Raspberry Leaf Blotch Virus (RLBV) sequencing. RLBV

Total RNA was isolated from leaves of four separate raspberry plants showing symptoms of RLBV infection. The cDNA samples were amplified in separate LA Taq reactions and then combined in pairs so that samples SMF2_S1 and SMF3_S2 were each derived from two different infected plants. For amplification of complete RLBV RNAs, the degenerate 5’ and 3’ terminal emaravirus primers T13(5) and T13(3) (Mielke & Muehlbach, 2007) were used in combination to prime RLBV cDNA synthesis, using total RNA isolated from infected plants as the template and Superscript III reverse transcriptase (Invitrogen). The cDNA was amplified using the same primer pair and LA Taq (TaKaRa), with a programme of 94oC 3min x 1 cycle; 94oC 15 sec, 45oC 30 sec, 68oC 2 min 30 sec x 40 cycles; 68oC 5 min x 1 cycle, producing clearly defined DNA fragments in a size range of approximately 1 to 2.5 kb. The amplified cDNA was sheared using a Covaris M220 Ultrasonicator using recommended settings to an average size of ~400 bp and converted to an Illumina sequencing-compatible DNA library using the TruSeq ChIP Sample Preparation Kit (Illumina), before being sequenced with a 2x250 bp v2 kit on a MiSeq (Illumina) machine.