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The DNA concentration was measured with a Qubit 2.0 fluorometer (Thermo Fisher Scientific, Waltham, MA, USA). Each genomic DNA sample was adjusted to the concentration of 5 ng/μL, and 20 samples (2.5 ng each) of individuals with esotropia, those with exotropia, and normal individuals were mixed into one sequencing library, separately [20 (link)]. Sample preparation and exome enrichment for next generation sequencing (NGS)-based locus mapping was performed by exome capture technique with Nextera Rapid Capture Exome Kit (8 rxn × 3 plex, Illumina, San Diego, CA, USA), and sequencing of the libraries was performed by multiplexing three libraries per lane with variant filter cutoff 30 on MiSeq according to the MiSeq System User’s Guide (Illumina, San Diego, CA, USA). The sequencing processes of the same pooled libraries were repeated, and then, the first and second sets of data in each group, esotropia, exotropia, and normal group, were combined to increase the read depth. The combined data for esotropia group and exotropia group were further combined to obtain the data for strabismus group, which was then compared with the normal group.

10 μL of ligation reaction was added to 5 μL of 5× Q5 Reaction buffer (NEB), 3.75 μL of dH2O, 1 μL of each Nextera XT index (unique i5 and i7, Illumina), 2 μL of dNTPs (2.5 mM each, Bioline), and 0.25 μL of Q5 Hot Start High-Fidelity DNA Polymerase (NEB). The PCR was performed as follows: 30 seconds at 98°C for initial denaturation, followed by 25 cycles of 10 seconds at 98°C, 15 seconds at 63°C, and 20 seconds at 72°C, followed 2 minutes at 72°C for final extension.
The libraries were cleaned using standard Agencourt AmpureXP beads (Beckman Coulter) procedure with DNA to bead ratio of 1:0.9, and eluted in 20 μL of ddH₂O.
Library quality control was performed by analysing the library fragment size distribution on a 2% agarose gel, and molar concentrations were calculated from the concentrations obtained by using the Qubit dsDNA HS Assay Kit. Libraries were normalised to 2 nM concentration, then pooled and denatured according to the manufacturer’s instructions (Preparing Libraries for Sequencing on the MiSeq, #15039740, Revision D, Illumina). They were then sequenced using MiSeq v2 300-cycle kit (Illumina) at 15 pM final concentration according to the MiSeq System User Guide (#15027617, Revision M, Illumina), with 150 separate reaction libraries per run.