Rémy Bruggmann

The Sorghum bicolor genome and the diversification of grasses

Sorghum, an African grass related to sugar cane and maize, is grown for food, feed, fibre and fuel. We present an initial analysis of the ∼730-megabase Sorghum bicolor (L.) Moench genome, placing ∼98% of genes in their chromosomal context using whole-genome shotgun sequence validated by genetic, physical and syntenic information. Genetic recombination is largely confined to about one-third of the sorghum genome with gene order and density similar to those of rice. Retrotransposon accumulation in recombinationally recalcitrant heterochromatin explains the ∼75% larger genome size of sorghum compared with rice. Although gene and repetitive DNA distributions have been preserved since palaeopolyploidization ∼70 million years ago, most duplicated gene sets lost one member before the sorghum–rice divergence. Concerted evolution makes one duplicated chromosomal segment appear to be only a few million years old. About 24% of genes are grass-specific and 7% are sorghum-specific. Recent gene and microRNA duplications may contribute to sorghum’s drought tolerance.

The wheat powdery mildew genome shows the unique evolution of an obligate biotroph

Wheat powdery mildew, Blumeria graminis forma specialis tritici, is a devastating fungal pathogen with a poorly understood evolutionary history. Here we report the draft genome sequence of wheat powdery mildew, the resequencing of three additional isolates from different geographic regions and comparative analyses with the barley powdery mildew genome. Our comparative genomic analyses identified 602 candidate effector genes, with many showing evidence of positive selection. We characterize patterns of genetic diversity and suggest that mildew genomes are mosaics of ancient haplogroups that existed before wheat domestication. The patterns of diversity in modern isolates suggest that there was no pronounced loss of genetic diversity upon formation of the new host bread wheat 10,000 years ago. We conclude that the ready adaptation of B. graminis f.sp. tritici to the new host species was based on a diverse haplotype pool that provided great genetic potential for pathogen variation.

Identification of a SIRT1 mutation in a family with type 1 diabetes

Type 1 diabetes is caused by autoimmune-mediated β cell destruction leading to insulin deficiency. The histone deacetylase SIRT1 plays an essential role in modulating several age-related diseases. Here we describe a family carrying a mutation in the SIRT1 gene, in which all five affected members developed an autoimmune disorder: four developed type 1 diabetes, and one developed ulcerative colitis. Initially, a 26-year-old man was diagnosed with the typical features of type 1 diabetes, including lean body mass, autoantibodies, T cell reactivity to β cell antigens, and a rapid dependence on insulin. Direct and exome sequencing identified the presence of a T-to-C exchange in exon 1 of SIRT1, corresponding to a leucine-to-proline mutation at residue 107. Expression of SIRT1-L107P in insulin-producing cells resulted in overproduction of nitric oxide, cytokines, and chemokines. These observations identify a role for SIRT1 in human autoimmunity and unveil a monogenic form of type 1 diabetes.

Insight into the evolution of the Solanaceae from the parental genomes of Petunia hybrida

Petunia hybrida is a popular bedding plant that has a long history as a genetic model system. We report the whole-genome sequencing and assembly of inbred derivatives of its two wild parents, P. axillaris N and P. inflata S6. The assemblies include 91.3% and 90.2% coverage of their diploid genomes (1.4 Gb; 2n = 14) containing 32,928 and 36,697 protein-coding genes, respectively. The genomes reveal that the Petunia lineage has experienced at least two rounds of hexaploidization: the older gamma event, which is shared with most Eudicots, and a more recent Solanaceae event that is shared with tomato and other solanaceous species. Transcription factors involved in the shift from bee to moth pollination reside in particularly dynamic regions of the genome, which may have been key to the remarkable diversity of floral colour patterns and pollination systems. The high-quality genome sequences will enhance the value of Petunia as a model system for research on unique biological phenomena such as small RNAs, symbiosis, self-incompatibility and circadian rhythms.

