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Genetic mapping on fully sequenced individuals is transforming understanding of the relationship between molecular variation and variation in complex traits. Here we report a combined sequence and genetic mapping analysis in outbred rats that maps 355 quantitative trait loci for 122 phenotypes. We identify 35 causal genes involved in 31 phenotypes, implicating new genes in models of anxiety, heart disease and multiple sclerosis. The relationship between sequence and genetic variation is unexpectedly complex: at approximately 40% of quantitative trait loci, a single sequence variant cannot account for the phenotypic effect. Using comparable sequence and mapping data from mice, we show that the extent and spatial pattern of variation in inbred rats differ substantially from those of inbred mice and that the genetic variants in orthologous genes rarely contribute to the same phenotype in both species.

Background: Paired-tag sequencing approaches are commonly used for the analysis of genome structure. However, mammalian genomes have a complex organization with a variety of repetitive elements that complicate comprehensive genome-wide analyses., Results: Here, we systematically assessed the utility of paired-end and mate-pair (MP) next-generation sequencing libraries with insert sizes ranging from 170 bp to 25 kb, for genome coverage and for improving scaffolding of a mammalian genome (Rattus norvegicus). Despite a lower library complexity, large insert MP libraries (20 or 25 kb) provided very high physical genome coverage and were found to efficiently span repeat elements in the genome. Medium-sized (5, 8 or 15 kb) MP libraries were much more efficient for genome structure analysis than the more commonly used shorter insert paired-end and 3 kb MP libraries. Furthermore, the combination of medium- and large insert libraries resulted in a 3-fold increase in N50 in scaffolding processes. Finally, we show that our data can be used to evaluate and improve contig order and orientation in the current rat reference genome assembly., Conclusions: We conclude that applying combinations of mate-pair libraries with insert sizes that match the distributions of repetitive elements improves contig scaffolding and can contribute to the finishing of draft genomes.

The use of a priori knowledge in the alignment of targeted sequencing data is investigated using computational experiments. Adapting a Needleman-Wunsch algorithm to incorporate the genomic position information from the targeted capture, we demonstrate that alignment can be done to just the target region of interest. When in addition use is made of direct string comparison, an improvement of up to a factor of 8 in alignment speed compared to the fastest conventional aligner (Bowtie) is obtained. This results in a total alignment time in targeted sequencing of around 7 min for aligning approximately 56 million captured reads. For conventional aligners such as Bowtie, BWA or MAQ, alignment to just the target region is not feasible as experiments show that this leads to an additional 88% SNP calls, the vast majority of which are false positives (≈ 92%).

Background: With the advent of next generation sequencing it has become possible to detect genomic variation on a large scale. However, predicting which genomic variants are damaging to gene function remains a challenge, as knowledge of the effects of genomic variation on gene expression is still limited. Recombinant inbred panels are powerful tools to study the cis and trans effects of genetic variation on molecular phenotypes such as gene expression., Results: We generated a comprehensive inventory of genomic differences between the two founder strains of the rat HXB/BXH recombinant inbred panel: SHR/OlaIpcv and BN-Lx/Cub. We identified 3.2 million single nucleotide variants, 425,924 small insertions and deletions, 907 copy number changes and 1,094 large structural genetic variants. RNA-sequencing analyses on liver tissue of the two strains identified 532 differentially expressed genes and 40 alterations in transcript structure. We identified both coding and non-coding variants that correlate with differential expression and alternative splicing. Furthermore, structural variants, in particular gene duplications, show a strong correlation with transcriptome alterations., Conclusions: We show that the panel is a good model for assessing the genetic basis of phenotypic heterogeneity and for providing insights into possible underlying molecular mechanisms. Our results reveal a high diversity and complexity underlying quantitative and qualitative transcriptional differences.

Routine methods for assaying steady-state mRNA levels such as RNA-seq and micro-arrays are commonly used as readouts to study the role of transcription factors (TFs) in gene expression regulation. However, cellular RNA levels do not solely depend on activity of TFs and subsequent transcription by RNA polymerase II (Pol II), but are also affected by RNA turnover rate. Here, we demonstrate that integrated analysis of genome-wide TF occupancy, Pol II binding and steady-state RNA levels provide important insights in gene regulatory mechanisms. Pol II occupancy, as detected by Pol II ChIP-seq, was found to correlate better with TF occupancy compared to steady-state RNA levels and is thus a more precise readout for the primary transcriptional mechanisms that are triggered by signal transduction. Furthermore, analysis of differential Pol II occupancy and RNA-seq levels identified genes with high Pol II occupancy and relatively low RNA levels and vice versa. These categories are strongly enriched for genes from different functional classes. Our results demonstrate a complementary value in Pol II chip-seq and RNA-seq approaches for better understanding of gene expression regulation.