The first genome for the Cape Primrose Streptocarpus rexii (Gesneriaceae), a model plant for studying meristem-driven shoot diversity.

Cape Primroses (Streptocarpus, Gesneriaceae) are an ideal study system for investigating the genetics underlying species diversity in angiosperms. Streptocarpus rexii has served as a model species for plant developmental research for over five decades due to its unusual extended meristem activity present in the leaves. In this study, we sequenced and assembled the complete nuclear, chloroplast, and mitochondrial genomes of S. rexii using Oxford Nanopore Technologies long read sequencing. Two flow cells of PromethION sequencing resulted in 32 billion reads and were sufficient to generate a draft assembly including the chloroplast, mitochondrial and nuclear genomes, spanning 776 Mbp. The final nuclear genome assembly contained 5,855 contigs, spanning 766 Mbp of the 929-Mbp haploid genome with an N50 of 3.7 Mbp and an L50 of 57 contigs. Over 70% of the draft genome was identified as repeats. A genome repeat library of Gesneriaceae was generated and used for genome annotation, with a total of 45,045 genes annotated in the S. rexii genome. Ks plots of the paranomes suggested a recent whole genome duplication event, shared between S. rexii and Primulina huaijiensis. A new chloroplast and mitochondrial genome assembly method, based on contig coverage and identification, was developed, and successfully used to assemble both organellar genomes of S. rexii. This method was developed into a pipeline and proved widely applicable. The nuclear genome of S. rexii and other datasets generated and reported here will be invaluable resources for further research to aid in the identification of genes involved in morphological variation underpinning plant diversification.

A telomere-to-telomere assembly of Oscheius tipulae and the evolution of rhabditid nematode chromosomes.

Eukaryotic chromosomes have phylogenetic persistence. In many taxa, each chromosome has a single functional centromere with essential roles in spindle attachment and segregation. Fusion and fission can generate chromosomes with no or multiple centromeres, leading to genome instability. Groups with holocentric chromosomes (where centromeric function is distributed along each chromosome) might be expected to show karyotypic instability. This is generally not the case, and in Caenorhabditis elegans, it has been proposed that the role of maintenance of a stable karyotype has been transferred to the meiotic pairing centers, which are found at one end of each chromosome. Here, we explore the phylogenetic stability of nematode chromosomes using a new telomere-to-telomere assembly of the rhabditine nematode Oscheius tipulae generated from nanopore long reads. The 60-Mb O. tipulae genome is resolved into six chromosomal molecules. We find the evidence of specific chromatin diminution at all telomeres. Comparing this chromosomal O. tipulae assembly with chromosomal assemblies of diverse rhabditid nematodes, we identify seven ancestral chromosomal elements (Nigon elements) and present a model for the evolution of nematode chromosomes through rearrangement and fusion of these elements. We identify frequent fusion events involving NigonX, the element associated with the rhabditid X chromosome, and thus sex chromosome-associated gene sets differ markedly between species. Despite the karyotypic stability, gene order within chromosomes defined by Nigon elements is not conserved. Our model for nematode chromosome evolution provides a platform for investigation of the tensions between local genome rearrangement and karyotypic evolution in generating extant genome architectures.

Author Correction: A transcriptomic analysis of the phylum Nematoda.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Maintaining their genetic distance: Little evidence for introgression between widely hybridizing species of Geum with contrasting mating systems.

Within the plant kingdom, many genera contain sister lineages with contrasting outcrossing and inbreeding mating systems that are known to hybridize. The evolutionary fate of these sister lineages is likely to be influenced by the extent to which they exchange genes. We measured gene flow between outcrossing Geum rivale and selfing Geum urbanum, sister species that hybridize in contemporary populations. We generated and used a draft genome of G. urbanum to develop dd-RAD data scorable in both species. Coalescent analysis of RAD data from allopatric populations indicated that the species diverged 2-3 Mya, and that historical gene flow between them was extremely low (1 migrant every 25 generations). Comparison of genetic divergence between species in sympatry and allopatry, together with an analysis of allele frequencies in potential parental and hybrid populations, provided no evidence of contemporary introgression in sympatric populations. Cluster- and species-specific marker analyses revealed that, apart from four early-generation hybrids, individuals in sympatric populations fell into two genetically distinct groups that corresponded exactly to their morphological species classification with maximum individual admixture estimates of only 1-3%. However, we did observe joint segregation of four putatively introgressed SNPs across two scaffolds in the G. urbanum population that was associated with significant morphological variation, interpreted as tentative evidence for rare, recent interspecific gene flow. Overall, our results indicate that despite the presence of hybrids in contemporary populations, genetic exchange between G. rivale and G. urbanum has been extremely limited throughout their evolutionary history.

