Helen Lindsay

PyCogent: a toolkit for making sense from sequence

We have implemented in Python the COmparative GENomic Toolkit, a fully integrated and thoroughly tested framework for novel probabilistic analyses of biological sequences, devising workflows, and generating publication quality graphics. PyCogent includes connectors to remote databases, built-in generalized probabilistic techniques for working with biological sequences, and controllers for third-party applications. The toolkit takes advantage of parallel architectures and runs on a range of hardware and operating systems, and is available under the general public license from http://sourceforge.net/projects/pycogent.

Robustly detecting differential expression in RNA sequencing data using observation weights

A popular approach for comparing gene expression levels between (replicated) conditions of RNA sequencing data relies on counting reads that map to features of interest. Within such count-based methods, many flexible and advanced statistical approaches now exist and offer the ability to adjust for covariates (e.g. batch effects). Often, these methods include some sort of ‘sharing of information’ across features to improve inferences in small samples. It is important to achieve an appropriate tradeoff between statistical power and protection against outliers. Here, we study the robustness of existing approaches for count-based differential expression analysis and propose a new strategy based on observation weights that can be used within existing frameworks. The results suggest that outliers can have a global effect on differential analyses. We demonstrate the effectiveness of our new approach with real data and simulated data that reflects properties of real datasets (e.g. dispersion-mean trend) and develop an extensible framework for comprehensive testing of current and future methods. In addition, we explore the origin of such outliers, in some cases highlighting additional biological or technical factors within the experiment. Further details can be downloaded from the project website: http://imlspenticton.uzh.ch/robinson_lab/edgeR_robust/.

Maximizing mutagenesis with solubilized CRISPR-Cas9 ribonucleoprotein complexes.

CRISPR-Cas9 enables efficient sequence-specific mutagenesis for creating somatic or germline mutants of model organisms. Key constraints in vivo remain the expression and delivery of active Cas9-sgRNA ribonucleoprotein complexes (RNPs) with minimal toxicity, variable mutagenesis efficiencies depending on targeting sequence, and high mutation mosaicism. Here, we apply in vitro assembled, fluorescent Cas9-sgRNA RNPs in solubilizing salt solution to achieve maximal mutagenesis efficiency in zebrafish embryos. MiSeq-based sequence analysis of targeted loci in individual embryos using CrispRVariants, a customized software tool for mutagenesis quantification and visualization, reveals efficient bi-allelic mutagenesis that reaches saturation at several tested gene loci. Such virtually complete mutagenesis exposes loss-of-function phenotypes for candidate genes in somatic mutant embryos for subsequent generation of stable germline mutants. We further show that targeting of non-coding elements in gene regulatory regions using saturating mutagenesis uncovers functional control elements in transgenic reporters and endogenous genes in injected embryos. Our results establish that optimally solubilized, in vitro assembled fluorescent Cas9-sgRNA RNPs provide a reproducible reagent for direct and scalable loss-of-function studies and applications beyond zebrafish experiments that require maximal DNA cutting efficiency in vivo. Summary: Maximal mutagenesis efficiency is achieved in vivo in zebrafish embryos using salt-solubilized, fluorescently labelled Cas9-sgRNA complexes.

Statistical methods for detecting differentially methylated loci and regions.

DNA methylation, the reversible addition of methyl groups at CpG dinucleotides, represents an important regulatory layer associated with gene expression. Changed methylation status has been noted across diverse pathological states, including cancer. The rapid development and uptake of microarrays and large scale DNA sequencing has prompted an explosion of data analytic methods for processing and discovering changes in DNA methylation across varied data types. In this mini-review, we present a compact and accessible discussion of many of the salient challenges, such as experimental design, statistical methods for differential methylation detection, critical considerations such as cell type composition and the potential confounding that can arise from batch effects. From a statistical perspective, our main interests include the use of empirical Bayes or hierarchical models, which have proved immensely powerful in genomics, and the procedures by which false discovery control is achieved.

