Murray P. Cox

SolexaQA: At-a-glance quality assessment of Illumina second-generation sequencing data

BackgroundIlluminas second-generation sequencing platform is playing an increasingly prominent role in modern DNA and RNA sequencing efforts. However, rapid, simple, standardized and independent measures of run quality are currently lacking, as are tools to process sequences for use in downstream applications based on read-level quality data.ResultsWe present SolexaQA, a user-friendly software package designed to generate detailed statistics and at-a-glance graphics of sequence data quality both quickly and in an automated fashion. This package contains associated software to trim sequences dynamically using the quality scores of bases within individual reads.ConclusionThe SolexaQA package produces standardized outputs within minutes, thus facilitating ready comparison between flow cell lanes and machine runs, as well as providing immediate diagnostic information to guide the manipulation of sequence data for downstream analyses.

Plant-symbiotic fungi as chemical engineers: multi-genome analysis of the clavicipitaceae reveals dynamics of alkaloid loci

The fungal family Clavicipitaceae includes plant symbionts and parasites that produce several psychoactive and bioprotective alkaloids. The family includes grass symbionts in the epichloae clade (Epichloë and Neotyphodium species), which are extraordinarily diverse both in their host interactions and in their alkaloid profiles. Epichloae produce alkaloids of four distinct classes, all of which deter insects, and some—including the infamous ergot alkaloids—have potent effects on mammals. The exceptional chemotypic diversity of the epichloae may relate to their broad range of host interactions, whereby some are pathogenic and contagious, others are mutualistic and vertically transmitted (seed-borne), and still others vary in pathogenic or mutualistic behavior. We profiled the alkaloids and sequenced the genomes of 10 epichloae, three ergot fungi (Claviceps species), a morning-glory symbiont (Periglandula ipomoeae), and a bamboo pathogen (Aciculosporium take), and compared the gene clusters for four classes of alkaloids. Results indicated a strong tendency for alkaloid loci to have conserved cores that specify the skeleton structures and peripheral genes that determine chemical variations that are known to affect their pharmacological specificities. Generally, gene locations in cluster peripheries positioned them near to transposon-derived, AT-rich repeat blocks, which were probably involved in gene losses, duplications, and neofunctionalizations. The alkaloid loci in the epichloae had unusual structures riddled with large, complex, and dynamic repeat blocks. This feature was not reflective of overall differences in repeat contents in the genomes, nor was it characteristic of most other specialized metabolism loci. The organization and dynamics of alkaloid loci and abundant repeat blocks in the epichloae suggested that these fungi are under selection for alkaloid diversification. We suggest that such selection is related to the variable life histories of the epichloae, their protective roles as symbionts, and their associations with the highly speciose and ecologically diverse cool-season grasses.

Recombination-filtered genomic datasets by information maximization

UNLABELLED With the increasing amount of DNA sequence data available from natural populations, new computational methods are needed to efficiently process raw sequences into formats that are applicable to a variety of analytical methods. One highly successful approach to inferring aspects of demographic history is grounded in coalescent theory. Many of these methods restrict themselves to perfectly tree-like genealogies (i.e. regions with no observed recombination), because theoretical difficulties prevent ready statistical evaluation of recombining regions. However, determining which recombination-filtered dataset to analyze from a larger recombination-rich genomic region is a non-trivial problem. Current applications primarily aim to quantify recombination rates (rather than produce optimal recombination-filtered blocks), require significant manual intervention, and are impractical for multiple genomic datasets in high-throughput, automated research environments. Here, we present a fast, simple and automatable command-line program that extracts optimal recombination-filtered blocks (no four-gamete violations) from recombination-rich genomic re-sequence data. AVAILABILITY http://hammerlab.biosci.arizona.edu/software.html.

The genomes of the fungal plant pathogens Cladosporium fulvum and Dothistroma septosporum reveal adaptation to different hosts and lifestyles but also signatures of common ancestry.

