Deep sequencing of SRMV isolate Nigeria 75/1: Vero 69
Viral RNA extraction was performed with a KingFisherTM mL extraction robot and the ID kit Gene Mag Universal Extraction (IDvet, Grabels, France), following the manufacturers instructions. cDNA synthesis and PCR amplification of the complete PPRV genome was performed on five overlapping fragments: F1 (4050 pb), F2 (3663 pb), F3 (3816 pb), F4 (3800 pb) and F5 (3335 pb). cDNA synthesis was performed with a Reverse Transcriptase (RT) (RevertAid RT kit, Thermo Scientific, Courtaboeuf, France) and the specific primers of each fragment (F1 to F5), following the instructions provided with the kit. First-strand cDNA of each fragment was then amplified by PCR according to the kit protocol KAPA DNA Polymerase 250 unit (KAPA HiFi PCR Kit Kapa Biosystems, Champagne-au-Mont-dOr, France). The PCR products were then separated by electrophoresis on 1.5% agarose gel and the fragments of the expected sizes excised and purified using the NucleoSpin Gel and PCR Clean-up kit purification kit following the manufacturers instructions (Machery-Nagel, Hoerdt, France). The different amplified fragments were pooled in a single tube in an equimolar manner. The pool was used to prepare sequencing libraries. Library preparation was performed according to the instructions provided in the Nextera XT kit (Illumina). The libraries were prepared in dual index, i.e., using two indexes for each sample. Sequencing was done on a MiSeq Illumina machine at the AGAP sequencing platform (CIRAD, Montpellier, France).
SUB6168896
Small Ruminants Morbillivirus isolate Nigeria 75/1 Vero69