Library preparation and sequencing
DNA samples are first sheared to an average of 350 bp using Covaris S2 focused ultrasonicator. Next the fragmented DNA undergoes a number of enzymatic reactions, a size selection step and several clean up steps to yield a final product that is an adaptor-ligated library with a unique identifier, index, for each sample. The library preparation is carried out using the Illumina TruSeq DNA PCR-free LT library preparation kit (FC-121-3001). The concentration of the library is determined using a quantitative PCR assay.
DNA samples are first sheared to an average of 300 bp using Covaris S2 focused ultrasonicator. Next the fragmented DNA undergoes a number of enzymatic reactions, a size selection, enrichment and several clean-up steps. The adapter-ligated DNA library is then mixed in a temperature controlled environment with specific RNA probes, targeting transcripts or exonic DNA sequences in the genome. The hybridized probe/sample construct are then enriched, followed by a tagging procedure to yield a unique index for each sample. The library preparation and enrichment is carried out using Agilent SureSelect version 5.0. The concentration of the library is determined using a quantitative PCR assay.
Libraries are normalized, pooled and denatured. Cluster generation is performed using bridge amplification to enrich for the adapter-ligated library. Sequencing is performed according to Sequencing by Synthesis (SBS) technology on the Illumina HiSeq2500 using the Rapid mode chemistry.
In each sequencing run there is the possibility to read one or two index reads. This enables parallel analysis of up to 96 samples per run, depending on the sequencing depth needed for each sample. According to manufacturer’s specification, a Rapid mode flow cell generates 300 M read pairs of raw data (at least 80% passing filter with Q30 >80%) for a 2x100bp run. If required, two flow cells can be run in parallel and the read length can be extended to 150 bp.
Each sample is sequenced to the depth required for the specific application and as ordered.