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A multi-gene region targeted capture approach to detect plant DNA in environmental samples: A case study using coastal environments
This project tests a targeted capture approach on mock DNA mixtures using a bait set that targets 20 chloroplast gene regions. Sensitivity and discrimination trials were conducted on mock DNA mixtures to test how well this approach can recover multiple species and trace DNA from environmental samples. The sensitivity trials consisted of a combination of DNA extracts from three coastal plant species; Avicenna marina (grey mangrove), Tecticornia flabelliformis (saltmarsh/samphire) and Zostera marina (seagrass). These DNA extracts were standardised by volume not concentration and combined into a single mixture. This mixture was then diluted in triplicate to 1ng/uL, 0.1ng/uL, 0.01ng/uL, 0.001ng/uL and 0.0001ng/uL. The raw, demultiplexed sequence data are available for these mixtures.
The discriminatory trial consisted of iteratively adding DNA extracts from species together until 10 species was reached. Again species were standardised by volume not concentration. The species added are outlined in the pre-print manuscript. Raw, demultiplexed sequence data is available for these files.
Finally, this method was then tested on real sediment samples of unknown composition and raw demultiplxed data files are available for these samples.
All data was sequenced on an Illumina HiSeq X Ten using 2x150 chemistry. Data was demultiplexed using Illumina Bcl2fastq v2.18.0. The output Read 1 and Read 2 fastq.gz files were then demultiplexed based on the Y-adapter internal barcodes using AdapterRemoval v2 (Schubert, Lindgreen & Orlando, 2016). The raw files have not undergone any trimming or filtering.
The script file for processing samples is also provided