Experimental Workflow

   The SureSelect Target Enrichment workflow is solution-based system utilizing ultra-long - 120 mer biotinylated cRNA baits - to capture regions of interest, enriching them out of a NGS genomic fragment library.

   To generate standard exome capture libraries, we use the Agilent SureSelectXT Low Input Target Enrichment protocol for Illumina paired-end sequencing library with 1ug of input gDNA. The DNA quantity and quality is measured by PicoGreen and agarose gel electrophoresis, respectively. We use 1μg of each cell line's genomic DNA diluted with EB Buffer and sheared to a target peak size of 150–200 bp with the Covaris LE220 focused-ultrasonicator (Covaris, Woburn, MA) according to the manufacturer's recommendations. Fragmentation is followed by end-repair and the addition of an ‘A’ tail. Agilent adapters are then ligated to the fragments. After assessing the efficiency of ligation, the adapter ligated product is PCR amplified. The final purified product is quantified by TapeStation DNA screentape D1000 (Agilent). For exome capture, 250 ng of DNA library is mixed with hybridization buffers, blocking mixes, RNase block and 5 µl of SureSelect all exon capture library, according to the standard Agilent SureSelect Target Enrichment protocol. The captured DNA is washed and amplified. Then final purified product is quantified by qPCR according to the qPCR Quantification Protocol Guide Guide (KAPA Library Quantificatoin kits for Illumina Sequecing platforms) and qualified by the TapeStation DNA screentape D1000 (Agilent).

   Illumina utilizes a unique amplification reaction that occurs on the surface of the flow cell. A flow cell containing millions of unique clusters is loaded into the Illumina platform for automated cycles of extension and imaging. Sequencing-by-Synthesis utilizes four proprietary nucleotides possessing reversible fluorophore and termination properties. Each sequencing cycle occurs in the presence of all four nucleotides leading to higher accuracy than methods where only one nucleotide is present in the reaction mix at a time. This cycle is repeated, one base at a time, generating a series of images each representing a single base extension at a specific cluster.

   The Illumina platform generates raw images and base calling through integrated primary analysis software called RTA(Real Time Analysis). The base calling files which are expressed in binary are converted into FASTQ by Illumina package bcl2fastq v2.20.0. The demultiplexing option (--barcode-mismatches) is set to as value : 0