Massively-parallel cDNA sequencing has opened the way to deep and efficient probing of transcriptomes. Current approaches for transcript reconstruction from such data often rely on aligning reads to a reference genome, and are thus unsuitable for samples with a partial or missing reference genome. Here, we present the Trinity methodology for de novo full-length transcriptome reconstruction, and evaluate it on samples from fission yeast, mouse, and whitefly – an insect whose genome has not yet been sequenced. Trinity fully reconstructs a large fraction of the transcripts present in the data, also reporting alternative splice isoforms and transcripts from recently duplicated genes. In all cases, Trinity performs better than other available de novo transcriptome assembly programs, and its sensitivity is comparable to methods relying on genome alignments. Our approach provides a unified and general solution for transcriptome reconstruction in any sample, especially in the complete absence of a reference genome.

The fission yeast clade, comprising Schizosaccharomyces pombe, S. octosporus, S. cryophilus and S. japonicus, occupies the basal branch of Ascomycete fungi and is an important model of eukaryote biology. A comparative annotation of these genomes identified a near extinction of transposons and the associated innovation of transposon-free centromeres. Expression analysis established that meiotic genes are subject to antisense transcription during vegetative growth, suggesting a mechanism for their tight regulation. In addition, trans-acting regulators control new genes within the context of expanded functional modules for meiosis and stress response. Differences in gene content and regulation also explain why, unlike the Saccharomycotina, fission yeasts cannot use ethanol as a primary carbon source. These analyses elucidate the genome structure and gene regulation of fission yeast and provide tools for investigation across the Schizosaccharomyces clade.

Strand-specific, massively-parallel cDNA sequencing (RNA-Seq) is a powerful tool for novel transcript discovery, genome annotation, and expression profiling. Despite multiple published methods for strand-specific RNA-Seq, no consensus exists as to how to choose between them. Here, we developed a comprehensive computational pipeline to compare library quality metrics from any RNA-Seq method. Using the well-annotated Saccharomyces cerevisiae transcriptome as a benchmark, we compared seven library construction protocols, including both published and our own novel methods. We found marked differences in strand-specificity, library complexity, evenness and continuity of coverage, agreement with known annotations, and accuracy for expression profiling. Weighing each method’s performance and ease, we identify the dUTP second strand marking and the Illumina RNA ligation methods as the leading protocols, with the former benefitting from the current availability of paired-end sequencing. Our analysis provides a comprehensive benchmark, and our computational pipeline is applicable for assessment of future protocols in other organisms.

RNA-Seq is an effective method to study the transcriptome, but can be difficult to apply to scarce or degraded RNA from fixed clinical samples, rare cell populations, or cadavers. Recent studies have proposed several methods for RNA-Seq of low quality and/or low quantity samples, but their relative merits have not been systematically analyzed. Here, we compare five such methods using metrics relevant to transcriptome annotation, transcript discovery, and gene expression. Using a single human RNA sample, we constructed and sequenced ten libraries with these methods and two control libraries. We find that the RNase H method performed best for low quality RNA, and confirmed this with actual degraded samples. RNase H can even effectively replace oligo (dT) based methods for standard RNA-Seq. SMART and NuGEN had distinct strengths for low quantity RNA. Our analysis allows biologists to select the most suitable methods and provides a benchmark for future method development.

The fungal kingdom is the source of almost all industrial enzymes in use for lignocellulose bioprocessing. We developed a systems-level approach that integrates transcriptomic sequencing, proteomics, phenotype, and biochemical studies of relatively unexplored basal fungi. Anaerobic gut fungi isolated from herbivores produce a large array of biomass-degrading enzymes that synergistically degrade crude, untreated plant biomass and are competitive with optimized commercial preparations from Aspergillus and Trichoderma. Compared to these model platforms, gut fungal enzymes are unbiased in substrate preference due to a wealth of xylan-degrading enzymes. These enzymes are universally catabolite-repressed and are further regulated by a rich landscape of noncoding regulatory RNAs. Additionally, we identified several promising sequence-divergent enzyme candidates for lignocellulosic bioprocessing.

A comparative genomics study maps nucleosomes across the entire genomes of 12 fungal species, identifying multiple distinct mechanisms linking changes in chromatin architecture to evolution of gene regulation.

Mitochondrial dysfunction is associated with a spectrum of human disorders, ranging from rare, inborn errors of metabolism to common, age-associated diseases such as neurodegeneration. How these lesions give rise to diverse pathology is not well understood, partly because their proximal consequences have not been well-studied in mammalian cells. Here we provide two lines of evidence that mitochondrial respiratory chain dysfunction leads to alterations in one-carbon metabolism pathways. First, using hypothesis-generating metabolic, proteomic, and transcriptional profiling, followed by confirmatory experiments, we report that mitochondrial DNA depletion leads to an ATF4-mediated increase in serine biosynthesis and transsulfuration. Second, we show that lesioning the respiratory chain impairs mitochondrial production of formate from serine, and that in some cells, respiratory chain inhibition leads to growth defects upon serine withdrawal that are rescuable with purine or formate supplementation. Our work underscores the connection between the respiratory chain and one-carbon metabolism with implications for understanding mitochondrial pathogenesis.DOI: http://dx.doi.org/10.7554/eLife.10575.001

Candida albicans is both a member of the healthy human microbiome and a major pathogen in immunocompromised individuals. Infections are typically treated with azole inhibitors of ergosterol biosynthesis often leading to drug resistance. Studies in clinical isolates have implicated multiple mechanisms in resistance, but have focused on large-scale aberrations or candidate genes, and do not comprehensively chart the genetic basis of adaptation. Here, we leveraged next-generation sequencing to analyze 43 isolates from 11 oral candidiasis patients. We detected newly selected mutations, including single-nucleotide polymorphisms (SNPs), copy-number variations and loss-of-heterozygosity (LOH) events. LOH events were commonly associated with acquired resistance, and SNPs in 240 genes may be related to host adaptation. Conversely, most aneuploidies were transient and did not correlate with drug resistance. Our analysis also shows that isolates also varied in adherence, filamentation, and virulence. Our work reveals new molecular mechanisms underlying the evolution of drug resistance and host adaptation.DOI: http://dx.doi.org/10.7554/eLife.00662.001