The world of next-generation sequencing has exploded! And in doing so it has given us the option to sequence almost anything. You name it and there’s a seq for that. A giant confusing mess of some of the most complicated biological methods the world has ever seen! Have no fear, you are not alone in this feeling of confusion. Here we look at a couple of the new sequencing techniques out there and give our spin on what they truly mean.
DIP, ChIP, DRIP, RIP, CLIP… all the IPs!
You take a thing that binds to another thing and you sequence it! Simples. These techniques require the use of antibodies hence the immunoprecipitation (IP) part of their names. Once you have your glorious, specific antibody of choice; the IP sequencing world is your oyster. Maybe you are looking for modifications on DNA. Opt for a DNA IP (DIP). If RNA is more your thing, go for a RIP-seq or CLIP-seq (like RIP but with extra cross-linking). Looking at DNA-protein binding in its natural chromatin surrounding environment? Use ChIP-seq. Or if you are feeling really crazy, you can look at DNA-RNA binding to form hybrids! Mind blown. For this, you would use DRIP-seq. There you have it: if you have a thing you know binds to another thing and you want to know what thing number two is, there is an IP sequencing method out there for you.
Is it closed chromatin? Open chromatin? Who knows?! But you can find out with ATAC-seq. Or its full name Assay for Transposase-Accessible Chromatin using sequencing. Rolls off the tongue. This method relies on the fast, efficient cutting of transposase enzymes within exposed regions of DNA. Followed by the usual adapter ligation, next-gen sequencing procedure you’ve all grown to know and love! But why use ATAC over other methods designed to sequence open chromatin such as DNAse or FAIRE-seq? It’s easier, faster, and simpler! And that’s that really. But you can always do all three methods if you are the suspicious type.
3C, 4C, 5C, Hi-C – however many Cs you fancy
This is what you do when you have a region within the genome (a locus) that you think binds to another region of the genome and you want to know when, how, what, why, or how much! It sounds crazy but it’s quite common for one genomic locus to interact with another found hundreds of kilobases apart. It does this by folding itself correctly in three-dimensional space. Like folding Great Britain in half to travel from London to Edinburgh. Starting off small with 3C, also known as Chromatin Conformation Capture (get it! There are 3 Cs. Genius). 3C looks at the interaction of one locus to another individual locus. The more Cs you add, the more information and/or complex confusion you get out. 4C lets you see where one locus is binding anywhere in the genome. 5C lets you see where a few loci bind in the genome. And finally, the ultimate, mind-blowing of all the Cs: Hi-C lets you see where any regions of the genome are binding to any other region of the genome. Warning! Hi-C is not for the faint-hearted and should only be attempted with help from an extremely skilled bioinformatician. You have been warned.
If DNA methylation and epigenetics is your bag you will have heard about bisulfite sequencing. To cut a long story short, this sequencing method tells you where methylation is found throughout the genome. Why do we care? Because methylation is so important! And not to mention hip and cool right now. Methylation is typically associated with gene repression. However, there is so much more to it than that. All kinds of cool new DNA modifications are being discovered that play unknown and exciting roles in gene regulation. Some of these start off as methylated cytosine (5mC) and get converted into something new and mind-blowing. There are even sequencing techniques available to look at some of these new guys. Variations of bisulfite sequencing such as TAB-seq to look at 5hmC or MAB-seq to look at 5fC/5caC. Endless epigenetic fun times!
There are obviously more seq methods out there. Some need no further explanation, such as RNA-seq and small RNA-seq. These do what they say on the tin. Others, as we have seen, can be terribly complicated and named in the most confusing way possible. New sequencing methods are being invented all the time, allowing us to delve deeper and deeper into the maze of molecular genetics. We’ve done our best to make sense of a few of these. If there are others out there that you think we should summarize, get in touch! We will do our best to put a humorous spin on the most agonizing of seqs!