 well Thermal Cycler (Applied Biosystems), and diluted to a final loading concentration of 1.5 pM. Final libraries with 1% PhiX control were sequenced at a paired-end 150 bp (2 × 150 bp) read length on the MiniSeq platform (Illumina, San Diego, CA, USA). The FASTQ file containing the sequence data was analyzed using NGeneAnalySys v1.6.4 software (NGeneBio).