Abstract
BackgroundThe rat genome was sequenced in 2004 with the aim to improve human health altered by disease and environmental influences through gene discovery and animal model validation. Here, we report development and testing of a probe set for whole exome sequencing (WES) to detect sequence variants in exons and UTRs of the rat genome. Using an in-silico approach, we designed probes targeting the rat exome and compared captured mutations in cancer-related genes from four chemically induced rat tumor cell lines (C6, FAT7, DSL-6A/C1, NBTII) to validated cancer genes in the human database, Catalogue of Somatic Mutations in Cancer (COSMIC) as well as normal rat DNA. Paired, fresh frozen (FF) and formalin-fixed, paraffin-embedded (FFPE) liver tissue from naive rats were sequenced to confirm known dbSNP variants and identify any additional variants.ResultsInformatics analysis of available gene annotation from rat RGSC6.0/rn6 RefSeq and Ensembl transcripts provided 223,636 unique exons representing a total of 26,365 unique genes and untranslated regions. Using this annotation and the Rn6 reference genome, an in-silico probe design generated 826,878 probe sequences of which 94.2% were uniquely aligned to the rat genome without mismatches. Further informatics analysis revealed 25,249 genes (95.8%) covered by at least one probe and 23,603 genes (93.5%) had every exon covered by one or more probes. We report high performance metrics from exome sequencing of our probe set and Sanger validation of annotated, highly relevant, cancer gene mutations as cataloged in the human COSMIC database, in addition to several exonic variants in cancer-related genes.ConclusionsAn in-silico probe set was designed to enrich the rat exome from isolated DNA. The platform was tested on rat tumor cell lines and normal FF and FFPE liver tissue. The method effectively captured target exome regions in the test DNA samples with exceptional sensitivity and specificity to obtain reliable sequencing data representing variants that are likely chemically induced somatic mutations. Genomic discovery conducted by means of high throughput WES queries should benefit investigators in discovering rat genomic variants in disease etiology and in furthering human translational research.
Highlights
The rat genome was sequenced in 2004 with the aim to improve human health altered by disease and environmental influences through gene discovery and animal model validation
Fresh frozen tissue samples served as the “gold standard” for the rat whole exome sequencing (WES) evaluation to compare with paired FFPE tissue and the chemically induced, tumor cell lines with documented mutations selected as positive controls for obtaining exonic mutations in cancer-related genes
Enriched DNA fragments were sequenced on an Illumina HiSeq2500® platform from four paired fresh frozen (FF) and FFPE rat liver samples and four chemically induced, rat tumor cell lines
Summary
The rat genome was sequenced in 2004 with the aim to improve human health altered by disease and environmental influences through gene discovery and animal model validation. We report development and testing of a probe set for whole exome sequencing (WES) to detect sequence variants in exons and UTRs of the rat genome. A recent report on a rat probe set called TargetEC [8], based on the rat reference genome from assembly Rnor_5 was designed to capture coding exonic regions and conserved non-coding regulatory sequences from 13 vertebrate species. The large target size of capture probes at 146.8 Mb may limit the utility of this platform due to the sequence depth required for the detection of low frequency or rare sequence variants. Other novel approaches are needed to achieve sufficient performance that may enable evaluation of potentially human-relevant disease causing mutations in the rat genome
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