SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing

The lion's share of bacteria in various environments cannot be cloned in the laboratory and thus cannot be sequenced using existing technologies. A major goal of single-cell genomics is to complement gene-centric metagenomic data with whole-genome assemblies of uncultivated organisms. Assembly of single-cell data is challenging because of highly non-uniform read coverage as well as elevated levels of sequencing errors and chimeric reads. We describe SPAdes, a new assembler for both single-cell and standard (multicell) assembly, and demonstrate that it improves on the recently released E+V-SC assembler (specialized for single-cell data) and on popular assemblers Velvet and SoapDeNovo (for multicell data). SPAdes generates single-cell assemblies, providing information about genomes of uncultivatable bacteria that vastly exceeds what may be obtained via traditional metagenomics studies. SPAdes is available online ( http://bioinf.spbau.ru/spades ). It is distributed as open source software.     

Accuracy of de novo assembly of DNA sequences from double‐digest libraries varies substantially among software

Advances in DNA sequencing have made it feasible to gather genomic data for non‐model organisms and large sets of individuals, often using methods for sequencing subsets of the genome. Several of these methods sequence DNA associated with endonuclease restriction sites (various RAD and GBS methods). For use in taxa without a reference genome, these methods rely on de novo assembly of fragments in the sequencing library. Many of the software options available for this application were originally developed for other assembly types and we do not know their accuracy for reduced representation libraries. To address this important knowledge gap, we simulated data from the Arabidopsis thaliana and Homo sapiens genomes and compared de novo assemblies by six software programs that are commonly used or promising for this purpose (ABySS , CD‐HIT , Stacks , Stacks2 , Velvet and VSEARCH ). We simulated different mutation rates and types of mutations, and then applied the six assemblers to the simulated data sets, varying assembly parameters. We found substantial variation in software performance across simulations and parameter settings. ABySS failed to recover any true genome fragments, and Velvet and VSEARCH performed poorly for most simulations. Stacks and Stacks2 produced accurate assemblies of simulations containing SNPs, but the addition of insertion and deletion mutations decreased their performance. CD‐HIT was the only assembler that consistently recovered a high proportion of true genome fragments. Here, we demonstrate the substantial difference in the accuracy of assemblies from different software programs and the importance of comparing assemblies that result from different parameter settings.     

基于Illumina RNA Seq短序列的转录组从头组装软件比较与优化

利用公共数据库中果蝇F1代和栽培水稻基于高通量Illumina测序平台的RNA Seq短序列数据,比较了8个 (ABySS, Velvet, SOAPdenovo, Oases, Trinity, Multiple k, T IDBA and Trans ABySS) 转录组从头组装软件。结果显示,在基于单一k mer和多重k mer方法的两类软件中,Trinity和Trans ABySS分别表现出最好的组装性能,而其它软件性能比较接近。我们还发现基于多重k mer比单一k mer可以组装获得更多的总碱基数目,但是即使利用最好的多重k mer组装软件,所获得的数据质量也比研究人员所期望的要低。鉴于此,我们提出了“ETM”优化方法,将多重k mer方法组合到Trinity中,使其在具有最好的组装性能的基础上兼具了多重k mer的优势,测试结果显示了该方法具有一定的优越性。我们的研究结果为用户选择合适的软件提供了依据,对推动基于高通量Illumina测序的转录组研究具有重要意义。     

A novel assembly pipeline and functional annotations for targeted sequencing: A case study on the globally threatened Margaritiferidae (Bivalvia: Unionida)

The proliferation of genomic sequencing approaches has significantly impacted the field of phylogenetics. Target capture approaches provide a cost-effective, fast and easily applied strategy for phylogenetic inference of non-model organisms. However, several existing target capture processing pipelines are incapable of incorporating whole genome sequencing (WGS). Here, we develop a new pipeline for capture and de novo assembly of the targeted regions using whole genome re-sequencing reads. This new pipeline captured targeted loci accurately, and given its unbiased nature, can be used with any target capture probe set. Moreover, due to its low computational demand, this new pipeline may be ideal for users with limited resources and when high-coverage sequencing outputs are required. We demonstrate the utility of our approach by incorporating WGS data into the first comprehensive phylogenomic reconstruction of the freshwater mussel family Margaritiferidae. We also provide a catalogue of well-curated functional annotations of these previously uncharacterized freshwater mussel-specific target regions, representing a complementary tool for scrutinizing phylogenetic inferences while expanding future applications of the probe set.     

