Agilent sureselect target enrichment system kit

The research protocol was approved by the ethics review board of the Peking University School of Medicine. The study procedures were carried out in accordance with institutional guidelines and the Declaration of Helsinki. Informed consent was obtained from all patients after a full explanation of the procedures.
Thirty suspected male patients were enrolled in this study from 2004 to 2016. Patients underwent a comprehensive ophthalmological examination including slit-lamp biomicroscopy, funduscopic examinations, measurements of best corrected visual acuity (VA) with a logarithmic VA chart, OCT examinations (Spectralis; Heidelberg Engineering, Heidelberg, Germany) and ERG tests (RETIport, ROLAND CONSULT, Germany). Clinical therapies were recorded during the follow-up visit. Molecular genetic examinations of the RS1 gene were performed in the 30 males, 51 parents, and 100 control subjects (normal vision and without any eye diseases). Genomic DNA was extracted using an Agilent SureSelect Target Enrichment System Kit (Agilent, USA). Polymerase chain reaction (PCR) was performed to amplify six exons of the RS1 gene. Samples were sequenced directly by loading the sequencing reaction product onto an NEXTSEQ500 (Illumina, USA). T test was carried out on the process of clinical data comparison (SPSS 16.0). P value of 0.05 was considered to be statistically significant.

Genomic DNA was extracted from peripheral blood of ROP patients using an Agilent SureSelect Target Enrichment System Kit (Agilent, USA). Polymerase chain reaction (PCR) was performed using Goldstar Taq MasterMix (Cwbio, PRC) to amplify the exons of the FZD4, TSPAN12, and NDP genes. The samples were sequenced and analyzed by NEXTSEQ500 (Illumina, USA) as described previously [18 (link)].
The possible pathogenicity of novel missense mutations would be evaluated by using SIFT (http://sift.jcvi.org/), PolyPhen-2 (http://genetics.bwh.harvard.edu/pph2/), and Mutation Taster (http://www.mutationtaster.org) prediction software and via evolutionary conservation analysis. The minor allele frequencies (MAF) of variants in participants were checked using Exome Aggregation Consortium database (http://exac.broadinstitute.org/faq) and Genome Aggregation database (http://gnomad-sg.org/); All mutations were also evaluated regarding pathogenicity according to the standards and guidelines of American College of Medical Genetics and Genomics (ACMG) [19 (link)].