doi: 10.1038/s41598-020-58025-3.
Building de novo reference genome assemblies of complex eukaryotic microorganisms from single nuclei
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- PMID:31992756
- PMCID: PMC6987183
- DOI: 10.1038/s41598-020-58025-3
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Building de novo reference genome assemblies of complex eukaryotic microorganisms from single nuclei
Merce Montoliu-Nerin et al. Sci Rep..
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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.
Conflict of interest statement
The authors declare no competing interests.
Figures
(a) Schematic representation of the life-cycle in AM fungi. A spore detects a plant root in the vicinity and grows hyphae towards it. The hyphae penetrate the plant cell wall and form the characteristically branching haustoria with the shape of arbuscules. The arbuscules are used to exchange nutrients with the plant. New spores are produced in other hyphal terminations, bud off upon maturity and remain in dormant state until the cycle starts again, while the first spore dies and the fungi retracts from the plant cell. (b) Schematic representation of a spore containing nuclei, lipid vesicles and endosymbiotic bacteria. The hyphae have very reduced compartmentalization with incomplete septa and nuclei appear to move freely.
From a soil sample to AM fungal genome assemblies. (a) Whole inoculum from the culture collection INVAM is blended with water and (b) poured into a set of sieves; the material stuck in the 38 μm sieve is placed into a (c) tube that contains a solution of 60% sucrose, then centrifuged for 1 min. The supernatant is run through a 38 μm sieve and washed with water. (d) The sieve content is placed in a Petri dish for the spores to be manually picked using a glass pipette. (e) After cleaning the spores with ddH2O, these are placed one-by-one into tubes and crushed with a pestle. (f) The DNA from a broken spore is stained with SYBR Green, giving a strong fluorescent signal for the nuclei and a lighter signal for the background, organelles and microbes. (g) The stained spore content is loaded on the FACS, where the sample moves inside a constant flow of buffer and crosses a laser beam. An excitation laser of 488 nm and 530/40 band pass filter was used for the SYBR Green fluorescence detection. In addition, scattered light, forward scatter (FSC) and side scatter (SSC) were used as proxy for size and granularity to identify the nuclei. (h) The signals can be interpreted in a scatterplot, and particles of a selected cloud (e.g., R1, blue-box) can be sorted individually or pooled (i) into individual wells of a 96-well plate by directing them with a charge. (j) The content of each well is whole genome amplified using MDA. (k) The amplified products are tested for fungi and bacteria by PCR screening with specific rDNA primers. The products confirmed to be from fungal nuclei are sequenced with (l) Illumina HiSeqX, for single nuclei; and (m) Oxford Nanopore, for pools of nuclei.(n) In workflow 1, Illumina reads are assembled separately for individual nuclei using MaSuRCA.(o) In workflow 2, reads from individual nuclei are normalized and assembled with SPADES.(q) In workflow 3 reads from all nuclei are combined, then normalized and finally assembled with SPADES.(p) Lingon is used to produce a consensus assembly from individual nuclei assemblies in both workflows 1 and 2.(r) Nanopore data is assembled with Canu, polished with Pilon using the Illumina raw-reads and used to(s) scaffold the three assemblies generated with workflows 1, 2 and 3 using Chromosemble, of Satsuma.
Summary statistics for different number of assembled nuclei (1–24) using three different assembly workflows. BUSCO estimates of completeness for (a) workflow 1: raw reads of individual nuclei assembled using MaSuRCa, consensus assembly using Lingon (b) workflow 2: normalized reads of individual nuclei assembled using SPADES, consensus assembly using Lingon and (c) workflow 3: reads from individual nuclei are pooled and normalized before assembling with SPADES. Percentage of single copy core genes detected as single copy (S: grey), duplicated (D: light grey) or fragmented (F: black). Average of 3–6 replicate assemblies up to 12 nuclei with error bars indicating SEM. In (d) assembly size (dashed lines) and N50 (solid lines) for the three methods 1 (black), 2 (grey) and 3 (light grey).
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