GENOTYPING BY SEQUENCING OF CANIS FAMILIARIS USING iGENOMX RIPTIDE™ DNA LIBRARY PREPARATION

Azeem Siddique,1,3 Gaia Suckow,1,3 Nils Homer,2 Phillip Ordoukhanian,1,3 Steve Head,1,3 Keith Brown3 ([email protected]), Paul Doran5, Matt Huentelman6, Joseph Pickrell5, Alvaro Gonzalo Hernandez 4, 1The Scripps Research Institute, La Jolla, CA; 2Fulcrum Genomics, Somerville, MA; 3iGenomX, Carlsbad, CA; 4University of Illinois at Urbana-Champaign, Urbana, IL; 5Gencove, New York, NY; 6Tgen, Phoenix, AZ

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Abstract

Dogs have been living with humans for approximately 15000 years. Selective breeding has created a multitude of dog breeds with distinct characteristics. Great interest exists in understanding how selection has affected the modern dog genome and what variants are linked to specific canine breed characteristics. Dogs are also susceptible to a number of diseases that have counterparts in humans. Their unique population structure, relatively limited heterogeneity within breeds, greater genome sequence identity to humans than mice, and their sharing of a common environment with humans make them an excellent model organism for certain human diseases.

The iGenomX RipTide library prep is a high throughput DNA library prep for next generation sequencing that has been used to prepare libraries for a variety of applications where large numbers of samples require library preparation at low cost. One such application is genotyping by sequencing. Here we show the use of the RipTide library prep in a case control GWAS study, generating over 30 million biallelic SNPs per sample on a cohort of West Highland White Terriers. After filtering, more than 5.2 million SNPs were identified with a minor allele frequency of >5%. PCA analysis showed that the variants permitted the accurate identification of breeds. The data also showed a novel genetic association with Westie lung disease, the canine equivalent of chronic obstructive pulmonary disease in humans.

The iGenomX RipTide library prep combined with Illumina sequencing generated more variants in less time and at lower cost than the standard microarray-based genotyping experiment.

Schematic of the RipTide Workflow

A) Random primers with 5’ barcoded Illumina adapter sequences are annealed to a denatured DNA template (Figure 1). A polymerase extends each primer, generating a copy of the DNA template. Polymerization is terminated with a biotinylated dideoxynucleotide of which there is a small fraction in the nucleotide mix. B) Primer-extended products are captured on streptavidin coated magnetic beads. The beads are washed to remove excess reactants. C) A second 5’ adapter-tailed random primer is used with a strand-displacing polymerase to convert the captured DNA strands to a dual adapter library. D) The beads are washed once again to remove excess reactants and displaced primer-extended products. E) PCR is used to amplify the products and add an index barcode. In the high throughput version of the library prep, individual samples are uniquely labelled in a 96-well plate with the use of a uniquely barcoded random primer in each well of the plate. After the initial labelling step, products from all wells are pooled and all subsequent steps are performed in a single tube. An optional plate barcode is added during the PCR step to allow for multiple 96-sample plates to be sequenced simultaneously.

Discussion Points

  • Genotyping Rate (GR) over 95% in our study sample results in 29.7M SNPs
  • Over 5.2M SNPs have a study-wide (e.g. case and control) minor allele frequency (MAF) of
    greater than 5%. This category of SNPs are the most useful for GWAS. This is over 50X more
    variants for analysis vs. the leading canine microarray.
  • Current canine arrays can result in high amounts (30-60%) of monomorphic SNPs depending on
    the breed under study. This is because the array relies on static content – RIPTIDE avoids this
    and can therefore generate higher numbers of informative markers.
  • RIPTIDE results in a more comprehensive SNP set for analysis that was generated in less time
    and with less cost than standard array genotyping.

Conclusions

• Sequencing based genotyping is a logical replacement to microarrays for population
genetics research.
• More markers, and more samples improve statistical power.
• Sequencing avoids ascertainment bias from a fixed content microarray.
• Cost and throughput dramatically improved with RIPTIDE.

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