Sequencing DNA is reading the string of “letters” in the genetic code: the bases A, C, G, and T. Since the 1980s we’ve used Sanger sequencing to read up to 1,000 bases of DNA at a time. We still use Sanger sequencing today, but in the early 2000s, a wave of new DNA sequencing technologies began to emerge that allow us to read hundreds of thousands, or even millions of bases at a time, faster and cheaper than ever before. We can now sequence entire chromosomes, or even entire genomes billions of bases in length, in a single run. We call these technologies Next Generation Sequencing, or NGS.

There are several NGS technologies and each works a bit differently, but they all share one thing in common: instead of sequencing stretches of DNA one at a time, NGS can read many different pieces of DNA simultaneously, and then put them back together like pieces of a puzzle. The first step in NGS is to shred the DNA sample that we want to read (say, a two-million base-pair bacterial chromosome) randomly into smaller fragments, to create a DNA library. Fragments in the DNA library will have overlaps, as each of the starting DNA molecules (in our case, many copies of a bacterial chromosome) randomly break at different points in the nucleotide chain. The second step in NGS is to read each of the smaller DNA fragments in the library in parallel with the others. In today’s prevailing NGS technology, the fragments are arrayed onto a small glass slide such that individual library fragments attach to discrete spots on the slide; in our example, the bacterial chromosome is now represented by many thousands of smaller DNA spots, each containing a unique fragment. In each spot, a DNA polymerase (an enzyme that copies DNA) replicates the library fragment by adding complementary nucleotides, where the As, Cs, Gs, and Ts are fluorescently labeled each with a different color. As the DNA replicates, an optical beam scans the slide, and reads which fluorescent color (i.e. which base) is being incorporated into the nucleotide chain for each spot. As a third step, a computer program puts together, or assembles, the overall sequence of the starting sample (our bacterial chromosome) by identifying areas where the sequences overlap. Assembling the overall sequence is easier when the small fragment sequences are aligned to a known sample used as a scaffold (e.g. the full sequence of a similar bacterial chromosome.)

In other words, Next Generation Sequencing works by breaking DNA into smaller pieces to make each piece more manageable to read, then putting those pieces back together to read a complete sequence. The starting DNA sample can be very large – even as large as the entire human genome!