Accessing the genomic information of unculturable oceanic picoeukaryotes by combining multiple single cells

Jean-François Mangot,Pablo Sánchez,Michael E Sieracki,Patrick Wincker,Colomban De Vargas,Olivier Jaillon,Ramiro Logares,Ramon Massana,Yoann Seeleuthner,Fran Latorre,Samuel Mondy

doi:10.1038/srep41498

Abstract

Pico-sized eukaryotes play key roles in the functioning of marine ecosystems, but we still have a limited knowledge on their ecology and evolution. The MAST-4 lineage is of particular interest, since it is widespread in surface oceans, presents ecotypic differentiation and has defied culturing efforts so far. Single cell genomics (SCG) are promising tools to retrieve genomic information from these uncultured organisms. However, SCG are based on whole genome amplification, which normally introduces amplification biases that limit the amount of genomic data retrieved from a single cell. Here, we increase the recovery of genomic information from two MAST-4 lineages by co-assembling short reads from multiple Single Amplified Genomes (SAGs) belonging to evolutionary closely related cells. We found that complementary genomic information is retrieved from different SAGs, generating co-assembly that features >74% of genome recovery, against about 20% when assembled individually. Even though this approach is not aimed at generating high-quality draft genomes, it allows accessing to the genomic information of microbes that would otherwise remain unreachable. Since most of the picoeukaryotes still remain uncultured, our work serves as a proof-of-concept that can be applied to other taxa in order to extract genomic data and address new ecological and evolutionary questions.

Highlights

As a powerful approach, but the lack of sufficient genomic DNA material due to cells’ unculturability prevents traditional shotgun sequencing and genome assembly
A total of 22 pico-heterotrophic cells affiliating to MAST-4A (n = 13) and MAST-4E (n = 9) were isolated during the Tara Oceans expedition
All MAST-4E cells and most MAST-4A cells were isolated from the same station in the Mediterranean Sea, whereas two additional MAST-4A cells derived from the Indian Ocean (Supplementary Tables S1 and S2)

Summary

Introduction

As a powerful approach, but the lack of sufficient genomic DNA material due to cells’ unculturability prevents traditional shotgun sequencing and genome assembly. Only 15% of the completed or ongoing projects in the Genomes OnLine Database (GOLD; https://gold.jgi.doe.gov)[15] concern protistan taxa, and most of them come from cultured phototrophic[16,17,18] or parasitic species[19], resulting in a biased view of the full eukaryotic diversity[20] In this context, a natural option is single cell genomics (SCG), which produce Single Amplified Genomes (SAGs) that can later be sequenced. Roy and colleagues[26] retrieved about one third of the conserved eukaryotic protein coding genes, used as proxy for genome completeness, in their MAST-4 SAG assembly This limited recovery is likely produced by the bias introduced during the whole-genome amplification, which seems to preferentially amplify certain genomic regions[27]. Our approach allowed recovering genomic functions from genomes that were previously unknown, and which will be pivotal to understand the ecological role of these uncultured flagellates in the ocean

Methods

Results

Conclusion