We sequenced and compared the genomes of the Dothideomycete fungal plant pathogens Cladosporium fulvum (Cfu) (syn. Passalora fulva) and Dothistroma septosporum (Dse) that are closely related phylogenetically, but have different lifestyles and hosts. Although both fungi grow extracellularly in close contact with host mesophyll cells, Cfu is a biotroph infecting tomato, while Dse is a hemibiotroph infecting pine. The genomes of these fungi have a similar set of genes (70% of gene content in both genomes are homologs), but differ significantly in size (Cfu >61.1-Mb; Dse 31.2-Mb), which is mainly due to the difference in repeat content (47.2% in Cfu versus 3.2% in Dse). Recent adaptation to different lifestyles and hosts is suggested by diverged sets of genes. Cfu contains an α-tomatinase gene that we predict might be required for detoxification of tomatine, while this gene is absent in Dse. Many genes encoding secreted proteins are unique to each species and the repeat-rich areas in Cfu are enriched for these species-specific genes. In contrast, conserved genes suggest common host ancestry. Homologs of Cfu effector genes, including Ecp2 and Avr4, are present in Dse and induce a Cf-Ecp2- and Cf-4-mediated hypersensitive response, respectively. Strikingly, genes involved in production of the toxin dothistromin, a likely virulence factor for Dse, are conserved in Cfu, but their expression differs markedly with essentially no expression by Cfu in planta. Likewise, Cfu has a carbohydrate-degrading enzyme catalog that is more similar to that of necrotrophs or hemibiotrophs and a larger pectinolytic gene arsenal than Dse, but many of these genes are not expressed in planta or are pseudogenized. Overall, comparison of their genomes suggests that these closely related plant pathogens had a common ancestral host but since adapted to different hosts and lifestyles by a combination of differentiated gene content, pseudogenization, and gene regulation.

Sex-Biased Evolutionary Forces Shape Genomic Patterns of Human Diversity

Comparisons of levels of variability on the autosomes and X chromosome can be used to test hypotheses about factors influencing patterns of genomic variation. While a tremendous amount of nucleotide sequence data from across the genome is now available for multiple human populations, there has been no systematic effort to examine relative levels of neutral polymorphism on the X chromosome versus autosomes. We analyzed ∼210 kb of DNA sequencing data representing 40 independent noncoding regions on the autosomes and X chromosome from each of 90 humans from six geographically diverse populations. We correct for differences in mutation rates between males and females by considering the ratio of within-human diversity to human-orangutan divergence. We find that relative levels of genetic variation are higher than expected on the X chromosome in all six human populations. We test a number of alternative hypotheses to explain the excess polymorphism on the X chromosome, including models of background selection, changes in population size, and sex-specific migration in a structured population. While each of these processes may have a small effect on the relative ratio of X-linked to autosomal diversity, our results point to a systematic difference between the sexes in the variance in reproductive success; namely, the widespread effects of polygyny in human populations. We conclude that factors leading to a lower male versus female effective population size must be considered as important demographic variables in efforts to construct models of human demographic history and for understanding the forces shaping patterns of human genomic variability.

A recent bottleneck of Y chromosome diversity coincides with a global change in culture

It is commonly thought that human genetic diversity in non-African populations was shaped primarily by an out-of-Africa dispersal 50-100 thousand yr ago (kya). Here, we present a study of 456 geographically diverse high-coverage Y chromosome sequences, including 299 newly reported samples. Applying ancient DNA calibration, we date the Y-chromosomal most recent common ancestor (MRCA) in Africa at 254 (95% CI 192-307) kya and detect a cluster of major non-African founder haplogroups in a narrow time interval at 47-52 kya, consistent with a rapid initial colonization model of Eurasia and Oceania after the out-of-Africa bottleneck. In contrast to demographic reconstructions based on mtDNA, we infer a second strong bottleneck in Y-chromosome lineages dating to the last 10 ky. We hypothesize that this bottleneck is caused by cultural changes affecting variance of reproductive success among males.

Major East–West Division Underlies Y Chromosome Stratification across Indonesia

The early history of island Southeast Asia is often characterized as the story of two major population dispersals: the initial Paleolithic colonization of Sahul approximately 45 ka ago and the much later Neolithic expansion of Austronesian-speaking farmers approximately 4 ka ago. Here, in the largest survey of Indonesian Y chromosomes to date, we present evidence for multiple genetic strata that likely arose through a series of distinct migratory processes. We genotype an extensive battery of Y chromosome markers, including 85 single-nucleotide polymorphisms/indels and 12 short tandem repeats, in a sample of 1,917 men from 32 communities located across Indonesia. We find that the paternal gene pool is sharply subdivided between western and eastern locations, with a boundary running between the islands of Bali and Flores. Analysis of molecular variance reveals one of the highest levels of between-group variance yet reported for human Y chromosome data (e.g., Phi(ST) = 0.47). Eastern Y chromosome haplogroups are closely related to Melanesian lineages (i.e., within the C, M, and S subclades) and likely reflect the initial wave of colonization of the region, whereas the majority of western Y chromosomes (i.e., O-M119*, O-P203, and O-M95*) are related to haplogroups that may have entered Indonesia during the Paleolithic from mainland Asia. In addition, two novel markers (P201 and P203) provide significantly enhanced phylogenetic resolution of two key haplogroups (O-M122 and O-M119) that are often associated with the Austronesian expansion. This more refined picture leads us to put forward a four-phase colonization model in which Paleolithic migrations of hunter-gatherers shape the primary structure of current Indonesian Y chromosome diversity, and Neolithic incursions make only a minor impact on the paternal gene pool, despite the large cultural impact of the Austronesian expansion.

