Shanlin Liu

SOAPdenovo-Trans: De novo transcriptome assembly with short RNA-Seq reads

MOTIVATION Transcriptome sequencing has long been the favored method for quickly and inexpensively obtaining a large number of gene sequences from an organism with no reference genome. Owing to the rapid increase in throughputs and decrease in costs of next- generation sequencing, RNA-Seq in particular has become the method of choice. However, the very short reads (e.g. 2 × 90 bp paired ends) from next generation sequencing makes de novo assembly to recover complete or full-length transcript sequences an algorithmic challenge. RESULTS Here, we present SOAPdenovo-Trans, a de novo transcriptome assembler designed specifically for RNA-Seq. We evaluated its performance on transcriptome datasets from rice and mouse. Using as our benchmarks the known transcripts from these well-annotated genomes (sequenced a decade ago), we assessed how SOAPdenovo-Trans and two other popular transcriptome assemblers handled such practical issues as alternative splicing and variable expression levels. Our conclusion is that SOAPdenovo-Trans provides higher contiguity, lower redundancy and faster execution. AVAILABILITY AND IMPLEMENTATION Source code and user manual are available at http://sourceforge.net/projects/soapdenovotrans/.

Ultra-deep sequencing enables high-fidelity recovery of biodiversity for bulk arthropod samples without PCR amplification

BackgroundNext-generation-sequencing (NGS) technologies combined with a classic DNA barcoding approach have enabled fast and credible measurement for biodiversity of mixed environmental samples. However, the PCR amplification involved in nearly all existing NGS protocols inevitably introduces taxonomic biases. In the present study, we developed new Illumina pipelines without PCR amplifications to analyze terrestrial arthropod communities.ResultsMitochondrial enrichment directly followed by Illumina shotgun sequencing, at an ultra-high sequence volume, enabled the recovery of Cytochrome c Oxidase subunit 1 (COI) barcode sequences, which allowed for the estimation of species composition at high fidelity for a terrestrial insect community. With 15.5 Gbp Illumina data, approximately 97% and 92% were detected out of the 37 input Operational Taxonomic Units (OTUs), whether the reference barcode library was used or not, respectively, while only 1 novel OTU was found for the latter. Additionally, relatively strong correlation between the sequencing volume and the total biomass was observed for species from the bulk sample, suggesting a potential solution to reveal relative abundance.ConclusionsThe ability of the new Illumina PCR-free pipeline for DNA metabarcoding to detect small arthropod specimens and its tendency to avoid most, if not all, false positives suggests its great potential in biodiversity-related surveillance, such as in biomonitoring programs. However, further improvement for mitochondrial enrichment is likely needed for the application of the new pipeline in analyzing arthropod communities at higher diversity.

Genomes of the rice pest brown planthopper and its endosymbionts reveal complex complementary contributions for host adaptation

BackgroundThe brown planthopper, Nilaparvata lugens, the most destructive pest of rice, is a typical monophagous herbivore that feeds exclusively on rice sap, which migrates over long distances. Outbreaks of it have re-occurred approximately every three years in Asia. It has also been used as a model system for ecological studies and for developing effective pest management. To better understand how a monophagous sap-sucking arthropod herbivore has adapted to its exclusive host selection and to provide insights to improve pest control, we analyzed the genomes of the brown planthopper and its two endosymbionts.ResultsWe describe the 1.14 gigabase planthopper draft genome and the genomes of two microbial endosymbionts that permit the planthopper to forage exclusively on rice fields. Only 40.8% of the 27,571 identified Nilaparvata protein coding genes have detectable shared homology with the proteomes of the other 14 arthropods included in this study, reflecting large-scale gene losses including in evolutionarily conserved gene families and biochemical pathways. These unique genomic features are functionally associated with the animal’s exclusive plant host selection. Genes missing from the insect in conserved biochemical pathways that are essential for its survival on the nutritionally imbalanced sap diet are present in the genomes of its microbial endosymbionts, which have evolved to complement the mutualistic nutritional needs of the host.ConclusionsOur study reveals a series of complex adaptations of the brown planthopper involving a variety of biological processes, that result in its highly destructive impact on the exclusive host rice. All these findings highlight potential directions for effective pest control of the planthopper.