Mechanisms That Determine the Internal Environment of the Developing Brain: A Transcriptomic, Functional and Ultrastructural Approach

We provide comprehensive identification of embryonic (E15) and adult rat lateral ventricular choroid plexus transcriptome, with focus on junction-associated proteins, ionic influx transporters and channels. Additionally, these data are related to new structural and previously published permeability studies. Results reveal that most genes associated with intercellular junctions are expressed at similar levels at both ages. In total, 32 molecules known to be associated with brain barrier interfaces were identified. Nine claudins showed unaltered expression, while two claudins (6 and 8) were expressed at higher levels in the embryo. Expression levels for most cytoplasmic/regulatory adaptors (10 of 12) were similar at the two ages. A few junctional genes displayed lower expression in embryos, including 5 claudins, occludin and one junctional adhesion molecule. Three gap junction genes were enriched in the embryo. The functional effectiveness of these junctions was assessed using blood-delivered water-soluble tracers at both the light and electron microscopic level: embryo and adult junctions halted movement of both 286Da and 3kDa molecules into the cerebrospinal fluid (CSF). The molecular identities of many ion channel and transporter genes previously reported as important for CSF formation and secretion in the adult were demonstrated in the embryonic choroid plexus (and validated with immunohistochemistry of protein products), but with some major age-related differences in expression. In addition, a large number of previously unidentified ion channel and transporter genes were identified for the first time in plexus epithelium. These results, in addition to data obtained from electron microscopical and physiological permeability experiments in immature brains, indicate that exchange between blood and CSF is mainly transcellular, as well-formed tight junctions restrict movement of small water-soluble molecules from early in development. These data strongly indicate the brain develops within a well-protected internal environment and the exchange between the blood, brain and CSF is transcellular and not through incomplete barriers.

Estimates of the Effect of Natural Selection on Protein-Coding Content

Analysis of natural selection is key to understanding many core biological processes, including the emergence of competition, cooperation, and complexity, and has important applications in the targeted development of vaccines. Selection is hard to observe directly but can be inferred from molecular sequence variation. For protein-coding nucleotide sequences, the ratio of nonsynonymous to synonymous substitutions (ω) distinguishes neutrally evolving sequences (ω = 1) from those subjected to purifying (ω < 1) or positive Darwinian (ω > 1) selection. We show that current models used to estimate ω are substantially biased by naturally occurring sequence compositions. We present a novel model that weights substitutions by conditional nucleotide frequencies and which escapes these artifacts. Applying it to the genomes of pathogens causing malaria, leprosy, tuberculosis, and Lyme disease gave significant discrepancies in estimates with ∼10–30% of genes affected. Our work has substantial implications for how vaccine targets are chosen and for studying the molecular basis of adaptive evolution.

Pitfalls of the most commonly used models of context dependent substitution

BackgroundNeighboring nucleotides exert a striking influence on mutation, with the hypermutability of CpG dinucleotides in many genomes being an exemplar. Among the approaches employed to measure the relative importance of sequence neighbors on molecular evolution have been continuous-time Markov process models for substitutions that treat sequences as a series of independent tuples. The most widely used examples are the codon substitution models. We evaluated the suitability of derivatives of the nucleotide frequency weighted (hereafter NF) and tuple frequency weighted (hereafter TF) models for measuring sequence context dependent substitution. Critical properties we address are their relationships to an independent nucleotide process and the robustness of parameter estimation to changes in sequence composition. We then consider the impact on inference concerning dinucleotide substitution processes from application of these two forms to intron sequence alignments from primates.ResultsWe prove that the NF form always nests the independent nucleotide process and that this is not true for the TF form. As a consequence, using TF to study context effects can be misleading, which is shown by both theoretical calculations and simulations. We describe a simple example where a context parameter estimated under TF is confounded with composition terms unless all sequence states are equi-frequent. We illustrate this for the dinucleotide case by simulation under a nucleotide model, showing that the TF form identifies a CpG effect when none exists. Our analysis of primate introns revealed that the effect of nucleotide neighbors is over-estimated under TF compared with NF. Parameter estimates for a number of contexts are also strikingly discordant between the two model forms.ConclusionOur results establish that the NF form should be used for analysis of independent-tuple context dependent processes. Although neighboring effects in general are still important, prominent influences such as the elevated CpG transversion rate previously identified using the TF form are an artifact. Our results further suggest as few as 5 parameters may account for ~85% of neighboring nucleotide influence.ReviewersThis article was reviewed by Dr Rob Knight, Dr Josh Cherry (nominated by Dr David Lipman) and Dr Stephen Altschul (nominated by Dr David Lipman).

Influx mechanisms in the embryonic and adult rat choroid plexus: a transcriptome study.

