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.
Matteo Girardi;Laura Parducci
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