Transcriptome‐based target‐enrichment baits for stony corals (Cnidaria: Anthozoa: Scleractinia)

Despite the ecological and economic significance of stony corals (Scleractinia), a robust understanding of their phylogeny remains elusive due to patchy taxonomic and genetic sampling, as well as the limited availability of informative markers. To increase the number of genetic loci available for phylogenomic analyses in Scleractinia, we designed 15,919 DNA enrichment baits targeting 605 orthogroups (mean 565 ± SD 366 bp) over 1,139 exon regions. A further 236 and 62 barcoding baits were designed for COI and histone H3 genes respectively for quality and contamination checks. Hybrid capture using these baits was performed on 18 coral species spanning the presently understood scleractinian phylogeny, with two corallimorpharians as outgroup. On average, 74% of all loci targeted were successfully captured for each species. Barcoding baits were matched unambiguously to their respective samples and revealed low levels of cross‐contamination in accordance with expectation. We put the data through a series of stringent filtering steps to ensure only scleractinian and phylogenetically informative loci were retained, and the final probe set comprised 13,479 baits, targeting 452 loci (mean 531 ± SD 307 bp) across 865 exon regions. Maximum likelihood, Bayesian and species tree analyses recovered maximally supported, topologically congruent trees consistent with previous phylogenomic reconstructions. The phylogenomic method presented here allows for consistent capture of orthologous loci among divergent coral taxa, facilitating the pooling of data from different studies and increasing the phylogenetic sampling of scleractinians in the future.     

Cross‐platform compatibility of de novo‐aligned SNPs in a nonmodel butterfly genus

High‐throughput sequencing methods for genotyping genome‐wide markers are being rapidly adopted for phylogenetics of nonmodel organisms in conservation and biodiversity studies. However, the reproducibility of SNP genotyping and degree of marker overlap or compatibility between datasets from different methodologies have not been tested in nonmodel systems. Using double‐digest restriction site‐associated DNA sequencing, we sequenced a common set of 22 specimens from the butterfly genus Speyeria on two different Illumina platforms, using two variations of library preparation. We then used a de novo approach to bioinformatic locus assembly and SNP discovery for subsequent phylogenetic analyses. We found a high rate of locus recovery despite differences in library preparation and sequencing platforms, as well as overall high levels of data compatibility after data processing and filtering. These results provide the first application of NGS methods for phylogenetic reconstruction in Speyeria and support the use and long‐term viability of SNP genotyping applications in nonmodel systems.     

Assessment of urban microbiome assemblies with the help of targeted in silico gold standards

Background

Microbial communities play a crucial role in our environment and may influence human health tremendously. Despite being the place where human interaction is most abundant we still know little about the urban microbiome. This is highlighted by the large amount of unclassified DNA reads found in urban metagenome samples. The only in silico approach that allows us to find unknown species, is the assembly and classification of draft genomes from a metagenomic dataset. In this study we (1) investigate the applicability of an assembly and binning approach for urban metagenome datasets, and (2) develop a new method for the generation of in silico gold standards to better understand the specific challenges of such datasets and provide a guide in the selection of available software.

Results

We applied combinations of three assembly (Megahit, SPAdes and MetaSPAdes) and three binning tools (MaxBin, MetaBAT and CONCOCT) to whole genome shotgun datasets from the CAMDA 2017 Challenge. Complex in silico gold standards with a simulated bacterial fraction were generated for representative samples of each surface type and city. Using these gold standards, we found the combination of SPAdes and MetaBAT to be optimal for urban metagenome datasets by providing the best trade-off between the number of high-quality genome draft bins (MIMAG standards) retrieved, the least amount of misassemblies and contamination. The assembled draft genomes included known species like Propionibacterium acnes but also novel species according to respective ANI values.

Conclusions

In our work, we showed that, even for datasets with high diversity and low sequencing depth from urban environments, assembly and binning-based methods can provide high-quality genome drafts. Of vital importance to retrieve high-quality genome drafts is sequence depth but even more so a high proportion of the bacterial sequence fraction too achieve high coverage for bacterial genomes. In contrast to read-based methods relying on database knowledge, genome-centric methods as applied in this study can provide valuable information about unknown species and strains as well as functional contributions of single community members within a sample. Furthermore, we present a method for the generation of sample-specific highly complex in silico gold standards.

Reviewers

This article was reviewed by Craig Herbold, Serghei Mangul and Yana Bromberg.

