Exome Sequencing Banner

Exons of protein coding or important regulatory RNA genes make up only 1-2% of the three billion nucleotides of sequence in the human genome. The protein-coding regions alone contain 80-90% of the known disease-related variants with large effects on traits in human1. Likewise, in many important agriculture and research model species the total genome length dwarfs the exonic regions. By targeting only the coding regions of genomic DNA, exome sequencing has emerged as a lower cost and targeted next-generation sequencing approach to the identification of novel and known pathogenic or disease-associated polymorphisms in patient samples for the purposes of clinical diagnosis, companion-diagnostics development, and disease research. Exome sequencing is based on hybridization-based capture of sample DNA molecules using a library of oligonucleotide probes designed to target the exonic regions of the genome. Using this approach, the direct cost of sequencing can be reduced 50-fold at a given coverage depth, relative to whole genome sequencing, while not sacrificing the detection of sequence variants with important phenotypic associations. Additionally, by ignoring the 98% of DNA that is under relatively less selective pressure then coding exons, statistical power for identification of polymorphisms associated with a variety of phenotypic classes (e.g. disease prognosis, treatment efficacy, disease resistance) increases.

The high depth of coverage possible with exome sequencing makes it an ideal tool for studying single-nucleotide variants, insertions, and deletions associated with diseases. Additional targeted human exome panels have recently been developed which provide full coverage of protein-coding exons from specific sets of genes (e.g. oncology-associated), further lowering the cost and complexity of identifying phenotype-associated polymorphisms. Moreover, exome panels have also become available for common research animals and for important agricultural plant and animal species. ORB offers exome sequencing services for a wide variety of species, sample types, and exome panels, some of which are described below.

Project work flow

Fig. 1 Hybridization Explanation.

Figure 1. : Simplified illustration hybridization target selection.

  1. DNA Isolation: Genomic DNA (gDNA) is isolated from each sample. The extracted gDNA is quantified to evaluate quality of the isolated DNA.
  2. Fragmentation: Fragmentation of gDNA is performed using dsDNA Fragmentase. The degree of fragmentation is evaluated to ensure that the size is within a desired range.
  3. Library preparation: Fragmented gDNA is used to generate DNA libraries using the kit best suited to the sample input.
  4. Exome Selection: DNA library pools are created from the samples and are hybridized and selected for exonic sequences. The size distribution and general yield of the exome-selected library pools are measured prior to sequencing.
  5. Sequencing: The exome-selected library pools are combined to produce a final pool. Libraries are diluted, denatured and sequenced using Illumina instrumentation.
  6. Post Sequencing Data Processing: The reads are trimmed of adapter sequences, and low-quality bases are removed from the read termini. Reads are then aligned to the hg38 reference genome. Duplicate reads are removed, and quality tables are recalculated. Variant calling is performed and the found variants are filtered and annotated.
Fig. 1 Hybridization Explanation.

Figure 2. : Flow chart showing a few key step in the overall sample handling process.

