EGU21-13501
https://doi.org/10.5194/egusphere-egu21-13501
EGU General Assembly 2021
© Author(s) 2021. This work is distributed under
the Creative Commons Attribution 4.0 License.

Shotgun barcode baiting: capturing barcoding genes from environmental samples for species identification

Kevin Nota1, Ludovic Orlando2,3, Alexis Marchesini4, Matteo Girardi4, Cristiano Vernesi4, and Laura Parducci1,5
Kevin Nota et al.
  • 1Department of Ecology and Genetics, Evolutionary Biology Centre, Uppsala University, Uppsala, Sweden
  • 2Laboratoire d’Anthropobiologie Moléculaire et d’Imagerie de Synthèse, CNRS UMR 5288, Université de Toulouse, France
  • 3Section for Geogenetics, GLOBE Institut, University of Copenhagen, Copenhagen, Denmark
  • 4Research and Innovation Centre, Fondazione Edmund Mach, S. Michele all’Adige (TN), Italy
  • 5Department of Environmental Biology, Sapienza University of Rome, Rome, Italy

The current main methodologies for identifying DNA fragments in ancient environmental samples are metabarcoding and shotgun sequencing which present strong advantages and limitations. Target capture is a promising method for enriching shotgun libraries for target organisms and might be able to combine the advantages from metabarcoding and shotgun sequencing into a single method. Target capture operates by hybridising DNA fragments in a sample to synthetic RNA-baits which share enough homology (≥85 %). These RNA-baits contain a magnetic molecule which is used to pull the hybridised fragments of interest to a magnet, allowing for the non-hybridised molecules to be washed away. The RNA-baits are designed according to prior knowledge of target sequences. Target capture does not require a PCR amplification step to amplify fragments using taxon-specific primers, and it might, therefore, be less prone to PCR amplification biases.

We designed a bait-set for capturing two barcoding plastidial genes matK and rbcL for all the species in four major plant orders: Asterales, Fagales, Pinales, and Poales. These orders are species-rich and/or difficult to identify to low taxonomic levels (family, genus or species) using metabarcoding. Our objectives were: 1) to design a universal method for trimming and selecting sequences for bait design using online sequence repositories, 2) to investigate a potential capture bias of species with a low GC-content (proportion of guanine and cytosine nucleotides) and 3) to investigate taxonomic resolution of target capture compared to metabarcoding. Because species with an overall low GC content (<32%) might be less efficiently captured than species with a higher GC content, we used mock communities with a known proportion of amplicons fragment size distribution and GC content. Further, we used sequence data simulations to investigate taxonomic resolution using varies species pools.

How to cite: Nota, K., Orlando, L., Marchesini, A., Girardi, M., Vernesi, C., and Parducci, L.: Shotgun barcode baiting: capturing barcoding genes from environmental samples for species identification, EGU General Assembly 2021, online, 19–30 Apr 2021, EGU21-13501, https://doi.org/10.5194/egusphere-egu21-13501, 2021.

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