There is a bit of debate about this, but there are a few questions you can answer for yourself that I'll offer to you. 

First of all - next gen sequencing is expensive and sensitive. So you want to go in to your sequencing prep with as high quality and pure a sample as possible. Junk in/Junk out, as they say. And I'd be more worried about any abundant RNA (rRNA for example) interfering with the efficiency of my library prep reactions.

Secondly, share some details with us about your sample:

These questions will help point future responses in the right direction for you.

That being said - I can offer the following:

• Generally, there is a LOT of RNA left over in your sample after a gDNA prep. Depending on your prep or sample type, up to greater than 90% of your sample can be RNA. There is a LOT of RNA in cells... assuming they are alive or recently dead. Quantify your sample if at all possible. If you have access to a Qubit fluorescent quantification system - use their RNA quantification.https://www.thermofisher.com/au/en/home/life-science/laboratory-instruments/fluorometers/qubit.html
• If your feathers are old, you may not have that much RNA to begin with as it will degrade much faster than gDNA thanks to the abundance of RNAse. If your feathers are say 1-6 months or a year old (or older), then chances are there won't be much  RNA in there - unless they've been frozen or preserved somehow.
• Heat destroying your RNA doesn't happen instantly, and if you were to heat your sample to 58C, and then run  it on a gel, you would probably see a different band than untreated RNA - not because it has been degraded so much as its been partly denatured. For example, we heat denature RNA-probes for in situ hybridization (these are very expensive!) at 85C for a couple minutes prior to hybridization just to denature the probes. I wouldn't really rely on that to clean up my RNA.
• I would personally RNAse my samples - it is trivial, takes very little time, and can be cleaned up with very high recovery using either a mini-prep kit designed for college small amounts of gDNA, or a phenol:cholorform:IsoAmyl alcohol DNA extraction followed by an overnight 100etOH precipitation. (Practice this before attempting on your samples if you haven't already.)
• If possible, you can run a small aliquot of your sample on a gel (or better - us a Agilent BioAnalyser)  to check for RNA bands (See: https://www.quora.com/What-is-the-meaning-of-the-28S-18S-ribosomal-ratios). 

Best of luck with this. Its really tough working with rare samples - my lab handles extinct Thylacine (tasmanian tiger) gDNA regularly, so coming by samples is quite difficult. None-the-less, we've managed to sequence the genome and reconstruct it using very little sample. 

My best guess here is that you could probably move in to your library prep without any significant problems, but if you can spare losing a little bit of DNA (like 10% or less depending on your extraction technique), RNAse A your sample for 20 minutes and be done with it. But try to figure out whether or not there is any RNA in there to begin with. 

Post a response later with what you did and how it turned out for us to see.

I've attached a few links that you may find helpful.

All the best, 

Paul G

http://seqanswers.com/forums/showthread.php?t=18891

http://seqanswers.com/forums/showthread.php?t=18881

https://www.quora.com/What-is-the-meaning-of-the-28S-18S-ribosomal-ratios

http://bitesizebio.com/23105/quantifying-your-ngs-libraries/

https://www.thermofisher.com/au/en/home/life-science/laboratory-instruments/fluorometers/qubit.html

Thanks Paul for a detailed answer. My samples are more than six months old. They were preserved at room temperature (25-30 C) for two months when collected during the field work and then stored in a -20C fridge before extraction. All the samples were extracted using Qiagen DNAeasy Blood and Tissue Kit. When I did a nanodrop analysis, I found that on an average my samples have 3-5 ng/ul of DNA. I have 70 ul of volume for every sample. While extracting these samples, we had not given RNAse treatment. But, as you have pointed out correctly that it is 'better' to get rid off any RNA present in the sample, I am thinking to treat the extracts with RNAse. I will try to do a Bioanalyzer run and see the degree of RNA in my samples. 

In case if I find a higher concentration of RNA in my samples, do you think, it is a good idea to treat the DNA extracts with RNAse and elute the DNA following the Qiagen protocol?

regards,

Pankaj     

Pankaj, 

Your samples are pretty old, so there probably won't be much RNA left. That is also a large volume for such a low concentration. Have you double checked that your sequencing prep kit can accommodate that?

If you find that you need to concentrated your sample, then it may be a good time to RNAse... but again - there is probably no real worry with the RNA in this case. You may not want to risk losing any more DNA considering that you have such a tiny amount.

What is the required starting amount of gDNA for the sequencing method you are going to use?

With this information, I would suggest NOT RNAsing, and possibly concentrating your DNA sample by either phenol: Chloroform: Isoamyly alcohol extraction or a kit you know works really well and give you high yield - if you need to. If you want to use that DNA for anything else, you should probably try to not lose any. 

Hi Paul,

Thanks for the reply. We are aiming at Ultraconserved elements, which require minimum 20 ng of DNA (in total volume of the elute) to start with. I am going to run the sample on Bioanalyzer and let's see how it works. 

regards,

Pankaj

I ran few samples on Bioanalyzer, but there was no issue of RNA contamination. I guess, I do not have to worry about RNAse treatment now.