11ß-hydroxysteroid dehydrogenase-1 deficiency alters the gut microbiome response to Western diet.

The enzyme 11ß-hydroxysteroid dehydrogenase (11ß-HSD) interconverts active glucocorticoids and their intrinsically inert 11-keto forms. The type 1 isozyme, 11ß-HSD1, predominantly reactivates glucocorticoids in vivo and can also metabolise bile acids. 11ß-HSD1-deficient mice show altered inflammatory responses and are protected against the adverse metabolic effects of a high-fat diet. However, the impact of 11ß-HSD1 on the composition of the gut microbiome has not previously been investigated. We used high-throughput 16S rDNA amplicon sequencing to characterise the gut microbiome of 11ß-HSD1-deficient and C57Bl/6 control mice, fed either a standard chow diet or a cholesterol- and fat-enriched 'Western' diet. 11ß-HSD1 deficiency significantly altered the composition of the gut microbiome, and did so in a diet-specific manner. On a Western diet, 11ß-HSD1 deficiency increased the relative abundance of the family Bacteroidaceae, and on a chow diet, it altered relative abundance of the family Prevotellaceae Our results demonstrate that (i) genetic effects on host-microbiome interactions can depend upon diet and (ii) that alterations in the composition of the gut microbiome may contribute to the aspects of the metabolic and/or inflammatory phenotype observed with 11ß-HSD1 deficiency.

A single chromosome assembly of Bacteroides fragilis strain BE1 from Illumina and MinION nanopore sequencing data.

BACKGROUND: Second and third generation sequencing technologies have revolutionised bacterial genomics. Short-read Illumina reads result in cheap but fragmented assemblies, whereas longer reads are more expensive but result in more complete genomes. The Oxford Nanopore MinION device is a revolutionary mobile sequencer that can produce thousands of long, single molecule reads. RESULTS: We sequenced Bacteroides fragilis strain BE1 using both the Illumina MiSeq and Oxford Nanopore MinION platforms. We were able to assemble a single chromosome of 5.18 Mb, with no gaps, using publicly available software and commodity computing hardware. We identified gene rearrangements and the state of invertible promoters in the strain. CONCLUSIONS: The single chromosome assembly of Bacteroides fragilis strain BE1 was achieved using only modest amounts of data, publicly available software and commodity computing hardware. This combination of technologies offers the possibility of ultra-cheap, high quality, finished bacterial genomes.

poRe: an R package for the visualization and analysis of nanopore sequencing data.

MOTIVATION: The Oxford Nanopore MinION device represents a unique sequencing technology. As a mobile sequencing device powered by the USB port of a laptop, the MinION has huge potential applications. To enable these applications, the bioinformatics community will need to design and build a suite of tools specifically for MinION data. RESULTS: Here we present poRe, a package for R that enables users to manipulate, organize, summarize and visualize MinION nanopore sequencing data. As a package for R, poRe has been tested on Windows, Linux and MacOSX. Crucially, the Windows version allows users to analyse MinION data on the Windows laptop attached to the device. AVAILABILITY AND IMPLEMENTATION: poRe is released as a package for R at http://sourceforge.net/projects/rpore/. A tutorial and further information are available at https://sourceforge.net/p/rpore/wiki/Home/.

Estimation of population allele frequencies from next-generation sequencing data: pool-versus individual-based genotyping.

Molecular markers produced by next-generation sequencing (NGS) technologies are revolutionizing genetic research. However, the costs of analysing large numbers of individual genomes remain prohibitive for most population genetics studies. Here, we present results based on mathematical derivations showing that, under many realistic experimental designs, NGS of DNA pools from diploid individuals allows to estimate the allele frequencies at single nucleotide polymorphisms (SNPs) with at least the same accuracy as individual-based analyses, for considerably lower library construction and sequencing efforts. These findings remain true when taking into account the possibility of substantially unequal contributions of each individual to the final pool of sequence reads. We propose the intuitive notion of effective pool size to account for unequal pooling and derive a Bayesian hierarchical model to estimate this parameter directly from the data. We provide a user-friendly application assessing the accuracy of allele frequency estimation from both pool- and individual-based NGS population data under various sampling, sequencing depth and experimental error designs. We illustrate our findings with theoretical examples and real data sets corresponding to SNP loci obtained using restriction site-associated DNA (RAD) sequencing in pool- and individual-based experiments carried out on the same population of the pine processionary moth (Thaumetopoea pityocampa). NGS of DNA pools might not be optimal for all types of studies but provides a cost-effective approach for estimating allele frequencies for very large numbers of SNPs. It thus allows comparison of genome-wide patterns of genetic variation for large numbers of individuals in multiple populations.

