Integration of genomics, metagenomics, and metabolomics to identify interplay between susceptibility alleles and microbiota in adenoma initiation.

BACKGROUND: Colorectal cancer (CRC) is a multifactorial disease resulting from both genetic predisposition and environmental factors including the gut microbiota (GM), but deciphering the influence of genetic variants, environmental variables, and interactions with the GM is exceedingly difficult. We previously observed significant differences in intestinal adenoma multiplicity between C57BL/6 J-ApcMin (B6-Min/J) from The Jackson Laboratory (JAX), and original founder strain C57BL/6JD-ApcMin (B6-Min/D) from the University of Wisconsin. METHODS: To resolve genetic and environmental interactions and determine their contributions we utilized two genetically inbred, independently isolated ApcMin mouse colonies that have been separated for over 20 generations. Whole genome sequencing was used to identify genetic variants unique to the two substrains. To determine the influence of genetic variants and the impact of differences in the GM on phenotypic variability, we used complex microbiota targeted rederivation to generate two Apc mutant mouse colonies harboring complex GMs from two different sources (GMJAX originally from JAX or GMHSD originally from Envigo), creating four ApcMin groups. Untargeted metabolomics were used to characterize shifts in the fecal metabolite profile based on genetic variation and differences in the GM. RESULTS: WGS revealed several thousand high quality variants unique to the two substrains. No homozygous variants were present in coding regions, with the vast majority of variants residing in noncoding regions. Host genetic divergence between Min/J and Min/D and the complex GM additively determined differential adenoma susceptibility. Untargeted metabolomics revealed that both genetic lineage and the GM collectively determined the fecal metabolite profile, and that each differentially regulates bile acid (BA) metabolism. Metabolomics pathway analysis facilitated identification of a functionally relevant private noncoding variant associated with the bile acid transporter Fatty acid binding protein 6 (Fabp6). Expression studies demonstrated differential expression of Fabp6 between Min/J and Min/D, and the variant correlates with adenoma multiplicity in backcrossed mice. CONCLUSIONS: We found that both genetic variation and differences in microbiota influences the quantitiative adenoma phenotype in ApcMin mice. These findings demonstrate how the use of metabolomics datasets can aid as a functional genomic tool, and furthermore illustrate the power of a multi-omics approach to dissect complex disease susceptibility of noncoding variants.

Structural and diffusion weighted MRI demonstrates responses to ibrutinib in a mouse model of follicular helper (Tfh) T-cell lymphoma.

Recent analyses of the genetics of peripheral T-cell lymphoma (PTCL) have shown that a large proportion of cases are derived from normal follicular helper (Tfh) T-cells. The sanroque mouse strain bears a mutation that increases Tfh cell number and heterozygous animals (Roquinsan/+) develop lymphomas similar to human Tfh lymphoma. Here we demonstrate the usefulness of Roquinsan/+ animals as a pre-clinical model of Tfh lymphoma. Long latency of development and incomplete penetrance in this strain suggests the lymphomas are genetically diverse. We carried out preliminary genetic characterisation by whole exome sequencing and detected tumor specific mutations in Hsp90ab1, Ccnb3 and RhoA. Interleukin-2-inducible kinase (ITK) is expressed in Tfh lymphoma and is a potential therapeutic agent. A preclinical study of ibrutinib, a small molecule inhibitor of mouse and human ITK, in established lymphoma was carried out and showed lymphoma regression in 8/12 (67%) mice. Using T2-weighted MRI to assess lymph node volume and diffusion weighted MRI scanning as a measure of function, we showed that treatment increased mean apparent diffusion coefficient (ADC) suggesting cell death, and that change in ADC following treatment correlated with change in lymphoma volume. We suggest that heterozygous sanroque mice are a useful model of Tfh cell derived lymphomas in an immunocompetent animal.

Sixteen diverse laboratory mouse reference genomes define strain-specific haplotypes and novel functional loci.