The transcriptome of embryonic and adult rat lateral ventricular choroid plexus, using a combination of RNA-Sequencing and microarray data, was analyzed by functional groups of influx transporters, particularly solute carrier (SLC) transporters. RNA-Seq was performed at embryonic day (E) 15 and adult with additional data obtained at intermediate ages from microarray analysis. The largest represented functional group in the embryo was amino acid transporters (twelve) with expression levels 2–98 times greater than in the adult. In contrast, in the adult only six amino acid transporters were up-regulated compared to the embryo and at more modest enrichment levels (<5-fold enrichment above E15). In E15 plexus five glucose transporters, in particular Glut-1, and only one monocarboxylate transporter were enriched compared to the adult, whereas only two glucose transporters but six monocarboxylate transporters in the adult plexus were expressed at higher levels than in embryos. These results are compared with earlier published physiological studies of amino acid and monocarboxylate transport in developing rodents. This comparison shows correlation of high expression of some transporters in the developing brain with higher amino acid transport activity reported previously. Data for divalent metal transporters are also considered. Immunohistochemistry of several transporters (e.g., Slc16a10, a thyroid hormone transporter) gene products was carried out to confirm translational activity and to define cellular distribution of the proteins. Overall the results show that there is substantial expression of numerous influx transporters in the embryonic choroid plexus, many at higher levels than in the adult. This, together with immunohistochemical evidence and data from published physiological transport studies suggests that the choroid plexus in embryonic brain plays a major role in supplying the developing brain with essential nutrients.

Age-Dependent Transcriptome and Proteome Following Transection of Neonatal Spinal Cord of Monodelphis domestica (South American Grey Short-Tailed Opossum)

This study describes a combined transcriptome and proteome analysis of Monodelphis domestica response to spinal cord injury at two different postnatal ages. Previously we showed that complete transection at postnatal day 7 (P7) is followed by profuse axon growth across the lesion with near-normal locomotion and swimming when adult. In contrast, at P28 there is no axon growth across the lesion, the animals exhibit weight-bearing locomotion, but cannot use hind limbs when swimming. Here we examined changes in gene and protein expression in the segment of spinal cord rostral to the lesion at 24 h after transection at P7 and at P28. Following injury at P7 only forty genes changed (all increased expression); most were immune/inflammatory genes. Following injury at P28 many more genes changed their expression and the magnitude of change for some genes was strikingly greater. Again many were associated with the immune/inflammation response. In functional groups known to be inhibitory to regeneration in adult cords the expression changes were generally muted, in some cases opposite to that required to account for neurite inhibition. For example myelin basic protein expression was reduced following injury at P28 both at the gene and protein levels. Only four genes from families with extracellular matrix functions thought to influence neurite outgrowth in adult injured cords showed substantial changes in expression following injury at P28: Olfactomedin 4 (Olfm4, 480 fold compared to controls), matrix metallopeptidase (Mmp1, 104 fold), papilin (Papln, 152 fold) and integrin α4 (Itga4, 57 fold). These data provide a resource for investigation of a priori hypotheses in future studies of mechanisms of spinal cord regeneration in immature animals compared to lack of regeneration at more mature stages.

Highly efficient DNA-free gene disruption in the agricultural pest Ceratitis capitata by CRISPR-Cas9 ribonucleoprotein complexes

The Mediterranean fruitfly Ceratitis capitata (medfly) is an invasive agricultural pest of high economic impact and has become an emerging model for developing new genetic control strategies as an alternative to insecticides. Here, we report the successful adaptation of CRISPR-Cas9-based gene disruption in the medfly by injecting in vitro pre-assembled, solubilized Cas9 ribonucleoprotein complexes (RNPs) loaded with gene-specific single guide RNAs (sgRNA) into early embryos. When targeting the eye pigmentation gene white eye (we), a high rate of somatic mosaicism in surviving G0 adults was observed. Germline transmission rate of mutated we alleles by G0 animals was on average above 52%, with individual cases achieving nearly 100%. We further recovered large deletions in the we gene when two sites were simultaneously targeted by two sgRNAs. CRISPR-Cas9 targeting of the Ceratitis ortholog of the Drosophila segmentation paired gene (Ccprd) caused segmental malformations in late embryos and in hatched larvae. Mutant phenotypes correlate with repair by non-homologous end-joining (NHEJ) lesions in the two targeted genes. This simple and highly effective Cas9 RNP-based gene editing to introduce mutations in C. capitata will significantly advance the design and development of new effective strategies for pest control management.