     

Using phylogenomics to resolve mega-families: An example from Compositae

Next-generation sequencing and phylogenomics hold great promise for elucidating complex relationships among large plant families. Here, we performed targeted capture of low copy sequences followed by next-generation sequencing on the Illumina platform in the large and diverse angiosperm family Compositae (Asteraceae). The family is monophyletic, based on morphology and molecular data, yet many areas of the phylogeny have unresolved polytomies and interpreting phylogenetic patterns has been historically difficult. In order to outline a method and provide a framework and for future phylogenetic studies in the Compositae, we sequenced 23 taxa from across the family in which the relationships were well established as well as a member of the sister family Calyceraceae. We generated nuclear data from 795 loci and assembled chloroplast genomes from off-target capture reads enabling the comparison of nuclear and chloroplast genomes for phylogenetic analyses. We also analyzed multi-copy nuclear genes in our data set using a clustering method during orthology detection, and we applied a network approach to these clusters—analyzing all related locus copies. Using these data, we produced hypotheses of phylogenetic relationships employing both a conservative (restricted to only loci with one copy per targeted locus) and a multigene approach (including all copies per targeted locus). The methods and bioinformatics workflow presented here provide a solid foundation for future work aimed at understanding gene family evolution in the Compositae as well as providing a model for phylogenomic analyses in other plant mega-families.     

Common species affects the utility of non‐invasive genetic monitoring of a cryptic endangered mammal: The bridled nailtail wallaby

Non‐invasive methods of monitoring wild populations (such as genotyping faeces or hair) are now widely used and advocated. The potential advantages of such methods over traditional direct monitoring (such as live capture) are that accuracy improves because sampling of non‐trappable individuals may be possible, species in difficult and remote terrain can be surveyed more efficiently, and disturbance to animals is minimal. Few studies have assessed the effects of interactions between species on remote sampling success. We test the use of non‐invasive monitoring for the cryptic, forest‐dwelling, solitary and endangered bridled nailtail wallaby (Onychogalea fraenata) that is sympatric with the ecologically similar and more common black‐striped wallaby (Macropus dorsalis). Six types of hair traps were tested for 3668 trap days, and hairs were caught with about a 10% success rate. Camera traps showed that baited hair traps targeted both wallaby species. We microscopically identified hair as bridled nailtail wallaby or black‐striped wallaby. We compared these hairs and their genotypes (using seven microsatellite loci) with known bridled nailtail wallaby hairs and genotypes derived from animal trapping. Trapped bridled nailtail wallaby hairs had characteristics that could be mistaken for black‐stripe wallaby hairs; characteristics were not diagnostic. Genetic assignment tests consistently differentiated the known bridled nailtail wallaby samples from identified black‐striped wallaby samples, however genetic overlap between most of the microsatellite markers means that they are not suitable for species identification of single samples, with the possible exception of the microsatellite locus B151. With similar trapping effort and within the same area, live‐capture mark‐recapture techniques estimated 40–60 individuals and non‐invasive methods only detected 14 genotypes. A species‐specific genetic marker would allow more efficient targeting of bridled nailtail wallaby samples and increase capture rates.     

Potential and pitfalls of eukaryotic metagenome skimming: a test case for lichens

Whole‐genome shotgun sequencing of multispecies communities using only a single library layout is commonly used to assess taxonomic and functional diversity of microbial assemblages. Here, we investigate to what extent such metagenome skimming approaches are applicable for in‐depth genomic characterizations of eukaryotic communities, for example lichens. We address how to best assemble a particular eukaryotic metagenome skimming data, what pitfalls can occur, and what genome quality can be expected from these data. To facilitate a project‐specific benchmarking, we introduce the concept of twin sets, simulated data resembling the outcome of a particular metagenome sequencing study. We show that the quality of genome reconstructions depends essentially on assembler choice. Individual tools, including the metagenome assemblers Omega and MetaVelvet, are surprisingly sensitive to low and uneven coverages. In combination with the routine of assembly parameter choice to optimize the assembly N50 size, these tools can preclude an entire genome from the assembly. In contrast, MIRA, an all‐purpose overlap assembler, and SPAdes, a multisized de Bruijn graph assembler, facilitate a comprehensive view on the individual genomes across a wide range of coverage ratios. Testing assemblers on a real‐world metagenome skimming data from the lichen Lasallia pustulata demonstrates the applicability of twin sets for guiding method selection. Furthermore, it reveals that the assembly outcome for the photobiont Trebouxia sp. falls behind the a priori expectation given the simulations. Although the underlying reasons remain still unclear, this highlights that further studies on this organism require special attention during sequence data generation and downstream analysis.     

