Sequencing libraries and strategies for doing quality control
You can't do sequencing without libraries. But you don't know if you have libraries unless you look at them.
There's nothing more nerve wracking than loading your samples on a sequencer and waiting to see how the metrics look.
Way back in olden times, there was a thing called the 'first base report,' and after screwing in your reagent bottles, pulling your flowcell off the cBot, and oiling up your prism, you got to wait a couple hours for a window to pop up telling you if anything you spent the last days of work doing actually worked.
Kids these days just fill up the drawers and walk away BUT one way to know if your sequencing run might actually work is to do quality control checks throughout the preparation process!
Some vendors will tell you they've invented 'easy buttons' and between normalizer beads and their 'simple to use kits' you can skip most of these steps and pray that you don't blow $1,000-25,000 finding out that Joey added another round of 80% EtOH instead of water after the last bead clean.
Trust me, you want to know that before you load your 'samples.'
The key quality control steps during sample preparation that I've always implemented in all of my processes are:
Post-Extraction Quantification (Quant) - You sequence nucleic acids and to get nucleic acids you have to purify them from a sample. The sample can be anything from saliva to semen, but you want to know that you got the good bits out of that process before moving on to turning it into a library. For this quant I suggest using picogreen because it's quick, easy and pretty darn accurate. At this step it serves two purposes: 1) it tells you if your extraction worked, 2) you can use that number to normalize the amount of sample that goes into library prep!
Post-Library Fragment Analysis - It's debatable whether this should be done EVERY time. I've made the argument that it should because it saves a good number of failed runs but it adds a couple bucks to every sample. This is usually done using a Bioanalzyer, Tapestation or AATI HPLC system but basically what this does is tell you the fragment sizes that are contained within your library. For short-read sequencing, you generally want these to be peaking around 400-500bp for 150x150 runs. It also shows you the fragment size distribution.
Post-Libray and/or Post-Capture Quant - The quant you do before you load your samples on the sequencer should be the most reliable and accurate quant you can use. Good options here are qPCR or ddPCR based quants that use the sequencing primers as the targets for quantification because this tells you how much sequenceable library is in the sample and not just how much total DNA is in there. Those are two different things!
But chances are you're going to have a few 'where the F*$# did my library go?' moments.
Brian Krueger