Genomic analyses inform on migration events during the peopling of Eurasia

High-coverage whole-genome sequence studies have so far focused on a limited number of geographically restricted populations, or been targeted at specific diseases, such as cancer. Nevertheless, the availability of high-resolution genomic data has led to the development of new methodologies for inferring population history and refuelled the debate on the mutation rate in humans. Here we present the Estonian Biocentre Human Genome Diversity Panel (EGDP), a dataset of 483 high-coverage human genomes from 148 populations worldwide, including 379 new genomes from 125 populations, which we group into diversity and selection sets. We analyse this dataset to refine estimates of continent-wide patterns of heterozygosity, long- and short-distance gene flow, archaic admixture, and changes in effective population size through time as well as for signals of positive or balancing selection. We find a genetic signature in present-day Papuans that suggests that at least 2% of their genome originates from an early and largely extinct expansion of anatomically modern humans (AMHs) out of Africa. Together with evidence from the western Asian fossil record, and admixture between AMHs and Neanderthals predating the main Eurasian expansion, our results contribute to the mounting evidence for the presence of AMHs out of Africa earlier than 75,000 years ago.

What triggers grass endophytes to switch from mutualism to pathogenism

Symbioses between cool season grasses and fungi of the family Clavicipitaceae are an integral component of both natural and agricultural ecosystems. An excellent experimental model is the association between the biotrophic fungus Epichloë festucae and Lolium perenne (perennial ryegrass). The fungal partner produces a suite of secondary metabolites that protect the host from various biotic and abiotic stresses. The plant host provides a source of nutrients and a mechanism of dissemination via seed transmission. Crucial mechanisms that maintain a stable mutualistic association include signaling through the stress activated MAP kinase pathway and production of reactive oxygen species by the fungal NADPH oxidase (Nox) complex. Disruption of components of the Nox complex (NoxA, NoxR and RacA), or the stress-activated MAP kinase (SakA), leads to a breakdown in this finely balanced association, resulting in pathogenic infection instead of mutualism. Hosts infected with fungi lacking a functional Nox complex, or the stress-activated MAP kinase, display a stunted phenotype and undergo premature senescence, while the fungus switches from restricted to proliferative growth. To gain insight into the mechanisms that underlie these physiological changes, high throughput mRNA sequencing has been used to analyze the transcriptomes of both host and symbiont in wild-type and a mutant association. In the ΔsakA mutant association, a dramatic up-regulation of fungal hydrolases and transporters was observed, changes consistent with a switch from restricted symbiotic to proliferative pathogenic growth. Analysis of the plant transcriptome revealed dramatic changes in expression of host genes involved in pathogen defense, transposon activation and hormone biosynthesis and response. This review highlights how finely tuned grass-endophyte associations are, and how interfering with the signaling pathways involved in maintenance of these associations can trigger a change from mutualistic to pathogenic interaction.

Disruption of Signaling in a Fungal-Grass Symbiosis Leads to Pathogenesis

Symbiotic associations between plants and fungi are a dominant feature of many terrestrial ecosystems, yet relatively little is known about the signaling, and associated transcriptome profiles, that define the symbiotic metabolic state. Using the Epichloë festucae-perennial ryegrass (Lolium perenne) association as a model symbiotic experimental system, we show an essential role for the fungal stress-activated mitogen-activated protein kinase (sakA) in the establishment and maintenance of this mutualistic interaction. Deletion of sakA switches the fungal interaction with the host from mutualistic to pathogenic. Infected plants exhibit loss of apical dominance, premature senescence, and dramatic changes in development, including the formation of bulb-like structures at the base of tillers that lack anthocyanin pigmentation. A comparison of the transcriptome of wild-type and sakA associations using high-throughput mRNA sequencing reveals dramatic changes in fungal gene expression consistent with the transition from restricted to proliferative growth, including a down-regulation of several clusters of secondary metabolite genes and up-regulation of a large set of genes that encode hydrolytic enzymes and transporters. Analysis of the plant transcriptome reveals up-regulation of host genes involved in pathogen defense and transposon activation as well as dramatic changes in anthocyanin and hormone biosynthetic/responsive gene expression. These results highlight the fine balance between mutualism and antagonism in a plant-fungal interaction and the power of deep mRNA sequencing to identify candidate sets of genes underlying the symbiosis.