Sequencing and characterization of the guppy (Poecilia reticulata) transcriptome

Background: Next-generation sequencing is providing researchers with a relatively fast and affordable option for developing genomic resources for organisms that are not among the traditional genetic models. Here we present a de novo assembly of the guppy (Poecilia reticulata) transcriptome using 454 sequence reads, and we evaluate potential uses of this transcriptome, including detection of sex-specific transcripts and deployment as a reference for gene expression analysis in guppies and a related species. Guppies have been model organisms in ecology, evolutionary biology, and animal behaviour for over 100 years. An annotated transcriptome and other genomic tools will facilitate understanding the genetic and molecular bases of adaptation and variation in a vertebrate species with a uniquely well known natural history. Results: We generated approximately 336 Mbp of mRNA sequence data from male brain, male body, female brain, and female body. The resulting 1,162,670 reads assembled into 54,921 contigs, creating a reference transcriptome for the guppy with an average read depth of 28×. We annotated nearly 40% of this reference transcriptome by searching protein and gene ontology databases. Using this annotated transcriptome database, we identified candidate genes of interest to the guppy research community, putative single nucleotide polymorphisms (SNPs), and male-specific expressed genes. We also showed that our reference transcriptome can be used for RNA- sequencing-based analysis of differential gene expression. We identified transcripts that, in juveniles, are regulated differently in the presence and absence of an important predator, Rivulus hartii, including two genes implicated in stress response. For each sample in the RNA-seq study, >50% of high-quality reads mapped to unique sequences in the reference database with high confidence. In addition, we evaluated the use of the guppy reference transcriptome for gene expression analyses in a congeneric species, the sailfin molly (Poecilia latipinna). Over 40% of reads from the sailfin molly sample aligned to the guppy transcriptome. Conclusions: We show that next-generation sequencing provided a reliable and broad reference transcriptome. This resource allowed us to identify candidate gene variants, SNPs in coding regions, and sex-specific gene expression, and permitted quantitative analysis of differential gene expression

MEGASAT: automated inference of microsatellite genotypes from sequence data

MEGASAT is software that enables genotyping of microsatellite loci using next-generation sequencing data. Microsatellites are amplified in large multiplexes, and then sequenced in pooled amplicons. MEGASAT reads sequence files and automatically scores microsatellite genotypes. It uses fuzzy matches to allow for sequencing errors and applies decision rules to account for amplification artefacts, including nontarget amplification products, replication slippage during PCR (amplification stutter) and differential amplification of alleles. An important fea- ture of MEGASAT is the generation of histograms of the length–frequency distributions of amplification products for each locus and each individual. These histograms, analogous to electropherograms traditionally used to score microsatellite genotypes, enable rapid evaluation and editing of automatically scored genotypes. MEGASAT is written in Perl, runs on Windows, Mac OS X and Linux systems, and includes a simple graphical user interface. We demon- strate MEGASAT using data from guppy, Poecilia reticulata. We genotype 1024 guppies at 43 microsatellites per run on an Illumina MiSeq sequencer. We evaluated the accuracy of automatically called genotypes using two methods, based on pedigree and repeat genotyping data, and obtained estimates of mean genotyping error rates of 0.021 and 0.012. In both estimates, three loci accounted for a disproportionate fraction of genotyping errors; conversely, 26 loci were scored with 0–1 detected error (error rate ≤0.007). Our results show that with appropriate selection of loci, automated genotyping of microsatellite loci can be achieved with very high throughput, low genotyping error and very low genotyping costs

Measuring nickel masses in Type Ia supernovae using cobalt emission in nebular phase spectra