Service Specifications

  • Submitted Sample : ORB provides Genomic DNA (gDNA) isolation for multiple sample types. ORB is a Biosafety Level 2 (BSL-2) laboratory, and can accept samples which contain agents associated with human diseases.
    • FFPE: Submit a minimum of four 10 uM sections or two 20 uM sections having an area of at least 1 cm2 and containing at least 50% tissue in the section. Punches with a total weight of at least 5 mg may also be submitted. ORB also accepts paraffin sections on slides.
    • Serum or Plasma: Minimum sample size 4 mL for human. Please inquire about other species.
    • Frozen Tissue: Submit at least 3 mg of frozen tissue.
    • Whole blood: Submit a minimum of one PaxGene tube. Please inquire if submitting rat our mouse blood.
    • Other: ORB accepts many other sample types including PBMCs, flow-sorted cells, plant tissue, and more. Please contact us for more information.
  • Library Preparation : ORB utilizes the latest library preparation kits from Nugen, lllumina, and Takara to cover the range of projects from ultra-low (30 pg) to standard input (2 ug).
  • Exome Selection : ORB utilizes target selection kits from Kapa Biosystems (Roche Sequencing),Twist Bioscience, and Swift Biosciences. Some popular choices are listed below, but a large variety of panels are available including focused oncology panels covering 100 to 1000 genes. Please contact us to discuss which option would be best suited for your project.
    • Human:
      • Swift Exome: Panel covers 39 Mb target region with 429,826 probe sets. A total of 19,396 hg19 genes are covered. Designed to work with the low DNA mass available from cell-free DNA and to provide a high on-target rate to minimize sequencing cost.
      • Twist Human Core Exome: Probe set has been optimized to minimize the target region to 33 Mb while still covering 99% of ClinVar variants. The design is based on CCDS v20 coordinates on hg38.
      • Roche SeqCap EZ MedExome: Consists of a newer set of hybridization probes based on  the hg38 genome build. The 47 Mb design covers exons from multiple databases including CCDS v17, RefSeq CDS August 2014, Ensembl v76 CDS, VEGA v56 CDS, Gencode v20 CDS, and miRBase 21. Includes additional coverage of non-coding regions considered medically relevant.
      • Roche SeqCap EZ Exome + UTR Probes: An exhaustive set of hybridization probes including 64 Mb of core coding exons and miRNA regions plus an additional 32 Mb of 3'-UTR sequences. The core probe sets are based on the GRCh37 (hg19) genome sequence and cover the exons of more than 20,000 genes including the flanking regions of some coding exons and microRNAs. The UTR probe sets are based on NCBI RefSeq coordinates obtained from GRCh37 version 64.
    • Mouse:
      • Roche SeqCap EZ Design Mouse Exome: Panel is a design developed by Roche Sequencing in collaboration with a client. It is based on the UCSC mm9 version of the mouse genome. Probes cover 54.3 Mb of target sequence (primarily exons).
      • Twist Bioscience Mouse Exome Panel is a newer design with a smaller footprint (37.7 Mb) than the Roche panel. The design is based on the UCSC mm10 genome build and was built to provide coverage of exons from multiple major annotation databases.
    • Rat: Please inquire for current kit availability
    • Agriculture: Roche SeqCap EZ Design Exome for Wheat, Barley, Maize, Switchgrass, and Soy.
  • Sequencing : ORB performs sequencing using the NextSeq 500, HiSeq 4000, or NovaSeq 6000 depending on the project size and specific application. Please visit our Illumina sequencing page for details about these sequencing instruments, metrics, and the typical applications at ORB.

RNA Exome

A highly cited 2015 study from Cieslik et al demonstrated that exome capture of cDNA libraries prepared from severely degraded RNA extracted from formalin-fixed paraffin-embedded (FFPE) tissue greatly improved the quality of sequencing data obtained as compared to alternative cDNA library preparation techniques that relied upon polyA-capture or rRNA depletion to obtain libraries enriched in protein-coding cDNA sequences. Performance for hybridization-based capture of exon-containing cDNA library molecules greatly exceeded the other two methods in detection of variants, gene fusions, and splicing junctions in RNA from FFPE samples2. ORB scientists have tested and confirmed the superior performance of the Illumina TruSeq RNA Exome kit for performing gene expression analysis by sequencing as compared to alternative approaches using Takara’s SMARTer Stranded Total RNA-Seq Kit v2-Pico Input Mammalian and NuGen’s Ovation Human FFPE RNA-Seq Multiplex System 1-8 kits (not shown). ORB offers gene expression assays utilizing the TruSeq RNA Exome kit and can work with tissue blocks, and both mounted and unmounted sections. Please inquire for more information.

Demonstration Project

Click to see the results of the sample study.

To demonstrate and validate our capabilities, we performed exome sequencing on FFPE tissues derived from cancer patients aged 43-78 years. In this study both tumor and normal adjacent tissue (NAT) were isolated for each subject. The genomic DNA was isolated from two 20 µm sections of FFPE blocks collected from six subjects, and four 20 µm sections collected from two additional subjects. The DNA libraries were subsequently enriched for exonic regions using the Roche Prime Exome kit.


ORB has generated a set of proven SOPs for sequencing of specifically targeted regions of the genome using exome capture kits. Clients may submit isolated genomic DNA or alternatively ORB scientists can extract the DNA from a variety of sample types including frozen tissue, whole blood, PBMC's, flow sorted cells, FFPE blocks or sections, and cell-free biofluids (e.g. plasma / serum). Genomic DNA isolation can be performed from as little as two 20 µm sections from FFPE blocks and this enables the parsimonious use of archived disease tissue that are only available in limited quantities. The full suite of lab services is complemented by a regularly updated bioinformatic pipeline for variant calling that utilizes current databases. Reports are customized to meet the needs of clients. Whether the applications is disease association, oncology discovery, companion diagnostics development, or crop science, ORB can assist you to complete your variant analysis project in a timely and fruitful manner!