Genome sequence of dwarf birch (Betula nana) and cross-species RAD markers.

New sequencing technologies allow development of genome-wide markers for any genus of ecological interest, including plant genera such as Betula (birch) that have previously proved difficult to study due to widespread polyploidy and hybridization. We present a de novo reference genome sequence assembly, from 66× short read coverage, of Betula nana (dwarf birch) - a diploid that is the keystone woody species of subarctic scrub communities but of conservation concern in Britain. We also present 100 bp PstI RAD markers for B. nana and closely related Betula tree species. Assembly of RAD markers in 15 individuals by alignment to the reference B. nana genome yielded 44-86k RAD loci per individual, whereas de novo RAD assembly yielded 64-121k loci per individual. Of the loci assembled by the de novo method, 3k homologous loci were found in all 15 individuals studied, and 35k in 10 or more individuals. Matching of RAD loci to RAD locus catalogues from the B. nana individual used for the reference genome showed similar numbers of matches from both methods of RAD locus assembly but indicated that the de novo RAD assembly method may overassemble some paralogous loci. In 12 individuals hetero-specific to B. nana 37-47k RAD loci matched a catalogue of RAD loci from the B. nana individual used for the reference genome, whereas 44-60k RAD loci aligned to the B. nana reference genome itself. We present a preliminary study of allele sharing among species, demonstrating the utility of the data for introgression studies and for the identification of species-specific alleles.

Characterisation of QTL-linked and genome-wide restriction site-associated DNA (RAD) markers in farmed Atlantic salmon.

BACKGROUND: Restriction site-associated DNA sequencing (RAD-Seq) is a genome complexity reduction technique that facilitates large-scale marker discovery and genotyping by sequencing. Recent applications of RAD-Seq have included linkage and QTL mapping with a particular focus on non-model species. In the current study, we have applied RAD-Seq to two Atlantic salmon families from a commercial breeding program. The offspring from these families were classified into resistant or susceptible based on survival/mortality in an Infectious Pancreatic Necrosis (IPN) challenge experiment, and putative homozygous resistant or susceptible genotype at a major IPN-resistance QTL. From each family, the genomic DNA of the two heterozygous parents and seven offspring of each IPN phenotype and genotype was digested with the SbfI enzyme and sequenced in multiplexed pools. RESULTS: Sequence was obtained from approximately 70,000 RAD loci in both families and a filtered set of 6,712 segregating SNPs were identified. Analyses of genome-wide RAD marker segregation patterns in the two families suggested SNP discovery on all 29 Atlantic salmon chromosome pairs, and highlighted the dearth of male recombination. The use of pedigreed samples allowed us to distinguish segregating SNPs from putative paralogous sequence variants resulting from the relatively recent genome duplication of salmonid species. Of the segregating SNPs, 50 were linked to the QTL. A subset of these QTL-linked SNPs were converted to a high-throughput assay and genotyped across large commercial populations of IPNV-challenged salmon fry. Several SNPs showed highly significant linkage and association with resistance to IPN, and population linkage-disequilibrium-based SNP tests for resistance were identified. CONCLUSIONS: We used RAD-Seq to successfully identify and characterise high-density genetic markers in pedigreed aquaculture Atlantic salmon. These results underline the effectiveness of RAD-Seq as a tool for rapid and efficient generation of QTL-targeted and genome-wide marker data in a large complex genome, and its possible utility in farmed animal selection programs.

Deep resequencing of serial sputum isolates of Mycobacterium tuberculosis during therapeutic failure due to poor compliance reveals stepwise mutation of key resistance genes on an otherwise stable genetic background.