The light curves of Type Ia supernovae (SNe Ia) are powered by the radioactive decay of $^{56}$Ni to $^{56}$Co at early times, and the decay of $^{56}$Co to $^{56}$Fe from ~60 days after explosion. We examine the evolution of the [Co III] 5892 A emission complex during the nebular phase for SNe Ia with multiple nebular spectra and show that the line flux follows the square of the mass of $^{56}$Co as a function of time. This result indicates both efficient local energy deposition from positrons produced in $^{56}$Co decay, and long-term stability of the ionization state of the nebula. We compile 77 nebular spectra of 25 SN Ia from the literature and present 17 new nebular spectra of 7 SNe Ia, including SN2014J. From these we measure the flux in the [Co III] 5892 A line and remove its well-behaved time dependence to infer the initial mass of $^{56}$Ni ($M_{Ni}$) produced in the explosion. We then examine $^{56}$Ni yields for different SN Ia ejected masses ($M_{ej}$ - calculated using the relation between light curve width and ejected mass) and find the $^{56}$Ni masses of SNe Ia fall into two regimes: for narrow light curves (low stretch s~0.7-0.9), $M_{Ni}$ is clustered near $M_{Ni}$ ~ 0.4$M_\odot$ and shows a shallow increase as $M_{ej}$ increases from ~1-1.4$M_\odot$; at high stretch, $M_{ej}$ clusters at the Chandrasekhar mass (1.4$M_\odot$) while $M_{Ni}$ spans a broad range from 0.6-1.2$M_\odot$. This could constitute evidence for two distinct SN Ia explosion mechanisms.Comment: 16 pages, 12 figures (main text), plus data tables in appendix. Spectra released on WISeREP. Submitted to MNRAS, comments welcom

Advocacy training for young family doctors in primary mental health care: A report and global call to action

Background The World Health Organization (WHO) recognises the essential role of mental health in achieving health for all; its mental health action plan calls for more effective leadership for mental health and the provision of community-based, integrated care. 1 However, integrating mental health care into primary care is a challenging, transformational change that requires more than clinical knowledge. 2 It depends on strong advocacy, leadership, and change management: skills that can be learnt. 3,4 Project The Farley Health Policy Centre (FHPC) partnered with the World Organization of Family Doctors (WONCA) to develop and pilot a global curriculum to enable learners to lead practice transformation and be empowered with policy-influencing skills to advocate for their patients, to promote and enhance primary care mental health. We recruited 12 young family doctors, of whom seven were women and ten were from low- and middle-income countries (LMICs), as shown in Figure 1. The programme began with a survey of learners' needs and aspirations, and an expectation that each would self-identify a practice transformation goal. Faculty and learners took part in a two-phase learning evaluation. Funding from WONCA was provided for logistics and evaluation. A small stipend was offered to each learner on successful course completion. Faculty gave their time pro bono. The programme was conducted between March and October 2020. The evaluation process was approved by the University of Liverpool Health and Life Sciences Research Ethics Committee. The learners were divided into two learning cohorts. Sessions were facilitated by two leaders and supported by four mentors. The educational content was delivered twice (to accommodate differing time zones) in six 90-minute monthly virtual sessions. The topics were: • Introduction to mental health integratio

Measuring children’s involvement as an indicator of curriculum effectiveness : a curriculum evaluation of a selected child study centre in Singapore

This paper presents one aspect of a research project evaluating a curriculum model of a selected child study centre in Singapore. An issue of worldwide interest and concern is the ‘quality of learning’ debate as it relates to early childhood centres. In Singapore, the government is focusing on expansion in child care settings and increases in the amount of funded training. One of the issues surrounding prior-to-school education raises the question of how one measures the quality of teaching and learning, to describe the value of using, funding and promoting early education. The research reported in this study used a quasi experimental research paradigm to assess one aspect of the quality of a curriculum programme in a child study centre in Singapore. Children aged between 18 months and 6 years (N = 81) participated in the research. Using the observation scale of Laevers’ Child Involvement Scale, the active involvement of children in learning experiences was measured. The findings are presented and discussed

A 160-kilobit molecular electronic memory patterned at 10^(11) bits per square centimetre