Multiplex sequencing of pooled mitochondrial genomes—a crucial step toward biodiversity analysis using mito-metagenomics

The advent in high-throughput-sequencing (HTS) technologies has revolutionized conventional biodiversity research by enabling parallel capture of DNA sequences possessing species-level diagnosis. However, polymerase chain reaction (PCR)-based implementation is biased by the efficiency of primer binding across lineages of organisms. A PCR-free HTS approach will alleviate this artefact and significantly improve upon the multi-locus method utilizing full mitogenomes. Here we developed a novel multiplex sequencing and assembly pipeline allowing for simultaneous acquisition of full mitogenomes from pooled animals without DNA enrichment or amplification. By concatenating assemblies from three de novo assemblers, we obtained high-quality mitogenomes for all 49 pooled taxa, with 36 species >15 kb and the remaining >10 kb, including 20 complete mitogenomes and nearly all protein coding genes (99.6%). The assembly quality was carefully validated with Sanger sequences, reference genomes and conservativeness of protein coding genes across taxa. The new method was effective even for closely related taxa, e.g. three Drosophila spp., demonstrating its broad utility for biodiversity research and mito-phylogenomics. Finally, the in silico simulation showed that by recruiting multiple mito-loci, taxon detection was improved at a fixed sequencing depth. Combined, these results demonstrate the plausibility of a multi-locus mito-metagenomics approach as the next phase of the current single-locus metabarcoding method.

High-throughput monitoring of wild bee diversity and abundance via mitogenomics

Summary Bee populations and other pollinators face multiple, synergistically acting threats, which have led to population declines, loss of local species richness and pollination services, and extinctions. However, our understanding of the degree, distribution and causes of declines is patchy, in part due to inadequate monitoring systems, with the challenge of taxonomic identification posing a major logistical barrier. Pollinator conservation would benefit from a high‐throughput identification pipeline. We show that the metagenomic mining and resequencing of mitochondrial genomes (mitogenomics) can be applied successfully to bulk samples of wild bees. We assembled the mitogenomes of 48 UK bee species and then shotgun‐sequenced total DNA extracted from 204 whole bees that had been collected in 10 pan‐trap samples from farms in England and been identified morphologically to 33 species. Each sample data set was mapped against the 48 reference mitogenomes. The morphological and mitogenomic data sets were highly congruent. Out of 63 total species detections in the morphological data set, the mitogenomic data set made 59 correct detections (93·7% detection rate) and detected six more species (putative false positives). Direct inspection and an analysis with species‐specific primers suggested that these putative false positives were most likely due to incorrect morphological IDs. Read frequency significantly predicted species biomass frequency (R 2 = 24·9%). Species lists, biomass frequencies, extrapolated species richness and community structure were recovered with less error than in a metabarcoding pipeline. Mitogenomics automates the onerous task of taxonomic identification, even for cryptic species, allowing the tracking of changes in species richness and distributions. A mitogenomic pipeline should thus be able to contain costs, maintain consistently high‐quality data over long time series, incorporate retrospective taxonomic revisions and provide an auditable evidence trail. Mitogenomic data sets also provide estimates of species counts within samples and thus have potential for tracking population trajectories.

Networking our way to better ecosystem service provision

The ecosystem services (EcoS) concept is being used increasingly to attach values to natural systems and the multiple benefits they provide to human societies. Ecosystem processes or functions only become EcoS if they are shown to have social and/or economic value. This should assure an explicit connection between the natural and social sciences, but EcoS approaches have been criticized for retaining little natural science. Preserving the natural, ecological science context within EcoS research is challenging because the multiple disciplines involved have very different traditions and vocabularies (common-language challenge) and span many organizational levels and temporal and spatial scales (scale challenge) that define the relevant interacting entities (interaction challenge). We propose a network-based approach to transcend these discipline challenges and place the natural science context at the heart of EcoS research.

Mitochondrial capture enriches mito-DNA 100 fold, enabling PCR-free mitogenomics biodiversity analysis

Biodiversity analyses based on next‐generation sequencing (NGS) platforms have developed by leaps and bounds in recent years. A PCR‐free strategy, which can alleviate taxonomic bias, was considered as a promising approach to delivering reliable species compositions of targeted environments. The major impediment of such a method is the lack of appropriate mitochondrial DNA enrichment ways. Because mitochondrial genomes (mitogenomes) make up only a small proportion of total DNA, PCR‐free methods will inevitably result in a huge excess of data (>99%). Furthermore, the massive volume of sequence data is highly demanding on computing resources. Here, we present a mitogenome enrichment pipeline via a gene capture chip that was designed by virtue of the mitogenome sequences of the 1000 Insect Transcriptome Evolution project (1KITE, www.1kite.org). A mock sample containing 49 species was used to evaluate the efficiency of the mitogenome capture method. We demonstrate that the proportion of mitochondrial DNA can be increased by approximately 100‐fold (from the original 0.47% to 42.52%). Variation in phylogenetic distances of target taxa to the probe set could in principle result in bias in abundance. However, the frequencies of input taxa were largely maintained after capture (R2 = 0.81). We suggest that our mitogenome capture approach coupled with PCR‐free shotgun sequencing could provide ecological researchers an efficient NGS method to deliver reliable biodiversity assessment.

