amplicon of RYMV : Tg274_5674_A
The RYMV genome was amplified using a two step-process. Firstly, a specific full-length reverse transcription was performed using the primer DAS (Fargette et al., 2004) and PrimeScriptTM Reverse Transcriptase (Takara Bio Inc.). Secondly, the RYMV genome was amplified in two overlapping fragments using the primers AS/BAS and CS/DAS (Fargette et al., 2004) and ExTaq polymerase (Takara Bio Inc.). Libraries were constructed using 6-bp barcodes to allow for multiplexing as previously described (Mariac et al., 2014). Briefly, PCR fragments of the same sample were equimolary bulked and sheared to a mean target size of 400 bp. Fragments was then repaired, ligated, nicked fill-in and amplified in real-time PCR to complete adapters and to generate libraries ready for cluster generation and sequencing.