OBJECTIVES: It has generally been held that the repeated emergence of resistance in Mycobacterium tuberculosis is due to the effects of large population sizes, slow replication, and prolonged colonization and treatment. However, there have been suggestions that its emergence is facilitated by high mutation rates due to a lack of mismatch repair, error-prone polymerases, and a potentially mutagenic host niche. Genome re-sequencing has indicated higher variability in strains with emergent resistance, but these studies have not been performed in serial isolates in which drug resistance has emerged. We have used genome re-sequencing to address the mutational processes that occur during the evolution of drug resistance during a clinical infection. METHODS: Serial isolates from a patient obtained over a 12 month period, and spanning the transition of the colonizing population from fully drug sensitive, to isoniazid resistant, to isoniazid and rifampicin (multiply drug) resistant, spanning an estimated minimum of 100 generations within the host, were deep sequenced using Illumina sequencing. The genomes were compared, and all mutations in non-repetitive sequences were identified. RESULTS: Specific mutations conferring resistance were identified. No additional mutations in non-repetitive regions were present. The mutations observed were kat S315T and rpoB D516Y. CONCLUSIONS: M. tuberculosis is relatively stable genetically within the host, and demonstrates greater stability than is suggested by in vitro studies of emergent drug resistance, or by models of hypermutability. This indicates that it is primarily the nature and duration of the infection that are sufficient to lead to the repeated emergence of drug resistance in this infection if improperly managed, and that the selective pressure of the drugs limits additional diversification. This emphasizes the central importance of maintaining therapeutic concentrations of at least two effective antibiotics for the duration of treatment to prevent the emergence of resistance.

Experimental evolution, genetic analysis and genome re-sequencing reveal the mutation conferring artemisinin resistance in an isogenic lineage of malaria parasites.

BACKGROUND: Classical and quantitative linkage analyses of genetic crosses have traditionally been used to map genes of interest, such as those conferring chloroquine or quinine resistance in malaria parasites. Next-generation sequencing technologies now present the possibility of determining genome-wide genetic variation at single base-pair resolution. Here, we combine in vivo experimental evolution, a rapid genetic strategy and whole genome re-sequencing to identify the precise genetic basis of artemisinin resistance in a lineage of the rodent malaria parasite, Plasmodium chabaudi. Such genetic markers will further the investigation of resistance and its control in natural infections of the human malaria, P. falciparum. RESULTS: A lineage of isogenic in vivo drug-selected mutant P. chabaudi parasites was investigated. By measuring the artemisinin responses of these clones, the appearance of an in vivo artemisinin resistance phenotype within the lineage was defined. The underlying genetic locus was mapped to a region of chromosome 2 by Linkage Group Selection in two different genetic crosses. Whole-genome deep coverage short-read re-sequencing (Illumina Solexa) defined the point mutations, insertions, deletions and copy-number variations arising in the lineage. Eight point mutations arise within the mutant lineage, only one of which appears on chromosome 2. This missense mutation arises contemporaneously with artemisinin resistance and maps to a gene encoding a de-ubiquitinating enzyme. CONCLUSIONS: This integrated approach facilitates the rapid identification of mutations conferring selectable phenotypes, without prior knowledge of biological and molecular mechanisms. For malaria, this model can identify candidate genes before resistant parasites are commonly observed in natural human malaria populations.

Sequences of complete human cytomegalovirus genomes from infected cell cultures and clinical specimens.

We have assessed two approaches to sequencing complete human cytomegalovirus (HCMV) genomes (236 kbp) in DNA extracted from infected cell cultures (strains 3157, HAN13, HAN20 and HAN38) or clinical specimens (strains JP and 3301). The first approach involved amplifying genomes from the DNA samples as overlapping PCR products, sequencing these by the Sanger method, acquiring reads from a capillary instrument and assembling these using the Staden programs. The second approach involved generating sequence data from the DNA samples by using an Illumina Genome Analyzer (IGA), processing the filtered reads by reference-independent (de novo) assembly, utilizing the resulting sequence to direct reference-dependent assembly of the same data and finishing by limited PCR sequencing. Both approaches were successful. In particular, the investigation demonstrated the utility of IGA data for efficiently sequencing genomes from clinical samples containing as little as 3 % HCMV DNA. Analysis of the genome sequences obtained showed that each of the strains grown in cell culture was a mutant. Certain of the mutations were shared among strains from independent clinical sources, thus suggesting that they may have arisen in a common ancestor during natural infection. Moreover, one of the strains (JP) sequenced directly from a clinical specimen was mutated in two genes, one of which encodes a proposed immune-evasion function, viral interleukin-10. These observations imply that HCMV mutants exist in human infections.