Orthograph: a versatile tool for mapping coding nucleotide sequences to clusters of orthologous genes

BackgroundOrthology characterizes genes of different organisms that arose from a single ancestral gene via speciation, in contrast to paralogy, which is assigned to genes that arose via gene duplication. An accurate orthology assignment is a crucial step for comparative genomic studies. Orthologous genes in two organisms can be identified by applying a so-called reciprocal search strategy, given that complete information of the organisms’ gene repertoire is available. In many investigations, however, only a fraction of the gene content of the organisms under study is examined (e.g., RNA sequencing). Here, identification of orthologous nucleotide or amino acid sequences can be achieved using a graph-based approach that maps nucleotide sequences to genes of known orthology. Existing implementations of this approach, however, suffer from algorithmic issues that may cause problems in downstream analyses.ResultsWe present a new software pipeline, Orthograph, that addresses and solves the above problems and implements useful features for a wide range of comparative genomic and transcriptomic analyses. Orthograph applies a best reciprocal hit search strategy using profile hidden Markov models and maps nucleotide sequences to the globally best matching cluster of orthologous genes, thus enabling researchers to conveniently and reliably delineate orthologs and paralogs from transcriptomic and genomic sequence data. We demonstrate the performance of our approach on de novo-sequenced and assembled transcript libraries of 24 species of apoid wasps (Hymenoptera: Aculeata) as well as on published genomic datasets.ConclusionWith Orthograph, we implemented a best reciprocal hit approach to reference-based orthology prediction for coding nucleotide sequences such as RNAseq data. Orthograph is flexible, easy to use, open source and freely available at https://mptrsen.github.io/Orthograph. Additionally, we release 24 de novo-sequenced and assembled transcript libraries of apoid wasp species.

Phylogenetic Origin and Diversification of RNAi Pathway Genes in Insects

Abstract RNA interference (RNAi) refers to the set of molecular processes found in eukaryotic organisms in which small RNA molecules mediate the silencing or down-regulation of target genes. In insects, RNAi serves a number of functions, including regulation of endogenous genes, anti-viral defense, and defense against transposable elements. Despite being well studied in model organisms, such as Drosophila, the distribution of core RNAi pathway genes and their evolution in insects is not well understood. Here we present the most comprehensive overview of the distribution and diversity of core RNAi pathway genes across 100 insect species, encompassing all currently recognized insect orders. We inferred the phylogenetic origin of insect-specific RNAi pathway genes and also identified several hitherto unrecorded gene expansions using whole-body transcriptome data from the international 1KITE (1000 Insect Transcriptome Evolution) project as well as other resources such as i5K (5000 Insect Genome Project). Specifically, we traced the origin of the double stranded RNA binding protein R2D2 to the last common ancestor of winged insects (Pterygota), the loss of Sid-1/Tag-130 orthologs in Antliophora (fleas, flies and relatives, and scorpionflies in a broad sense), and confirm previous evidence for the splitting of the Argonaute proteins Aubergine and Piwi in Brachyceran flies (Diptera, Brachycera). Our study offers new reference points for future experimental research on RNAi-related pathway genes in insects.

Evolution of neuropeptides in non-pterygote hexapods

BackgroundNeuropeptides are key players in information transfer and act as important regulators of development, growth, metabolism, and reproduction within multi-cellular animal organisms (Metazoa). These short protein-like substances show a high degree of structural variability and are recognized as the most diverse group of messenger molecules. We used transcriptome sequences from the 1KITE (1K Insect Transcriptome Evolution) project to search for neuropeptide coding sequences in 24 species from the non-pterygote hexapod lineages Protura (coneheads), Collembola (springtails), Diplura (two-pronged bristletails), Archaeognatha (jumping bristletails), and Zygentoma (silverfish and firebrats), which are often referred to as “basal” hexapods. Phylogenetically, Protura, Collembola, Diplura, and Archaeognatha are currently placed between Remipedia and Pterygota (winged insects); Zygentoma is the sistergroup of Pterygota. The Remipedia are assumed to be among the closest relatives of all hexapods and belong to the crustaceans.ResultsWe identified neuropeptide precursor sequences within whole-body transcriptome data from these five hexapod groups and complemented this dataset with homologous sequences from three crustaceans (including Daphnia pulex), three myriapods, and the fruit fly Drosophila melanogaster. Our results indicate that the reported loss of several neuropeptide genes in a number of winged insects, particularly holometabolous insects, is a trend that has occurred within Pterygota. The neuropeptide precursor sequences of the non-pterygote hexapods show numerous amino acid substitutions, gene duplications, variants following alternative splicing, and numbers of paracopies. Nevertheless, most of these features fall within the range of variation known from pterygote insects. However, the capa/pyrokinin genes of non-pterygote hexapods provide an interesting example of rapid evolution, including duplication of a neuropeptide gene encoding different ligands.ConclusionsOur findings delineate a basic pattern of neuropeptide sequences that existed before lineage-specific developments occurred during the evolution of pterygote insects.