Ion 316 chip

The V3-V4 region of16S rDNA was amplified using a pair of universal primers (334F: CCTACGGGAGGCAGCAG and 519R: ATTACCGCGGCTGCTGG). The 50 μL PCR amplification reaction volume consisted of 2 μL Taq enzyme, 1 μL 10 μM primers, 5 μL genomic DNA (30 ng), and water up to 50 μL. The PCR protocol was as follows: initial denaturation at 98 °Cfor 45 s; eight cycles at 98 °C for 15 s, 57 °C for 30 s, and 72 °C for 60 s; and a final elongation step at 72 °C for 60 s. The amplified PCR products were purified using a Magnetic Stand-96 (Life Technologies, AM10027). Products were then quantified using a Qubit 2.0 fluorometer (Invitrogen, Q32866), and the lengths of the amplified fragments were detected using an Agilent 2100 Bioanalyzer (Agilent, G2939AA). The amplified products were purified using magnetic beads. The concentration was determined using a Qubit2.0, and fragment distribution was detected using an Agilent 2100 Bioanalyzer. The quantified PCR fragments were used to construct libraries using an Ion Plus Fragment Library kit (Life Technologies, USA) according to the manufacturer’s instructions. Library sequencing was performed using an Ion Personal Genome Machine (PGM) system (Life Technology, USA) with an Ion^™316 Chip and an Ion PGM Sequencing 200 Kit v2 (Life Technology, 4482006), according to the manufacturer’s guidelines.

The plasmid DNA of the anti-CDK2 second sort output was used as a template for the PCR targeting the HCDR3 region of the scFvs. A set of forward primers mapping to the framework region upstream of the HCDR3 and carrying one of the Ion Torrent sequencing adaptors were used in combination with a barcoded reverse primer mapping to the common SV5 tag region of the yeast display vector and carrying the second adaptor required for sequencing. The primer sequences and method are described in detail by D’Angelo et al. (61 (link)). Once amplified with the proofreading Phusion polymerase (NEB), gel extracted, and quantified (Q-bit, HS-DNA kit, Invitrogen), the amplicon libraries were processed using the Ion Xpress Amplicon library protocol and then prepared for sequencing on the Ion 316 Chip (Life Technologies). The sequences analysis was performed using the AbMining Toolbox as described by D’Angelo et al. (61 (link)).

WGA products of all the biopsied blastocysts were also processed for the NGS analysis in order to evaluate the reliability of rapid aCGH and compare the performances of difference platforms (i.e. rapid aCGH, regular aCGH and NGS). Approximately 1 μg of WGA DNA was used for library construction using Ion Xpress Plus gDNA Fragment Library Preparation Kit Set (Life technologies, California, USA) and following the manufacturer’s instructions. The quantity of library was determined using Qubit dsDNA HS assay kits (Life technologies) with Qubit fluorometer (Life technologies). The template-positive Ion Sphere Particles (ISPs) were generated using Ion PGM Hi-Q Template Kits (Life technologies) with the Ion OneTouch 2 Instrument (Life technologies) and then enriched with the Ion OneTouch ES Instrument (Life technologies). Sequencing was performed on the Ion Torrent PGM Instrument (Life technologies) platform using the Ion PGM Hi-Q Sequencing Kit and Ion 316 chip (Life technologies). PGS analysis for aneuploidy detection was performed by the Ion Reporter Server System (https://ionreporter.thermofisher.com/ir/).

The CHPv2 is a pool of PCR primers that target 207 amplicons for 2885 mutations in 50 cancer-associated genes [15 (link)] (Life Technologies, Carlsbad, CA). The entire list of genes is available through the supplier’s website (http://tools.invitrogen.com/downloads/cms_106003.csv). Approximately 10 ng of DNA per sample was used for amplicon production by multiplex PCR using the Ion AmpliSeq CHPv2 and Ion AmpliSeq Library Kit 2.0 (Life Technologies, Carlsbad, CA). The resulting multiplex PCR reaction pool was used for target sequence library preparation. Primer sequences for the multiplex PCR was partially digested to ligate barcode adapters (Ion P1 Adaptor and Ion XpressTM Bacode X, Life Technologies, Carlsbad, CA) followed by a bead-based nucleic acid purification system (AMPure® XP Reagent, Life Technologies, Carlsbad, CA). After confirmation that the final library fragment size peaked at 130 bp, the library fragments were clonally amplified by emulsion PCR (Ion PGM Template OT2, Life Technologies, Carlsbad, CA). The emulsion particles containing clonally amplified PCR fragments were then applied onto a semiconductor sequencing chip (Ion 316 Chip, Life Technologies, Carlsbad, CA) for massive parallel sequencing on an Ion PGM sequencer (Life Technologies, Carlsbad, CA).