Neurotropic Astrovirus in Cattle with Nonsuppurative Encephalitis in Europe

ABSTRACT Encephalitis is a frequently diagnosed condition in cattle with neurological diseases. Many affected animals present with a nonsuppurative inflammatory reaction pattern in the brain. While this pattern supports a viral etiology, the causative pathogen remains unknown in a large proportion of cases. Using viral metagenomics, we identified an astrovirus (bovine astrovirus [BoAstV]-CH13) in the brain of a cow with nonsuppurative encephalitis. Additionally, BoAstV RNA was detected with reverse transcription-PCR and in situ hybridization in about one fourth (5/22 animals) of cattle with nonsuppurative encephalitis of unknown etiology. Viral RNA was found primarily in neurons and at the site of pathology. These findings support the notion that BoAstV infection is a common cause of encephalitis in cattle. Phylogenetically, BoAstV-CH13 was closely related to rare astrovirus isolates from encephalitis cases in animals and a human patient. Future research needs to be directed toward the pathogenic mechanisms, epidemiology, and potential cross-species transmission of these neurotropic astroviruses.

Clinical sequencing: is WGS the better WES?

Current clinical next-generation sequencing is done by using gene panels and exome analysis, both of which involve selective capturing of target regions. However, capturing has limitations in sufficiently covering coding exons, especially GC-rich regions. We compared whole exome sequencing (WES) with the most recent PCR-free whole genome sequencing (WGS), showing that only the latter is able to provide hitherto unprecedented complete coverage of the coding region of the genome. Thus, from a clinical/technical point of view, WGS is the better WES so that capturing is no longer necessary for the most comprehensive genomic testing of Mendelian disorders.

Comparative sequence analysis of Solanum and Arabidopsis in a hot spot for pathogen resistance on potato chromosome V reveals a patchwork of conserved and rapidly evolving genome segments

BackgroundQuantitative phenotypic variation of agronomic characters in crop plants is controlled by environmental and genetic factors (quantitative trait loci = QTL). To understand the molecular basis of such QTL, the identification of the underlying genes is of primary interest and DNA sequence analysis of the genomic regions harboring QTL is a prerequisite for that. QTL mapping in potato (Solanum tuberosum) has identified a region on chromosome V tagged by DNA markers GP21 and GP179, which contains a number of important QTL, among others QTL for resistance to late blight caused by the oomycete Phytophthora infestans and to root cyst nematodes.ResultsTo obtain genomic sequence for the targeted region on chromosome V, two local BAC (bacterial artificial chromosome) contigs were constructed and sequenced, which corresponded to parts of the homologous chromosomes of the diploid, heterozygous genotype P6/210. Two contiguous sequences of 417,445 and 202,781 base pairs were assembled and annotated. Gene-by-gene co-linearity was disrupted by non-allelic insertions of retrotransposon elements, stretches of diverged intergenic sequences, differences in gene content and gene order. The latter was caused by inversion of a 70 kbp genomic fragment. These features were also found in comparison to orthologous sequence contigs from three homeologous chromosomes of Solanum demissum, a wild tuber bearing species. Functional annotation of the sequence identified 48 putative open reading frames (ORF) in one contig and 22 in the other, with an average of one ORF every 9 kbp. Ten ORFs were classified as resistance-gene-like, 11 as F-box-containing genes, 13 as transposable elements and three as transcription factors. Comparing potato to Arabidopsis thaliana annotated proteins revealed five micro-syntenic blocks of three to seven ORFs with A. thaliana chromosomes 1, 3 and 5.ConclusionComparative sequence analysis revealed highly conserved collinear regions that flank regions showing high variability and tandem duplicated genes. Sequence annotation revealed that the majority of the ORFs were members of multiple gene families. Comparing potato to Arabidopsis thaliana annotated proteins suggested fragmented structural conservation between these distantly related plant species.