Analysis of the genome sequences of three Drosophila melanogaster spontaneous mutation accumulation lines.

We inferred the rate and properties of new spontaneous mutations in Drosophila melanogaster by carrying out whole-genome shotgun sequencing-by-synthesis of three mutation accumulation (MA) lines that had been maintained by close inbreeding for an average of 262 generations. We tested for the presence of new mutations by generating alignments of each MA line to the D. melanogaster reference genome sequence and then compared these alignments base by base. We determined empirically that at least five reads at a site within each line are required for accurate single nucleotide mutation calling. We mapped a total of 174 single-nucleotide mutations, giving a single nucleotide mutation rate of 3.5 x 10(-9) per site per generation. There were no false positives in a random sample of 40 of these mutations checked by Sanger sequencing. Variation in the numbers of mutations among the MA lines was small and nonsignificant. Numbers of transition and transversion mutations were 86 and 88, respectively, implying that transition mutation rate is close to 2x the transversion rate. We observed 1.5x as many G or C --> A or T as A or T --> G or C mutations, implying that the G or C --> A or T mutation rate is close to 2x the A or T --> G or C mutation rate. The base composition of the genome is therefore not at an equilibrium determined solely by mutation. The predicted G + C content at mutational equilibrium (33%) is similar to that observed in transposable element remnants. Nearest-neighbor mutational context dependencies are nonsignificant, suggesting that this is a weak phenomenon in Drosophila. We also saw nonsignificant differences in the mutation rate between transcribed and untranscribed regions, implying that any transcription-coupled repair process is weak. Of seven short indel mutations confirmed, six were deletions, consistent with the deletion bias that is thought to exist in Drosophila.

A transcriptomic analysis of the phylum Nematoda.

The phylum Nematoda occupies a huge range of ecological niches, from free-living microbivores to human parasites. We analyzed the genomic biology of the phylum using 265,494 expressed-sequence tag sequences, corresponding to 93,645 putative genes, from 30 species, including 28 parasites. From 35% to 70% of each species' genes had significant similarity to proteins from the model nematode Caenorhabditis elegans. More than half of the putative genes were unique to the phylum, and 23% were unique to the species from which they were derived. We have not yet come close to exhausting the genomic diversity of the phylum. We identified more than 2,600 different known protein domains, some of which had differential abundances between major taxonomic groups of nematodes. We also defined 4,228 nematode-specific protein families from nematode-restricted genes: this class of genes probably underpins species- and higher-level taxonomic disparity. Nematode-specific families are particularly interesting as drug and vaccine targets.

NEMBASE: a resource for parasitic nematode ESTs.

NEMBASE (available at http://www.nematodes.org) is a publicly available online database providing access to the sequence and associated meta-data currently being generated as part of the Edinburgh-Wellcome Trust Sanger Institute parasitic nematode EST project. NEMBASE currently holds approximately 100 000 sequences from 10 different species of nematode. To facilitate ease of use, sequences have been processed to generate a non-redundant set of gene objects ('partial genome') for each species. Users may query the database on the basis of BLAST annotation, sequence similarity or expression profiles. NEMBASE also features an interactive Java-based tool (SimiTri) which allows the simultaneous display and analysis of the relative similarity relationships of groups of sequences to three different databases. NEMBASE is currently being expanded to include sequence data from other nematode species. Other developments include access to accurate peptide predictions, improved functional annotation and incorporation of automated processes allowing rapid analysis of nematode-specific gene families.

SNP genotyping on pooled DNAs: comparison of genotyping technologies and a semi automated method for data storage and analysis.

We have compared the accuracy, efficiency and robustness of three methods of genotyping single nucleotide polymorphisms on pooled DNAs. We conclude that (i) the frequencies of the two alleles in pools should be corrected with a factor for unequal allelic amplification, which should be estimated from the mean ratio of a set of heterozygotes (k); (ii) the repeatability of an assay is more important than pinpoint accuracy when estimating allele frequencies, and assays should therefore be optimised to increase the repeatability; and (iii) the size of a pool has a relatively small effect on the accuracy of allele frequency estimation. We therefore recommend that large pools are genotyped and replicated a minimum of four times. In addition, we describe statistical approaches to allow rigorous comparison of DNA pool results. Finally, we describe an extension to our ACeDB database that facilitates management and analysis of the data generated by association studies.