Whole genome sequencing is performed by shotgun sequencing of DNA fragments.  While the sample prep and sequencing is routine for whole genome projects, VANTAGE strongly recommends informatics consultation prior to undertaking any whole genome sequencing project.

Standard short-insert Paired-End libraries are generally adequate for whole genome coverage.  Mate-pair (or long-insert) 2-5 kb insert libraries can be generated at an added cost and are most effective for use on de novo genome sequencing.  The combination of short-insert and long-insert libraries greatly increase the physical coverage and ability to generate long contigs from the short reads.

For review of whole genome sequencing on the Illumina platform:

Bentley et al. "Accurate Whole Human Genome Sequencing using Reversible Terminator Chemistry", Nature 456(7218): 53-59 (2008).

Sample submission requirements:

  1. DNA: 1 to 5 ug genomic DNA, in < 100 ul nuclease-free water or TE.  DNA submitted will be assessed for quality, an acceptable sample should have an A260/280 ratio 1.8 - 2.0 and majority of high molecular weight with little migration when run on a 2% agarose gel. FFPE DNA accepted but must be noted on sample submission sheet.

Service Description:

  1. 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.
  2. Quality Assessment of DNA sample: Incoming DNA is measured for quantity using fluorometry (Qubit or Picogreen) and integrity by running on a 2% gel. 
  3. Library Construction: DNA samples are converted into a sequencing library suitable for subsequent target enrichment,  cluster generation and sequencing.
  4. 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.
  5. 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.
  6. Sequencing: Samples will be sequenced on the Illumina HiSeq 3000 in High Throughput or Rapid run mode.  
  7. 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.