Whole Exome Next Generation Sequencing & Bioinformatics

Supported by the Federal government’s super science Initiative, the APN has successfully built unique infrastructure to capture the potential of next-generation DNA sequencing and use it to solve the challenge of accurately detecting ENU-induced protein-changing DNA variants in mice with heritable phenotypes.

This technology leap, employing exome capture and massively parallel DNA sequencing on the Illumina platform, has dramatically accelerated the rate that users can identify DNA variants that change a protein and phenotype of key body systems. This has already greatly increased access to useful mouse models of human disease for their research.

The exome sequencing pipeline opens up a new dimension for integrating human phenomics, molecular biology, and systems biology at each step in the pipeline of new mouse model production. Adding this dimension will greatly expand the accessibility of ENU gene-variant mouse models to users across the biotechnology and biomedical spectrum to have a major impact upon translational medicine.

ENU-induced DNA variants occur at a rate of ~1 variant nucleotide per million bases. By contrast, there is ~1 variant nucleotide per thousand bases between any two human genomes. Our DNA sequencing and bioinformatics pipeline therefore has established sequence analysis strategies that reduce the rate of false positive DNA variant calls by several orders of magnitude, in order that the majority identified are true-breeding, protein-changing DNA variants.

Several technological innovations enabled this goal to be achieved in a short timeframe. One was to increase the sequence depth over the protein-coding parts of the genome (the “exome”). To do this, the APF collaborated with two different technology providers, Agilent Inc and Nimblegen Inc, to help develop and test DNA capture kits that process mouse genomic DNA and enrich the 1.5% of this DNA that comprises protein-coding exons. Exome capture kits had just been developed for the human genome, but the APF initiative served as an important catalyst for applying the technology to the mouse genome.

The second innovation is development of a bioinformatic analysis pipeline that brings together state-of-the-art alignment and SNV calling software tools, and then overlays them with unique tools developed by the APN to filter out common sources of false positive DNA variant calls. To develop the latter, the APN exploited unique advantages of ENU mutagenesis in inbred mice that were critical to extend the envelope of sensitivity and specificity.

As a result, the team has successfully developed a sequencing and bioinformatics pipeline that now reliably detects the majority of homozygous and heterozygous protein-changing DNA variants in each mouse taken from an ENU pedigree with a low false positive rate. This new capability has already reduced the time-line between identifying a mouse strain with a valuable phenotype and identifying the DNA variant causing this phenotypic change from 2-4 years to under 6 months, resulting in a significant increase in productivity for APF users.

Follow this link to proceed to the Ordering page to submit samples for whole exome NGS