NGS is still something a little scary for most operators. Mainly because of the price, which can make the pipette, in the hand of the best of us, shake a little with fear.
And even though the protocols have a tendency for getting simpler, faster, and more appropriate for a routine, there are still crucial steps that you should follow to obtain the best results.
So rest your pipette – I will tell you about three easy factors to consider for great NGS success.
1. Quality and Quantity of Your Sample
The importance of a good sample is crucial. If you start your protocol with a less than good sample your results will not be successful, and you might end up scratching your head for answers, and blaming your operator skills for no reason.
Always check for the 280/260 ratio. It might not be 100% accurate, but it will give you an idea on the sample’s purity – especially if it is DNA from an embedded-paraffin tissue. Remember – contaminants in your DNA don’t simply disappear because you wish them away.
Don’t use a quantification method like NanoDrop for the dilutions steps – even though it is basically your ally when doing a standard PCR. The truth is, NGS protocols are quite specific and they tell you exactly the quantity of DNA you should start with. The key word here is: exactly. NanoDrop may be quantifying degraded DNA or free nucleotides that aren’t going to do anything for you in the steps ahead.
Do use Qubit – even though it has a protocol of its own that demands a little bit more hands-on time, you won’t be sorry. You will know exactly how much DNA your sample has and exactly the input you start your protocol with.
2. Calibration of Pipettes –Can Make You or Break You
Pipettes are our resource every single day. In the lab bench they quickly become part of our hands, and we don’t even realize it. They truly are our best friends (I mean, imagine a world without them? What a sad and grey world that would be for all of us). So, of course we don´t think they could betray us. Well, think again.
Pipette calibration will be one of the most important concerns for you, once you start NGS. It starts with the dilution of your sample. And you do it once, and it’s not on target. You repeat, and still nothing. But you know the math is correct, and the protocol is being followed to the letter. You start to get nervous – after all, you probably don’t have limitless sample. So what could it be? Check your pipette – make sure that it is absolutely correct.
Of course pipette calibration will also be of the upmost importance for the rest of the protocol: the sample-bead ratio must be absolutely correct; all of the reagents must be measured precisely for the reaction to take place, etc.
Trust me: you will want to check your pipette’s calibration, and you will thank me later.
3. Follow the Protocol as Though it is Sacred (Unless They Tell You Not To)
Some protocols are extensive, and require a lot of attention and time. They have a lot of comments and bullet points that may not always make sense – especially if it is your first time doing it. But the protocols were designed, and experimented to exhaustion, so that we could have the best outcome possible of our library – so every single point they make, no matter how small it is, it’s there for a reason. Read it and re-read it until you know it almost by heart before you start.
And once in a while, go to the website of your product or talk to your salesman – make sure that there hasn’t been any development or alteration to your protocol. Although they experimented to exhaustion before it came out, they continue to look for better and simpler ways of getting the results, and sometimes little revisions may make your protocol easier and yield better results.
And remember, NGS is not to be feared, only understood.