WGS of Ambrosia Artemisiifolia sample
Leaf tissue was collected and stored in silica gel desiccants at room temperature until required for DNA extraction. Approximately 20 to 30 mg of dried leaf tissue from each sample were placed inside a 2.0-ml tube with a 3-mm stainless steel bead and ground with a TissueLyser II (QIAGEN). The DNA was extracted using a modified CTAB protocol adapted for a 96-well plate format using EconoSpin filter plates, and the DNA was suspended in 60 l of elution buffer. Extracted DNA was quantified using a Qubit 2.0 fluorometer (Invitrogen, Carlsbad, CA, USA) using dsDNA Quantitation HS (high sensitivity, 0.2 to 120 ng) kit . Extraction blanks were prepared alongside the samples to monitor possible contamination. Extracts were converted into blunt-end Illumina libraries as described above using BEST protocol, in which custom blunt-end adapters were ligated to the DNA fragments or single-stranded Illumina libraries using the Santa Cruz Reaction protocol. Dual-index libraries were generated using custom index primers during indexing PCR. Indexing PCR was carried out in a 50-l reaction using 10 l of library template, 0.2 M sample-specific forward index primer, 0.2 M sample-specific reverse index primer, 1 Platinum SuperFi PCR master mix and the rest of the volume filled up with molecular-grade water. The PCR was performed with an initial denaturation of 3 min at 95C, followed by 12 cycles of a 20-s denaturation at 98C, 60 s of annealing at 60C, and 60 s of extension at 72C, followed by a final extension for 5 min at 72C. Amplified libraries were purified with SPRI beads and eluted in 33 l of EBT buffer. The samples were sent to Genewiz and sequenced on one lane of Illumina NovaSeq 6000 PE150 which yielded 935Gbp of data.