Sureselect human all exon v7

We assessed the performance of exome captures of the selected 5’UTRs based on the designs provided by the kits which were mostly used for the generation of our in-house WES data, namely the SureSelect Human All Exon V6 and SureSelect Human All Exon V7 (Agilent Technologies), as well as a selection of the most recent versions of commonly used kits from 4 different providers: SureSelectXT Human All Exon V8 (Agilent Technologies), KAPA HyperExome V2 (Roche), Twist Exome 2.0 (Twist), and Illumina Exome Panel v1.2 (Illumina). BED files containing the genomic coordinates of the capture regions were downloaded from the corresponding design catalogs and intersected with the coordinates of the 5’UTRs of interest using bedtools intersect (v2.26.0) [66 (link)] with default parameters. For uniformity, only the captured regions were used to compare between the different designs. Additionally, for the SureSelect Human All Exon V6 and SureSelect Human All Exon V7 kits (Agilent Technologies), both the strict union of all regions covered by baits and a version padded by ±50bp extending into intronic regions were considered for the intersections. A custom Python (v.3.6.8) script was then used to compute for each IRD gene the length proportion (%) of its 5'UTR captured by these kits.

DNA libraries were prepared from fresh frozen tumor tissues and matched blood samples of 52 patients (N=52). 100 ng of DNA was used as input. SureSelect XT Low Input for Illumina and Enzymatic Fragmentation Kit (Agilent Technologies Inc.) were used for the preparation of libraries according to the manufacturer´s protocol. In each capture reaction, eight libraries were pooled equimolarly based on qPCR quantification (Kapa Library Quantification Kit, F.Hoffmann-La Roche AG). Hybridization was performed using SureSelect Human All Exon V7 (Agilent Technologies Inc.) according to standard protocol. Tumor and blood libraries were pooled in ratio 9:1 before sequencing. Sequencing was performed on the NovaSeq 6000 system (Illumina Inc.) using S4 chemistry (version 1.5) with 2 x 150 cycle setup.

Gene fusion detection was performed on mRNA isolated from a FFPE tumor sample using the Illumina NovaSeq platform (Illumina, Inc., San Diego, CA) and Agilent SureSelect Human All Exon V7 bait panel (Agilent Technologies, Santa Clara, CA). FFPE specimens underwent pathology review to diagnose percent tumor content and tumor size; a minimum of 10% of tumor content in the area for microdissection was required to enable enrichment and extraction of tumor-specific RNA. Qiagen RNA FFPE tissue extraction kit was used for extraction, and the RNA quality and quantity was determined using the Agilent TapeStation. Biotinylated RNA baits were hybridized to the synthesized and purified cDNA targets and the bait-target complexes were amplified in a post capture PCR reaction. The resultant libraries were quantified, normalized and the pooled libraries are denatured, diluted and sequenced; the reference genome used was GRCh37/hg19 and analytical validation of this test demonstrated ≥97% Positive Percent Agreement (PPA), ≥99% Negative Percent Agreement (NPA) and ≥99% Overall Percent Agreement (OPA) with a validated comparator method.