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. 2018 Jun 20;19(1):487.
doi: 10.1186/s12864-018-4858-8.

Whole exome sequencing in the rat

Julie F Foley  1 , Dhiral P Phadke  2 , Owen Hardy  3 , Sara Hardy  3 , Victor Miller  3 , Anup Madan  4 , Kellie Howard  5 , Kimberly Kruse  5 , Cara Lord  4 , Sreenivasa Ramaiahgari  6 , Gregory G Solomon  7 , Ruchir R Shah  2 , Arun R Pandiri  8 , Ronald A Herbert  8 , Robert C Sills  8 , B Alex Merrick  6
Affiliations

Whole exome sequencing in the rat

Julie F Foley et al. BMC Genomics. .

Abstract

Background: 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. 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.

Results: Informatics 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.

Conclusions: An 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.

Keywords: C6; COSMIC; DSL-6A/C1; FAT7; NBTII; Next generation sequencing; Sanger; Whole exome sequencing.

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Conflict of interest statement

Ethics approval

Experiments were performed according to the guidelines established in the NIH Guide for the Care and Use of Laboratory Animals (National Research Council, 2011). Animals were treated humanely for alleviation of potential suffering, as approved by the National Institute of Environmental Health Sciences Animal Care and Use Committee.

Competing interests

JFF, SR, GGS, RAH, RCS and BAM are employees of the National Institute of Environmental Health Sciences (NIEHS), National Institutes of Health (NIH), of the United States Government. The views expressed in this article are those of the authors and do not necessarily reflect the official policy or position of NIEHS, NIH or the United States Government. DPP and RRS are Bioinformaticians at Sciome, LLC and performed exome sequence location and annotation under NIEHS contract HHSN273201700001C under NIEHS supervision. OH, VM and SH comprised the team from Agilent Technologies that formulated the probe library for exome enrichment. JFF, SR, GGS, RAH, RCS and BAM supplied biological materials and isolated DNA to test the probe library for exome enrichment provided by Agilent. The NIEHS Epigenomics core facility (GGS) performed the exome sequencing. KH, KK, CL and AM are researchers at the Genomics Laboratory of LabCorp-Covance performed Sanger validation of targeted sequence variants under NIEHS contract HHSN273201500005I under NIEHS supervision. Neither Sciome, LabCorp-Covance or Agilent contributed financial funding for publication of this work. The authors adhere to policies on public sharing of data and materials.

Figures

Fig. 1
Fig. 1
Bioinformatic evaluation pipeline for variant detection. Initially, raw reads were mapped and trimmed followed by targeted coverage analysis, filtration and functional annotation of variant calls. The resulting calls were then compared to validated cancer gene variants in the human COSMIC database. The final analysis involved assessment of the mutational spectrum from the tested samples
Fig. 2
Fig. 2
Breadth of reference genome coverage. The percentage of target bases covering the rat Rn6 reference genome is shown at 1X, 10X, 20X, 30X and 50X depth of coverage. Rigourous testing at 50X demonstrated strong coverage of the rat reference genome by the sequenced fragments
Fig. 3
Fig. 3
Uniformity of coverage for up to 500 bp reads for the cell lines and paired FF-FFPE samples. a-d Depth of coverage distribution for the C6, FAT7, DSL-6A/C1 and NBTII cell lines. e-h Depth of coverage distribution for the fresh frozen (FF) liver tissue. i-l) Depth of coverage distribution for the formalin-fixed, paraffin-embedded (FFPE) liver tissue
Fig. 4
Fig. 4
Mutational spectrum of the rat exome-seq data. a All exonic variants captured across all samples in the 71 Mb design (50 Mb + UTRs) plus the dbSNP variants were plotted using the Kullback-Leibler divergence. a A high frequency of C > T and T > C mutations presented with minimal observed differences in the mutational spectrum across all samples and dbSNP. b Hierarchical clustering grouped dbSNP with the normal, FF-FFPE tissue and showed divergence of these groups from the tumor cell lines
Fig. 5
Fig. 5
Mutational spectrum of cell line specific exonic variants. a Filtered for FF-FFPE variants from the exome-seq data separated dbSNP from the tumor cell line samples. b Based on the hierarchical clustering of cell specific variants filtered from FF-FFPE and dbSNP variants, the mutational spectrum of the rat exome-seq data is closest to COSMIC Signatures 16, 5, 8 and 3
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References

    1. Gibbs RA, Weinstock GM, Metzker ML, Muzny DM, Sodergren EJ, Scherer S, et al. Genome sequence of the brown Norway rat yields insights in to mammalian evolution. Nat. 2004;428:493–521. doi: 10.1038/nature02426. - DOI - PubMed
    1. Sollner JF, Leparc G, Hildebrandt T, Klein H, Thomas L, Stopka E, et al. An RNA-Seq atlas of gene expression in mouse and rat normal tissues. Sci Data. 2017;4:170–185. doi: 10.1038/sdata.2017.185. - DOI - PMC - PubMed
    1. Shimoyama M, Smith JR, Bryda E, Kuramoto T, Saba L, Dwinell M. Rat genome and model resources. ILAR J. 2017;58:42–58. doi: 10.1093/ilar/ilw041. - DOI - PMC - PubMed
    1. Shimoyama M, Laulederkind SJ, De Pons J, Smith JR, Tutaj M, Petri V, Hayman GT, et al. Exploring human disease using the rat genome database. Dis Model Mech. 2016;9:1089–1095. doi: 10.1242/dmm.026021. - DOI - PMC - PubMed
    1. Meek S, Mashimo T, Burdon T. From engineering to editing the rat genome. Mamm Genome. 2017;28:302–314. doi: 10.1007/s00335-017-9705-8. - DOI - PMC - PubMed

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