There are multiple commercial providers that supply targeted enrichment kits and differ on cost and strategy. The most common target enrichment performed by VANTAGE is whole exome sequencing. We have over three years experience with Agilent, Illumina, and Nimblegen exome capture kits as well as custom content capture kits. Consultation with VANTAGE staff will allow investigators to choose the appropriate and cost effective method for their study.
Sample submission requirements:
- DNA: 1 to 3 ug genomic DNA, <100 ul in nuclease-free water or TE. FFPE DNA is also accepted but should be noted on the project creation sheet. FFPE DNA requires additional QC prior to being accepted for library creation.
- Project Planning and Consultation: It is recommended and encouraged to consult with VANTAGE staff prior to sample submission. This will allow both the user and VANTAGE staff to fully understand and agree on service being provided and costs incurred. Contac the VANGARD core for experimental design and analysis.
- Quality Assessment of DNA sample: Incoming DNA is measured for quantity using fluorometry (Qubit or Picogreen) and integrity by running on a 2% gel.
- Library Construction: DNA samples are converted into a sequencing library suitable for subsequent target enrichment, cluster generation and sequencing.
- qPCR quantification: Each library that passes the final QC undergoes qPCR using the KAPA Biosystems library quant kit on the Agilent Mx3005P. Using qPCR allows highly accurate quantification of library molecules that can seed clusters and allow target sample representation during pooling.
- Pooling (as requested): Multiplexed samples are pooled in equimolar or user-defined ratios to achieve the multiplexing level requested by the user. Up to 96-plex per lane is currently supported by Illumina.
- Sequencing: Samples will be sequenced on the Illumina HiSeq 2500 in High Throughput or Rapid run mode.
- Data processing and release: Sequencing data is processed through Illumina's CASAVA v1.8.2 pipeline. Users will be provided with de-multiplexed, sample specific data in a zipped FASTQ format.