Barley whole exome capture: a tool for genomic research in the genus Hordeum and beyond

Advanced resources for genome‐assisted research in barley (Hordeum vulgare) including a whole‐genome shotgun assembly and an integrated physical map have recently become available. These have made possible studies that aim to assess genetic diversity or to isolate single genes by whole‐genome resequencing and in silico variant detection. However such an approach remains expensive given the 5 Gb size of the barley genome. Targeted sequencing of the mRNA‐coding exome reduces barley genomic complexity more than 50‐fold, thus dramatically reducing this heavy sequencing and analysis load. We have developed and employed an in‐solution hybridization‐based sequence capture platform to selectively enrich for a 61.6 megabase coding sequence target that includes predicted genes from the genome assembly of the cultivar Morex as well as publicly available full‐length cDNAs and de novo assembled RNA‐Seq consensus sequence contigs. The platform provides a highly specific capture with substantial and reproducible enrichment of targeted exons, both for cultivated barley and related species. We show that this exome capture platform provides a clear path towards a broader and deeper understanding of the natural variation residing in the mRNA‐coding part of the barley genome and will thus constitute a valuable resource for applications such as mapping‐by‐sequencing and genetic diversity analyzes.     

Universal target‐enrichment baits for anthozoan (Cnidaria) phylogenomics: New approaches to long‐standing problems

Anthozoans (e.g., corals, anemones) are an ecologically important and diverse group of marine metazoans that occur from shallow to deep waters worldwide. However, our understanding of the evolutionary relationships among the ~7,500 species within this class is hindered by the lack of phylogenetically informative markers that can be reliably sequenced across a diversity of taxa. We designed and tested 16,306 RNA baits to capture 720 ultraconserved element loci and 1,071 exon loci. Library preparation and target enrichment were performed on 33 taxa from all orders within the class Anthozoa. Following Illumina sequencing and Trinity assembly, we recovered 1,774 of 1,791 targeted loci. The mean number of loci recovered from each species was 638 ± 222, with more loci recovered from octocorals (783 ± 138 loci) than hexacorals (475 ± 187 loci). Parsimony informative sites ranged from 26 to 49% for alignments at differing hierarchical taxonomic levels (e.g., Anthozoa, Octocorallia, Hexacorallia). The per cent of variable sites within each of three genera (Acropora, Alcyonium, and Sinularia) for which multiple species were sequenced ranged from 4.7% to 30%. Maximum‐likelihood analyses recovered highly resolved trees with topologies matching those supported by other studies, including the monophyly of the order Scleractinia. Our results demonstrate the utility of this target‐enrichment approach to resolve phylogenetic relationships from relatively old to recent divergences. Redesigning the baits with improved affinities to capture loci within each subclass will provide a valuable toolset to address systematic questions, further our understanding of the timing of diversifications and help resolve long‐standing controversial relationships in the class Anthozoa.     

On the independent gene trees assumption in phylogenomic studies

Multilocus coalescent methods for inferring species trees or historical demographic parameters typically require the assumption that gene trees for sampled SNPs or DNA sequence loci are conditionally independent given their species tree. In practice, researchers have used different criteria to delimit “independent loci.” One criterion identifies sampled loci as being independent of each other if they undergo Mendelian independent assortment (IA criterion). O'Neill et al. (2013, Molecular Ecology, 22, 111–129) used this approach in their phylogeographic study of North American tiger salamander species complex. In two other studies, researchers developed a pair of related methods that employ an independent genealogies criterion (IG criterion), which considers the effects of population‐level recombination on correlations between the gene trees of intrachromosomal loci. Here, I explain these three methods, illustrate their use with example data, and evaluate their efficacies. I show that the IA approach is more conservative, is simpler to use and requires fewer assumptions than the IG approaches. However, IG approaches can identify much larger numbers of independent loci than the IA method, which, in turn, allows researchers to obtain more precise and accurate estimates of species trees and historical demographic parameters. A disadvantage of the IG methods is that they require an estimate of the population recombination rate. Despite their drawbacks, IA and IG approaches provide molecular ecologists with promising a priori methods for selecting SNPs or DNA sequence loci that likely meet the independence assumption in coalescent‐based phylogenomic studies.