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

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.