Ion onetouch es

For the Ion Torrent sequencing system, we prepared sequencing templates (emulsion PCR and bead-enrichment) from sequencing libraries using an Ion PI Template OT2 200 Kit v2 or v3 (Life Technologies) and an Ion OneTouch system (Ion OneTouch Instrument and Ion OneTouch ES, Life Technologies) according to the manufacturer's protocol. Prepared templates were sequenced using an Ion PI Sequencing 200 Kit v2 or v3 and the Proton sequencer (Life Technologies). Torrent Suite 4.0 or 4.2 (Life Technologies) was used to convert the raw signals into base calls and to extract the FASTQ files of the sequencing reads. Sequencing data from the Illumina system were generated using a MiSeq system (Illumina, CA, USA) according to the manufacturer's protocol, and the FASTQ files of single-end reads were extracted.

The coding sequences of the EED, EZH2, and SUZ12 genes were analyzed using a targeted NGS approach. Experiments were performed on the NGS platform of the Cochin Hospital, Paris (Assistance Publique, Hopitaux de Paris, France). Briefly, the custom primer panel targeting the three genes (coding exons and IVS boundaries) was designed using AmpliSeq Designer (Life Technologies). For NGS library preparation, the Ion AmpliSeq 2.0 library kit was used according to the manufacturer's instructions. Amplified libraries were purified using Agencourt AMPure XP beads (Beckman Coulter). Prior to library pooling and sequencing sample preparation, amplified libraries were quantified using the Qubit fluorometer system (Agilent Technologies). Emulsion PCR and enrichment were performed on the Ion OneTouch and Ion OneTouch ES instruments with the Ion PGM template OT2 400 and Ion PGM sequencing 400 kits (Life Technologies). The template-positive ion sphere particles were loaded on Ion 318 chips and sequenced with an Ion PGM system (Life Technologies). Sequence alignment and extraction of SNPs and short insertions/deletions were performed using the Variant Caller plug-in on Ion Torrent suite version 4.4 and Ion Reporter version 4.4 (Life Technologies). DNA sequences were visualized using the Integrated Genomics Viewer (version 2.3.3) from the Broad Institute.

Whole exome sequencing was achieved using the Ion Proton platform (AmpliSeq kit). Briefly, 100 ng DNA from each sample were collected and the extracted DNA is then amplified using AmpliSeq HiFi mix (Life Technologies, Carlsbad, CA, USA) for 10 cycles. The resultant PCR products were then pooled followed by primer digestion using FuPa reagent (Life Technologies, Carlsbad, CA, USA). A ligation step was then conducted using Ion P1 and Ion Xpress Barcode adapters. After that the libraries were purified and quantified using qPCR and the Ion Library Quantification Kit (Life Technologies, Carlsbad, CA, USA). The next step included emulsion of the libraries using Ion OneTouch System to attach the DNA fragments to the Ion Sphere particles. The final step in the library preparation included enrichment of the Ion Sphere particles using Ion OneTouch ES (Life Technologies, Carlsbad, CA, USA). Once the library became ready, they are loaded on the sequencing chip which is then inserted into the Ion Proton instrument (Life Technologies, Carlsbad, CA, USA) for sequencing.

Emulsion PCR was performed using a OneTouch 2 instrument (Life Technologies) with an Ion PI Template OT2 200 Kit v2 following the manufacturer’s instructions. The enrichment of template-positive Ion Sphere Particles (ISP) in the Ion Proton I chip was achieved using the Ion OneTouch ES enrichment system (Life Technologies). Ion Proton I chip version 2 was prepared and loaded according to the manufacturer’s recommendations. The total base output as a criterion for a successful experiment was set as 9 GB. If a sample did not reach this criterion for the total base output in one experiment, we performed the sequencing again with the same library and merged the results before aligning the reads to the reference GRCh37/hg19 sequence.

All procedures for emulsion PCR and next-generation sequencing were performed with Ion Torrent equipment and Ion Torrent kits under the manufacturer specifications (Life Technologies, Carlsbad, CA, U.S.A.): emulsion PCR was performed with the Ion OneTouch 200 template kit in an Ion OneTouch. Enrichment of template positive Ionospheres (ISPs) was performed with an Ion OneTouch ES (Life Technologies, Carlsbad, CA, U.S.A.). Sequencing of enriched templates bound to ionospheres was done using the Ion PGM 200 sequencing kit in an Ion PGM Sequencer with either 314 or 316 chips. FASTQ files of each barcoded group of sequences were recovered from the Ion Torrent server for further analysis. Reads have been deposited in the Sequence Read Archive (SRA) from NCBI under study accession number: SRP052626.