Hi All,
I have been getting an unusual distinct band(size ~3kb) hanging down at the end of my gel with my genomic DNA. I was curious to find out what that is. My initial suspect was that it is a Plasmid so I gel extracted the band and illumina sequenced it. I assembled my data using SPADES denovo assembler and I fetched some contigs of the size of ~3kb, but I did not got high coverage which is expected if it is a Plasmid. So, in the end I am asking the same question, What is that unknown sequence? Could it be a dsRNA? If yes then is it possible that it resist the RNAse treatment and get itself sequenced on a next gen platform meant to sequenced genomes?
Yes, certain RNases cannot digest long double stranded RNAs. I know of at least one procedure for the expression and isolation of dsRNAs that uses RNase A to selectively degrade rRNA and mRNA. Indeed the stability of dsRNAs is great enough to survive otherwise inhospitable places like soil. As for what that band is, try melting it (95C) putting it on ice (getting single stranded nucleic-acid) and then adding RNase A and running it on a gel again to see if the band is degraded. Then try it with RNase H as well. That might (and that's a big might) answer your question. What is your hypothesis for the origin of a high concentration dsRNA in your sample? If you really think that is the case doing a dicer or DNase assay may answer your question as well. Take what you will from this answer but I would be very suspicious of a double stranded RNA being in such high concentrations unless it is a viral genome or genome segment.
You have sequenced it then try to do blast in nucleotide database probably you will get some idea from that.
Sachin,
I already did that. It is showing match against my study organisms reference genome.
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