title: Capture, enrichment and sequencing of genic sequences from populations of Lotus corniculatus from different climate treatments at the Buxton Climate Change Impacts Lab
library_construction_protocol: This experiment used a sequence capture assay to capture and enrich genic sequences from populations of the herbaceous perennial plant Lotus corniculatus that had been exposed to chronic climate treatments in the field at the Buxton Climate Change Impacts Lab. A custom Roche NimbleGen SeqCap EZ in-solution capture assay was designed for L. corniculatus based on transcript sequences from L. corniculatus and closely related Lotus species.  It included 108.2 Mbp of genic sequence information, and is likely to cover the majority of genes present in Lotus corniculatus. Living individuals of L. corniculatus were recovered from the Buxton Climate Change Impacts Lab in September 2011: 43 plants were recovered from control plots and 43 from drought-treated plots. Soil depth was measured at the point of plant collection. Collected plants were maintained as clonal lines at Ness Botanic Gardens, Wirral, Cheshire, UK. DNA was extracted from dried leaf tissue using the Qiagen DNeasy Plant kit, following the manufacturers protocol. DNA was extracted from each plant clonal line separately. DNA quality, quantity and integrity were checked via analysis on the nanodrop platform (ThermoFisher Scientific), via qubit assays (ThermoFisher Scientific), and using agarose gel electrophoresis. DNA samples were normalised and pooled to create four pooled samples: one sample representing control treatment plants that were growing in deep soil in the field, one sample representing control plants from shallow soils, one sample representing plants from the drought treatment growing on deep soils and one sample representing drought plants from shallow soils. The quality and quantity of pooled samples was checked using nanodrop and qubit assays, agarose gels, and the Agilent Fragment Analyzer platform. Lotus corniculatus gene-associated sequences were enriched from the four pooled DNA samples using the custom assay designed for L. corniculatus, using the manufacturers protocol (Roche Nimblegen). Quality control of samples, including qPCR, DNA quantitation and fragment length analysis followed the manufacturers protocol. One paired-end Illumina sequencing library was prepared for each pooled enriched sample. These four libraries were combined and sequenced using four lanes of a HiSeq2500 instrument, using 2 x 125 bp paired-end reads. Each pooled sample was represented on each of the sequencing lanes.