Deep sequencing

Two CFD associated DNA samples were prepared for deep sequencing. Individual RCA products obtained from virion preparations from samples 3, 7, 9, 11, 22 and 26 collected in 1988 and 1989 (Table ^S1) were pooled and designated sample pool CFD_1988/89. Individual RCA products obtained from total DNA extractions of 2013 from leaves 3, 5, 7, 8 and 10 (all leaves from one symptomatic palm) were pooled to yield sample pool CFD_2013. Individual RCA products obtained from total DNA extractions of three BBTV samples, BBTV-Hawaii_2013, BBTV-Nigeria_2013 and BBTV-Vietnam_2013, were also subjected to deep sequencing.

Barcoded sequencing libraries were prepared from 1 ng of RCA-enriched DNA for each sample using the Nextera XT DNA Library Prep kit (Illumina, San Diego, CA, USA), following the manufacturer's instructions. Libraries were paired-end sequenced (2 × 101 bp) on an Illumina HiSeq. 2000 DNA sequencer (Illumina, San Diego, CA, USA) at ANU, Canberra.

Total RNA prepared from leaves of a symptomatic coconut palm harvested in 2015, designated as CFD_2015 sample, was used for RNA deep sequencing. RNA sequencing was performed using Illumina RNA-Seq technology as described previously³². Briefly, from total RNA (see nucleic acid extracts) the ribosomal RNA was physically subtracted using a RiboMinus Plant Kit according to the manufacturer’s protocol (Life Technologies), and the resulting RNA served as the template for cDNA synthesis with random octamer primer using Revert Aid H Minus Reverse Transcriptase (Thermo Fisher Scientific). After a clean-up step second strand synthesis was done using the NEBNext mRNA Module (New England BioLabs Inc.). The sequencing library was prepared with the Nextera XT Library Kit (Illumina) and run after quality check on Illumina MiSeq as paired end reads (2 × 301 bp) (DSMZ, Germany).