DNA sequencing methods - an overview of Sanger, Illuminia, PacBio, and Oxford NanoPore

DNA sequencing methods - an overview… The first draft sequence of the human genome took a lot of time, innovation, and $$$ (we’re talking ~$300 million for that first draft, published in February 2001 - and it wasn’t even a complete draft), but now the technology has advanced so far that human genomes are routinely sequenced for under $1000. So what is this DNA-sequencing technology? Glad you asked! The traditional DNA sequencing method is Sanger Sequencing (aka chain termination method). It may be “old-gen” but it’s really accurate and is still the go-to if you have specific regions of DNA you want to read the sequence of. I’m going to explain this method in the most detail, but I also want to touch on the so-called “next-gen” sequencing methods including the 3 main ones, Illumina, PacBio, & NanoPore. blog form: http://bit.ly/DNAsequencingmethods note: I will go into more detail below on DNA & Sanger style, but I thought I’d start with an overall comparison of the methods. note: I’m not going to try to give exact numbers of maximum read lengths and accuracy percentages and stuff because they’re constantly changing - and genomics really isn’t my field, but people kept asking for this so… You’re probably familiar with the DNA double helix (thanks Rosalind Franklin!) That helix is 2 “complementary” (sequence-specifically-paired) DNA strands, each of which is composed of linked-together DNA letters which are called nucleotides (technically deoxynucleotides). There are 4 of these letters (A, T, C, & G) and they specifically base pair (A:T, G::C) so if you have one strand you can use it as a template to create the complementary strand, which can be used as a template for creating the “original” strand. This copying is often done with a technique called PCR, which uses an enzyme (reaction mediator/speed-upper) called DNA Polymerase (DNA Pol) to make lots of copies of stretches of DNA specified by short “starter” pieces of DNA called primers. So, basically, you just need to be able to read one of the strands and you’ll know it all. Sanger sequencing, Illumina, and PacBio read DNA as (or after) it gets written but with key differences, and NanoPore takes a wildly different approach - instead of making copies using labeled letters, it threads DNA through a pore and senses changes in electricity as the DNA passes through. Because the 4 DNA letters are slightly different, they produce different electricity changes, so the machine can tell what letter is going through the pore. Sanger: incorporates “chain terminator nucleotides” - these nucleotides are labeled, but they’re “dead ends” - so if they get incorporated, it’s the end of the line. By using small amounts of these labeled letters and larger amounts of normal letters, and then letting DNA Pol go to work you get a mix of fragments that all start at the same place but end in different places and you can read the last letter of each, so you can read out the sequence. You can get longer fragments by using lower levels of terminator, but you’re still limited to fairly short reads. If you want to get longer reads, you can use primers that start at different start points and then line up overlapping regions to figure out the bigger thing (keep this idea in mind for later). Sanger sequencing was used for the first whole-genome sequencing initiatives, but these days, if you want to sequence a whole genome, you typically turn to one of the “next-gen sequencing” (NGS) methods. Illumina: incorporates REVERSIBLE chain terminator nucleotides. When they get added, no more letters can get added *under the adding reaction conditions. This allows the machine dump in labeled letters → let the one that matches the template strand get added → wash away un-added letters → read which letter was added (the letters are labeled with different fluorophores so they glow different colors) → change the reaction conditions to remove the protective group from the labeled letter which was preventing further letters from being added. This thus “un-terminates” the chain and allows more letters to be added, so you can do this all again. and again. They call this “Sequencing by Synthesis” or SBS. Might not sound that fancy, but if you try looking up how Illumina works you get these kinda crazily complicated diagrams - most of the fancy stuff is happening in the “prep” phase. Basically you start by making a lot of copies of the DNA you want to sequence. And this copying is done in lots and lots and lots and lots of sites on a chip using something called isothermal amplification (unlike PCR, this copying takes place at a single temperature). finished in comments