After the Human Genome Project was completed in 2003, scientists were looking for faster, cheaper, and more efficient sequencing methods than the Sanger Sequencing utilized in the 13-year project. In 2005, researchers at 454 Life Sciences (later acquired by Roche) developed the first Next-Generation Sequencing method called pyrosequencing. Since then, many other companies have developed their own Next-Generation Sequencing methods. The main Next-Generation Sequencing methods developed in the last two decades include a newer, improved pyrosequencing called Roche 454, Illumina’s Solexa Genome Analyzer series, and Ion Torrent sequencing.
As scientists continue to develop newer and more efficient sequencing methods, it has opened the door for commercialized whole-genome sequencing. Today, we have commercialized genotyping kits, like 23andMe, ancestry.com, and those kits that tell you what specific configuration of mutt or house cat you have. These kits don’t actually sequence your genome; instead, they identify which variants of particular genes you have. Commercialized whole-genome sequencing has begun to be available though, at surprisingly affordable prices. Recently, the company Nebula Genomics has begun offering whole-genome sequencing for as little as $299. The commercialization of genomic data could give people the resources to identify and treat health issues more quickly, but it also raises issues of privacy. Exactly who should have access to your genetic makeup?
454 pyrosequencing was the first Next-Generation Sequencing method developed after the Human Genome Project. Capable of sequencing a third of the human genome in one day, this form of sequencing was far faster and cheaper than Sanger sequencing. It involves cutting the genomic DNA into 400-600 base pair fragments and attaching those fragments to tiny micrometer-scale beads (for comparison roughly 10 billion of these beads would fit inside the average tennis ball). The beads are moved into individual wells on a sequencing plate that contain the DNA polymerase and primer that enable the fragment to be copied. Free bases are added to the plate one group at a time (As, then Ts, then Gs, then Cs, for example), and if the base is incorporated into the copied strand by the polymerase, the well gives off light in proportional intensity to the number of bases added.
The strength of this method is that a significant portion of the genome can be sequenced in a single run because all of the fragments can be sequenced simultaneously, in parallel. The main limitation of this method is that it can be hard to determine the exact number of bases when a base is repeated (is it TTT or TTTT, the difference in light intensity may be difficult for even a machine to accurately distinguish).
Developed shortly after pyrosequencing, Illumina’s Solexa sequencers utilize a technique called “sequencing by synthesis.” In this process, fragments of the genomic DNA are bound to a tagged surface. The fragments can actually attach to the surface on both sides, allowing them to form a bridge from one surface tag to another. Polymerases use this bridging to create an entire cluster of fragment copies surrounding the original fragment and attached to the tagged surface. Each fragment is copied roughly a million times so that when sequencing starts, the fluorescent signal from the cluster is strong enough to be read by the device. Like in pyrosequencing, bases are added one group at a time in a cycle. The bases are fluorescently tagged with the machine recording the fluorescence coming off of each cluster as different bases are added and new strands are synthesized. The Solexa method also employs “reversible terminators” that can be removed once the synthesis is completed so that the process can be repeated for multiple runs.
This method of sequencing can produce enormous amounts of sequencing data quickly and reliably by sequencing many of these fragment clusters in parallel. Since its original release in 2007, the Illumina Solexa sequencer and all of its newer iterations have become some of the most popular forms of sequencing available.
Both pyrosequencing and the Solexa sequencing rely on accurate detection of light signals released during the synthesis phase. In contrast, Ion torrent sequencing relies on reading subtle changes in pH that occur when bases are added to a growing DNA chain. Check out this helpful video from ThermoFisher explaining how ion torrent sequencing works:
All of these Next-Generation Sequencing methods contributed to making whole-genome sequencing faster and far cheaper. But for a long time, it was still far too expensive to be a viable option for individuals seeking genome sequencing for health or curiosity. In the meantime, an industry popped up around genotyping, a process in which an individual can be screened for known gene variants. This process is much simpler than sequencing, but it also reveals much less information about a person’s genetic makeup.
Very recently, however, genome sequencing has broken through the 1000 dollar barrier, allowing companies to offer it directly to consumers. Nebula Genomics currently offers whole genome sequencing and interpretation for $299 (less than the newest iPhone). Nebula Genomics uses MGI Tech’s DNA nanoball sequencers (DNBSEQ-T7 is the model they use—coincidently, DNA nanoball sounds like the kind of thing a Doctor Who villain would release into the atmosphere to convert the human race into cybermen . . . don’t ask me where the metal comes from). A DNA nanoball (DNB) is created using a process called rolling circle amplification that takes a circularized fragment of the genome and replicates it into up to 1000 copies on one long strand that curls around itself like a tiny hairball (gross). All the DNBs get loaded onto a chip in an evenly distributed array (gotta keep your hairball collection organized). Primers get added to the chip followed by polymerase and fluorescently-labeled free bases. Each time a base is incorporated into the growing strands, a fluorescent signal is released that is specific to the type of base. The machine receives the signal and determines the base that was incorporated.
Nebula Genomics is unlikely to be the only company offering cheap whole-genome sequencing for very long. We’ve seen how the genotyping industry has grown exponentially in the past few years (you can buy 23andMe kits in Target now) demonstrating just how hungry the public is for information about themselves. As the industry grows, there is the growing problem of genomic data privacy. In the past couple of decades, a lot has gone into keeping our private digital data safe and protected from hackers with mixed results. But as important as our digital data is, nothing is more personal and private than our genetic make-up, and many are understandably nervous about storing that kind of information online.
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