I am working on the next generation sequencing of EAV from horse semen samples. As my attempts of isolating virus on RK13 cell cultures were unsuccessful I have decided to use RNA material directly extracted from semen. Before sequencing, I have tested all my samples in real time RT-PCR to confirm if viral RNA is present. As the lowest Ct values for EAV I have acquired using either Viral RNA mini kit (+ linear acrylamid) or Macherey Nucleo Spin RNA kit (+ linear acrylamid) I have chosen this kits as optimal extraction methods. Unfortunately most of my NGS attempts were unsuccessful as I was able to acquire only fragments of specific RNA genome. I suspected that the reason may be low proportion of viral RNA as overall percent of mapped reads was often lower than 0,1%. What could I do to improve this result?
I think you need to amplify your viral sample concentration by PCR then used nested PCR to get the perfect amount of virus to sequencer
Hi Wojtek,
have you tried to isolate EAV in BHK-21 or Vero cells? We once did the NGS of Aviadenovirus, but we needed high DNA levels (infected fibroblasts), and it only worked in some samples. I fear that in the semen you may have small amount of viral RNA.
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