Sureselectqxt target enrichment system

Patient analysis was performed using the abovementioned NGS panel. The custom design of our probes was realized using the web-based SureDesign application (https://earray.chem.agilent.com/suredesign accessed on 12 July 2020). A total of 50 ng of gDNA was processed through the SureSelect^QXT Target Enrichment system (Agilent Technologies, Santa Clara, CA, USA) for Illumina multiplexed sequencing. Briefly, gDNA was enzymatically fragmented and adaptor-tagged to obtain a pool of fragments that were amplified by PCR reaction. Then, the prepared DNA library amplicons were hybridized to the capture custom library, made up of our 92 genes, and purified by streptavidin-coated magnetic beads. The captured, targeted-enriched DNA library was amplified by PCR reaction by using dual index primers, which allowed us to univocally barcode each sample. Finally, SureSelect-enriched dual-indexed NGS samples were pooled together for multiplexed sequencing. Sequencing reactions were carried out on the MiSeq instrument (Illumina, San Diego, CA, USA) using a PE 150 × 2 flow cell, running 16 samples for each sequencing run to obtain an average coverage of about 200× (>95% of the gene’s target nucleotides are covered at >100 reads, with mapping quality score (MQ > 30) reads); 96% of the analyzable target regions were covered by at least 50×.

Samples were analyzed either by panel-based analysis of known retinopathy genes or by clinical exome sequencing. One sample underwent whole-exome sequencing. Genomic DNA was extracted from peripheral blood using the DNeasy Blood and Tissue Kit (QIAGEN) according to standard protocols. Targeted NGS analysis was performed using the RETplex targeted sequencing panel as previously described [30 (link)]. Clinical exome and whole-exome sequencing libraries were prepared using the ClearSeq Inherited Disease Panel (Agilent Technologies, Santa Clara, CA, USA) and the SureSelect Human All Exon V7 (Agilent Technologies, Santa Clara, CA, USA), respectively. Targeted regions were enriched using the SureSelectQXT Target Enrichment system (Agilent Technologies, Santa Clara, CA, USA). Libraries were run on a NextSeq500 sequencing platform (Illumina inc., San Diego, CA, USA). Sequencing data were analyzed using a previously described pipeline [43 (link)].

The method of detection of constitutional variants by capture and massive parallel sequencing on NextSeq550 has been validated and is used in our medical department (Genetic Diagnosis Laboratory, HUS, Strasbourg, France) in various diagnostic tests [15 (link),16 (link),17 (link)].
Libraries were prepared using SureSelectQXT Target Enrichment System (Agilent Technologies, Santa Clara, CA, USA), which uses RNA probes to capture known coding DNA. Briefly, 50 ng genomic DNA was fragmented enzymatically, and specific adaptor oligos were ligated to fragments of the DNA to be sequenced. The probes hybridized on the regions of interest were then captured by a magnetic system (beads coupled to streptavidin). After purification of the enriched DNA fragments, a PCR was carried out in order to increase the number of enriched libraries, which were then double-indexed by oligonucleotide barcodes (a unique barcode per patient within the same series) and pooled by 30 before sequencing on NextSeq 550 System (Illumina, San Diego, CA, USA) with 2 × 75 bp reads for a total 51 genes and 6 control SNPs.