We report full-length draft de novo genome assemblies for 16 widely used inbred mouse strains and find extensive strain-specific haplotype variation. We identify and characterize 2,567 regions on the current mouse reference genome exhibiting the greatest sequence diversity. These regions are enriched for genes involved in pathogen defence and immunity and exhibit enrichment of transposable elements and signatures of recent retrotransposition events. Combinations of alleles and genes unique to an individual strain are commonly observed at these loci, reflecting distinct strain phenotypes. We used these genomes to improve the mouse reference genome, resulting in the completion of 10 new gene structures. Also, 62 new coding loci were added to the reference genome annotation. These genomes identified a large, previously unannotated, gene (Efcab3-like) encoding 5,874 amino acids. Mutant Efcab3-like mice display anomalies in multiple brain regions, suggesting a possible role for this gene in the regulation of brain development.

Whole Genome Sequence of Two Wild-Derived Mus musculus domesticus Inbred Strains, LEWES/EiJ and ZALENDE/EiJ, with Different Diploid Numbers.

Wild-derived mouse inbred strains are becoming increasingly popular for complex traits analysis, evolutionary studies, and systems genetics. Here, we report the whole-genome sequencing of two wild-derived mouse inbred strains, LEWES/EiJ and ZALENDE/EiJ, of Mus musculus domesticus origin. These two inbred strains were selected based on their geographic origin, karyotype, and use in ongoing research. We generated 14× and 18× coverage sequence, respectively, and discovered over 1.1 million novel variants, most of which are private to one of these strains. This report expands the number of wild-derived inbred genomes in the Mus genus from six to eight. The sequence variation can be accessed via an online query tool; variant calls (VCF format) and alignments (BAM format) are available for download from a dedicated ftp site. Finally, the sequencing data have also been stored in a lossless, compressed, and indexed format using the multi-string Burrows-Wheeler transform. All data can be used without restriction.

Deep genome sequencing and variation analysis of 13 inbred mouse strains defines candidate phenotypic alleles, private variation and homozygous truncating mutations.

BACKGROUND: The Mouse Genomes Project is an ongoing collaborative effort to sequence the genomes of the common laboratory mouse strains. In 2011, the initial analysis of sequence variation across 17 strains found 56.7 M unique single nucleotide polymorphisms (SNPs) and 8.8 M indels. We carry out deep sequencing of 13 additional inbred strains (BUB/BnJ, C57BL/10J, C57BR/cdJ, C58/J, DBA/1J, I/LnJ, KK/HiJ, MOLF/EiJ, NZB/B1NJ, NZW/LacJ, RF/J, SEA/GnJ and ST/bJ), cataloguing molecular variation within and across the strains. These strains include important models for immune response, leukaemia, age-related hearing loss and rheumatoid arthritis. We now have several examples of fully sequenced closely related strains that are divergent for several disease phenotypes. RESULTS: Approximately 27.4 M unique SNPs and 5 M indels are identified across these strains compared to the C57BL/6 J reference genome (GRCm38). The amount of variation found in the inbred laboratory mouse genome has increased to 71 M SNPs and 12 M indels. We investigate the genetic basis of highly penetrant cancer susceptibility in RF/J finding private novel missense mutations in DNA damage repair and highly cancer associated genes. We use two highly related strains (DBA/1J and DBA/2J) to investigate the genetic basis of collagen-induced arthritis susceptibility. CONCLUSIONS: This paper significantly expands the catalogue of fully sequenced laboratory mouse strains and now contains several examples of highly genetically similar strains with divergent phenotypes. We show how studying private missense mutations can lead to insights into the genetic mechanism for a highly penetrant phenotype.

The Mouse Genomes Project: a repository of inbred laboratory mouse strain genomes.

