I am working with RNA virus. Extracted RNA from serum using Maxwell Total Nucleic Acid Purification Kit and got nucleic acid conc. 36-50ng/µl with nanodrop purity (260/280) ~1.3. After doing cDNA using GoScript Reverse Transcriptase kit got nucleic acid conc. 1030-1130ng/µl with nanodrop purity (260/280) ~1.5. Can I use this cDNA directly for whole genome sequencing or do I need to amplify using specific primers as well as need to further purify cDNA? If yes, what are the procedures?
You can proceed with WGS directly with the cDNA you have. How you proceed will depend on the platform you plan to use. If using Illumina you'll fragment the genome down to the appropriate size (
for direct sequence whole genom, i think you should use NGS, otherwise make 100bps overlaping primers for complete genome, and i do not know ur virus complete genome size?
I have used human serum as my samples and planning to use MiSeq to do my WGS. As mentioned earlier, I have used VIRAL total nucleic acid purification kit to extract viral RNA from serum. Still there possibilities to get host nucleic acid into my cDNA? I am expecting about 12kb viral ssRNA.
My target is to do WGS for phylogeny study of the virus. However, want to avoid amplification using specific primers. How to prepare my cDNA prior WGS using MiSeq?
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