Transcriptional changes in powdery mildew infected wheat and Arabidopsis leaves undergoing syringolin-triggered hypersensitive cell death at infection sites

Blumeria graminis f.sp. tritici, the causal agent of powdery mildew in wheat, is an obligate biotrophic fungus that exclusively invades epidermal cells. As previously shown, spraying of a solution of syringolin A, a circular peptide derivative secreted by the phytopathogenic bacterium Pseudomonas syringae pv. syringae, triggers hypersensitive cell death at infection sites in powdery mildew infected wheat. Thus, the fungus is essentially eradicated. Here we show that syringolin A also triggers hypersensitive cell death in Arabidopsis infected with the powdery mildew fungus Erysiphe cichoracearum. To monitor transcriptional changes associated with this effect, we cloned 307 cDNA clones representing 158 unigenes from powdery mildew infected, syringolin A sprayed wheat leaves by a suppression subtractive hybridization cloning procedure. These cDNAs were microarrayed onto glass slides together with 1088 cDNA-AFLP clones from powdery mildew-infected wheat. Microarray hybridization experiments were performed with probes derived from leaves, epidermal tissue, and mesophyll preparations of mildewed or uninfected wheat plants after syringolin A or control treatment. Similar experiments were performed in Arabidopsis using the Affymetrix ATH1 whole genome GeneChip. The results indicate a conserved mode of action of syringolin A as similar gene groups are induced in both species. Prominent groups include genes associated with the proteasomal degradation pathway, mitochondrial and other heat shock genes, genes involved in mitochondrial alternative electron pathways, and genes encoding glycolytic and fermentative enzymes. Surprisingly, in both species the observed transcriptional response to syringolin A was considerably weaker in infected plants as compared to uninfected plants. The results lead to the working hypothesis that cell death observed at infection sites may result from a parasite-induced suppression of the transcriptional response and thus to insufficient production of protective proteins necessary for the recovery of these cells from whatever insult is imposed by syringolin A.

Next-Generation Sequencing of HIV-1 RNA Genomes: Determination of Error Rates and Minimizing Artificial Recombination

Next-generation sequencing (NGS) is a valuable tool for the detection and quantification of HIV-1 variants in vivo. However, these technologies require detailed characterization and control of artificially induced errors to be applicable for accurate haplotype reconstruction. To investigate the occurrence of substitutions, insertions, and deletions at the individual steps of RT-PCR and NGS, 454 pyrosequencing was performed on amplified and non-amplified HIV-1 genomes. Artificial recombination was explored by mixing five different HIV-1 clonal strains (5-virus-mix) and applying different RT-PCR conditions followed by 454 pyrosequencing. Error rates ranged from 0.04–0.66% and were similar in amplified and non-amplified samples. Discrepancies were observed between forward and reverse reads, indicating that most errors were introduced during the pyrosequencing step. Using the 5-virus-mix, non-optimized, standard RT-PCR conditions introduced artificial recombinants in a fraction of at least 30% of the reads that subsequently led to an underestimation of true haplotype frequencies. We minimized the fraction of recombinants down to 0.9–2.6% by optimized, artifact-reducing RT-PCR conditions. This approach enabled correct haplotype reconstruction and frequency estimations consistent with reference data obtained by single genome amplification. RT-PCR conditions are crucial for correct frequency estimation and analysis of haplotypes in heterogeneous virus populations. We developed an RT-PCR procedure to generate NGS data useful for reliable haplotype reconstruction and quantification.

New insights into the performance of human whole-exome capture platforms.

Whole exome sequencing (WES) is increasingly used in research and diagnostics. WES users expect coverage of the entire coding region of known genes as well as sufficient read depth for the covered regions. It is, however, unknown which recent WES platform is most suitable to meet these expectations. We present insights into the performance of the most recent standard exome enrichment platforms from Agilent, NimbleGen and Illumina applied to six different DNA samples by two sequencing vendors per platform. Our results suggest that both Agilent and NimbleGen overall perform better than Illumina and that the high enrichment performance of Agilent is stable among samples and between vendors, whereas NimbleGen is only able to achieve vendor- and sample-specific best exome coverage. Moreover, the recent Agilent platform overall captures more coding exons with sufficient read depth than NimbleGen and Illumina. Due to considerable gaps in effective exome coverage, however, the three platforms cannot capture all known coding exons alone or in combination, requiring improvement. Our data emphasize the importance of evaluation of updated platform versions and suggest that enrichment-free whole genome sequencing can overcome the limitations of WES in sufficiently covering coding exons, especially GC-rich regions, and in characterizing structural variants.