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Building de novo reference genome assemblies of complex eukaryotic microorganisms from single nuclei.

ABSTRACT: The advent of novel sequencing techniques has unraveled a tremendous diversity on Earth. Genomic data allow us to understand ecology and function of organisms that we would not otherwise know existed. However, major methodological challenges remain, in particular for multicellular organisms with large genomes. Arbuscular mycorrhizal (AM) fungi are important plant symbionts with cryptic and complex multicellular life cycles, thus representing a suitable model system for method development. Here, we report a novel method for large scale, unbiased nuclear sorting, sequencing, and de novo assembling of AM fungal genomes. After comparative analyses of three assembly workflows we discuss how sequence data from single nuclei can best be used for different downstream analyses such as phylogenomics and comparative genomics of single nuclei. Based on analysis of completeness, we conclude that comprehensive de novo genome assemblies can be produced from six to seven nuclei. The method is highly applicable for a broad range of taxa, and will greatly improve our ability to study multicellular eukaryotes with complex life cycles.

SUBMITTER: Montoliu-Nerin M

PROVIDER: S-EPMC6987183 | biostudies-literature | 2020 Jan

REPOSITORIES: biostudies-literature

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Building de novo reference genome assemblies of complex eukaryotic microorganisms from single nuclei.

Montoliu-Nerin Merce M Sánchez-García Marisol M Bergin Claudia C Grabherr Manfred M Ellis Barbara B Kutschera Verena Esther VE Kierczak Marcin M Johannesson Hanna H Rosling Anna A

Scientific reports 20200128 1

The advent of novel sequencing techniques has unraveled a tremendous diversity on Earth. Genomic data allow us to understand ecology and function of organisms that we would not otherwise know existed. However, major methodological challenges remain, in particular for multicellular organisms with large genomes. Arbuscular mycorrhizal (AM) fungi are important plant symbionts with cryptic and complex multicellular life cycles, thus representing a suitable model system for method development. Here, ...[more]

PMID: 31992756

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Project description:Next-generation sequencing (NGS) technologies are arguably the most revolutionary technical development to join the list of tools available to molecular biologists since PCR. For researchers working with nonconventional model organisms one major problem with the currently dominant NGS platform (Illumina) stems from the obligatory fragmentation of nucleic acid material that occurs prior to sequencing during library preparation. This step creates a significant bioinformatic challenge for accurate de novo assembly of novel transcriptome data. This challenge becomes apparent when a variety of modern assembly tools (of which there is no shortage) are applied to the same raw NGS dataset. With the same assembly parameters these tools can generate markedly different assembly outputs.In this study we present an approach that generates an optimized consensus de novo assembly of eukaryotic coding transcriptomes. This approach does not represent a new assembler, rather it combines the outputs of a variety of established assembly packages, and removes redundancy via a series of clustering steps. We test and validate our approach using Illumina datasets from six phylogenetically diverse eukaryotes (three metazoans, two plants and a yeast) and two simulated datasets derived from metazoan reference genome annotations. All of these datasets were assembled using three currently popular assembly packages (CLC, Trinity and IDBA-tran). In addition, we experimentally demonstrate that transcripts unique to one particular assembly package are likely to be bioinformatic artefacts. For all eight datasets our pipeline generates more concise transcriptomes that in fact possess more unique annotatable protein domains than any of the three individual assemblers we employed. Another measure of assembly completeness (using the purpose built BUSCO databases) also confirmed that our approach yields more information.Our approach yields coding transcriptome assemblies that are more likely to be closer to biological reality than any of the three individual assembly packages we investigated. This approach (freely available as a simple perl script) will be of use to researchers working with species for which there is little or no reference data against which the assembly of a transcriptome can be performed.

Dataset Information

Building de novo reference genome assemblies of complex eukaryotic microorganisms from single nuclei.

Publications

Building de novo reference genome assemblies of complex eukaryotic microorganisms from single nuclei.

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