The primary metric for gauging progress in the various semiconductor integrated circuit technologies is the spacing, or pitch, between the most closely spaced wires within a dynamic random access memory (DRAM) circuit. Modern DRAM circuits have 140nm pitch wires and a memory cell size of 0.0408 μm^2. Improving integrated circuit technology will require that these dimensions decrease over time. However, at present a large fraction of the patterning and materials requirements that we expect to need for the construction of new integrated circuit technologies in 2013 have ‘no known solution’. Promising ingredients for advances in integrated circuit technology are nanowires, molecular electronics and defect-tolerant architectures, as demonstrated by reports of single devices and small circuits. Methods of extending these approaches to large-scale, high-density circuitry are largely undeveloped. Here we describe a 160,000-bit molecular electronic memory circuit, fabricated at a density of 10^(11) bits cm^(-2) (pitch 33 nm; memory cell size 0.0011 mm^2), that is, roughly analogous to the dimensions of a DRAM circuit projected to be available by 2020. A monolayer of bistable, [2]rotaxane molecules 10 served as the data storage elements. Although the circuit has large numbers of defects, those defects could be readily identified through electronic testing and isolated using software coding. The working bits were then configured to form a fully functional random access memory circuit for storing and retrieving information

The genome of the Trinidadian guppy, Poecilia reticulata, and variation in the Guanapo population

For over a century, the live bearing guppy, Poecilia reticulata, has been used to study sexual selection as well as local adaptation. Natural guppy populations differ in many traits that are of intuitively adaptive significance such as ornamentation, age at maturity, brood size and body shape. Water depth, light supply, food resources and predation regime shape these traits, and barrier waterfalls often separate contrasting environments in the same river. We have assembled and annotated the genome of an inbred single female from a high-preda- tion site in the Guanapo drainage. The final assembly comprises 731.6 Mb with a scaffold N50 of 5.3 MB. Scaffolds were mapped to linkage groups, placing 95% of the genome assembly on the 22 autosomes and the X-chromosome. To investigate genetic variation in the population used for the genome assembly, we sequenced 10 wild caught male individu- als. The identified 5 million SNPs correspond to an average nucleotide diversity (π) of 0.0025. The genome assembly and SNP map provide a rich resource for investigating adap- tation to different predation regimes. In addition, comparisons with the genomes of other Poeciliid species, which differ greatly in mechanisms of sex determination and maternal resource allocation, as well as comparisons to other teleost genera can begin to reveal how live bearing evolved in teleost fish

Improved reference genome uncovers novel sex-linked regions in the Guppy (Poecilia reticulata)

This is the author accepted manuscript. The final version is available on open access from Oxford University Press via the DOI in this recordData availability: Population genomics data are available on ENA: Study: PRJEB10680 PCR-free data are available on ENA: Study PRJEB36450 Genome assembly is available on ENA ID: PRJEB36704; ERP119926 All scripts and pipelines are available on github: https://github.com/bfrasercommits/guppy_genomeTheory predicts that the sexes can achieve greater fitness if loci with sexually antagonistic polymorphisms become linked to the sex determining loci, and this can favour the spread of reduced recombination around sex determining regions. Given that sex-linked regions are frequently repetitive and highly heterozygous, few complete Y chromosome assemblies are available to test these ideas. The guppy system (Poecilia reticulata) has long been invoked as an example of sex chromosome formation resulting from sexual conflict. Early genetics studies revealed that male colour patterning genes are mostly but not entirely Y-linked, and that X-linkage may be most common in low predation populations. More recent population genomic studies of guppies have reached varying conclusions about the size and placement of the Y-linked region. However, this previous work used a reference genome assembled from short-read sequences from a female guppy. Here, we present a new guppy reference genome assembly from a male, using long-read PacBio single-molecule real-time sequencing (SMRT) and chromosome contact information. Our new assembly sequences across repeat- and GC-rich regions and thus closes gaps and corrects mis-assemblies found in the short-read female-derived guppy genome. Using this improved reference genome, we then employed broad population sampling to detect sex differences across the genome. We identified two small regions that showed consistent male-specific signals. Moreover, our results help reconcile the contradictory conclusions put forth by past population genomic studies of the guppy sex chromosome. Our results are consistent with a small Y-specific region and rare recombination in male guppies.Max Planck SocietyEuropean Research Council (ERC)Natural Environment Research Council (NERC