The Mouse Genomes Project was initiated in 2009 with the goal of using next-generation sequencing technologies to catalogue molecular variation in the common laboratory mouse strains, and a selected set of wild-derived inbred strains. The initial sequencing and survey of sequence variation in 17 inbred strains was completed in 2011 and included comprehensive catalogue of single nucleotide polymorphisms, short insertion/deletions, larger structural variants including their fine scale architecture and landscape of transposable element variation, and genomic sites subject to post-transcriptional alteration of RNA. From this beginning, the resource has expanded significantly to include 36 fully sequenced inbred laboratory mouse strains, a refined and updated data processing pipeline, and new variation querying and data visualisation tools which are available on the project's website ( http://www.sanger.ac.uk/resources/mouse/genomes/ ). The focus of the project is now the completion of de novo assembled chromosome sequences and strain-specific gene structures for the core strains. We discuss how the assembled chromosomes will power comparative analysis, data access tools and future directions of mouse genetics.

Whole genome association study identifies regions of the bovine genome and biological pathways involved in carcass trait performance in Holstein-Friesian cattle.

BACKGROUND: Four traits related to carcass performance have been identified as economically important in beef production: carcass weight, carcass fat, carcass conformation of progeny and cull cow carcass weight. Although Holstein-Friesian cattle are primarily utilized for milk production, they are also an important source of meat for beef production and export. Because of this, there is great interest in understanding the underlying genomic structure influencing these traits. Several genome-wide association studies have identified regions of the bovine genome associated with growth or carcass traits, however, little is known about the mechanisms or underlying biological pathways involved. This study aims to detect regions of the bovine genome associated with carcass performance traits (employing a panel of 54,001 SNPs) using measures of genetic merit (as predicted transmitting abilities) for 5,705 Irish Holstein-Friesian animals. Candidate genes and biological pathways were then identified for each trait under investigation. RESULTS: Following adjustment for false discovery (q-value < 0.05), 479 quantitative trait loci (QTL) were associated with at least one of the four carcass traits using a single SNP regression approach. Using a Bayesian approach, 46 QTL were associated (posterior probability > 0.5) with at least one of the four traits. In total, 557 unique bovine genes, which mapped to 426 human orthologs, were within 500kbs of QTL found associated with a trait using the Bayesian approach. Using this information, 24 significantly over-represented pathways were identified across all traits. The most significantly over-represented biological pathway was the peroxisome proliferator-activated receptor (PPAR) signaling pathway. CONCLUSIONS: A large number of genomic regions putatively associated with bovine carcass traits were detected using two different statistical approaches. Notably, several significant associations were detected in close proximity to genes with a known role in animal growth such as glucagon and leptin. Several biological pathways, including PPAR signaling, were shown to be involved in various aspects of bovine carcass performance. These core genes and biological processes may form the foundation for further investigation to identify causative mutations involved in each trait. Results reported here support previous findings suggesting conservation of key biological processes involved in growth and metabolism.

Snpdat: easy and rapid annotation of results from de novo snp discovery projects for model and non-model organisms.

BACKGROUND: Single nucleotide polymorphisms (SNPs) are the most abundant genetic variant found in vertebrates and invertebrates. SNP discovery has become a highly automated, robust and relatively inexpensive process allowing the identification of many thousands of mutations for model and non-model organisms. Annotating large numbers of SNPs can be a difficult and complex process. Many tools available are optimised for use with organisms densely sampled for SNPs, such as humans. There are currently few tools available that are species non-specific or support non-model organism data. RESULTS: Here we present SNPdat, a high throughput analysis tool that can provide a comprehensive annotation of both novel and known SNPs for any organism with a draft sequence and annotation. Using a dataset of 4,566 SNPs identified in cattle using high-throughput DNA sequencing we demonstrate the annotations performed and the statistics that can be generated by SNPdat. CONCLUSIONS: SNPdat provides users with a simple tool for annotation of genomes that are either not supported by other tools or have a small number of annotated SNPs available. SNPdat can also be used to analyse datasets from organisms which are densely sampled for SNPs. As a command line tool it can easily be incorporated into existing SNP discovery pipelines and fills a niche for analyses involving non-model organisms that are not supported by many available SNP annotation tools. SNPdat will be of great interest to scientists involved in SNP discovery and analysis projects, particularly those with limited bioinformatics experience.