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A framework for in situ molecular characterization of coral holobionts using nanopore sequencing.

ABSTRACT: Molecular characterization of the coral host and the microbial assemblages associated with it (referred to as the coral holobiont) is currently undertaken via marker gene sequencing. This requires bulky instruments and controlled laboratory conditions which are impractical for environmental experiments in remote areas. Recent advances in sequencing technologies now permit rapid sequencing in the field; however, development of specific protocols and pipelines for the effective processing of complex microbial systems are currently lacking. Here, we used a combination of 3 marker genes targeting the coral animal host, its symbiotic alga, and the associated bacterial microbiome to characterize 60 coral colonies collected and processed in situ, during the Tara Pacific expedition. We used Oxford Nanopore Technologies to sequence marker gene amplicons and developed bioinformatics pipelines to analyze nanopore reads on a laptop, obtaining results in less than 24 h. Reef scale network analysis of coral-associated bacteria reveals broadly distributed taxa, as well as host-specific associations. Protocols and tools used in this work may be applicable for rapid coral holobiont surveys, immediate adaptation of sampling strategy in the field, and to make informed and timely decisions in the context of the current challenges affecting coral reefs worldwide.

SUBMITTER: Carradec Q

PROVIDER: S-EPMC7522235 | biostudies-literature | 2020 Sep

REPOSITORIES: biostudies-literature

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A framework for in situ molecular characterization of coral holobionts using nanopore sequencing.

Carradec Quentin Q Poulain Julie J Boissin Emilie E Hume Benjamin C C BCC Voolstra Christian R CR Ziegler Maren M Engelen Stefan S Cruaud Corinne C Planes Serge S Wincker Patrick P

Scientific reports 20200928 1

Molecular characterization of the coral host and the microbial assemblages associated with it (referred to as the coral holobiont) is currently undertaken via marker gene sequencing. This requires bulky instruments and controlled laboratory conditions which are impractical for environmental experiments in remote areas. Recent advances in sequencing technologies now permit rapid sequencing in the field; however, development of specific protocols and pipelines for the effective processing of compl ...[more]

PMID: 32985530

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Project description:BackgroundThe molecular characterization information of T-DNA integration is not only required by public risk assessors and regulators, but is also closely related to the expression of exogenous and endogenous genes. At present, with the development of sequencing technology, whole-genome resequencing has become an attractive approach to identify unknown genetically modified events and characterise T-DNA integration events.ResultsIn this study, we performed genome resequencing of Pb29, a transgenic high-resistance poplar 741 line that has been commercialized, using next-generation and Nanopore sequencing. The results revealed that there are two T-DNA insertion sites, located at 9,283,905-9,283,937 bp on chromosome 3 (Chr03) and 10,868,777-10,868,803 bp on Chr10. The accuracy of the T-DNA insertion locations and directions was verified using polymerase chain reaction amplification. Through sequence alignment, different degrees of base deletions were detected on the T-DNA left and right border sequences, and in the flanking sequences of the insertion sites. An unknown fragment was inserted between the Chr03 insertion site and the right flanking sequence, but the Pb29 genome did not undergo chromosomal rearrangement. It is worth noting that we did not detect the API gene in the Pb29 genome, indicating that Pb29 is a transgenic line containing only the BtCry1AC gene. On Chr03, the insertion of T-DNA disrupted a gene encoding TAF12 protein, but the transcriptional abundance of this gene did not change significantly in the leaves of Pb29. Additionally, except for the gene located closest to the T-DNA integration site, the expression levels of four other neighboring genes did not change significantly in the leaves of Pb29.ConclusionsThis study provides molecular characterization information of T-DNA integration in transgenic poplar 741 line Pb29, which contribute to safety supervision and further extensive commercial planting of transgenic poplar 741.

Dataset Information

A framework for in situ molecular characterization of coral holobionts using nanopore sequencing.

Publications

A framework for in situ molecular characterization of coral holobionts using nanopore sequencing.

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