Nucleotide Addition

Ion Torrent: The Dark Side of DNA Sequencing

Ion Torrent. Even the name is cool. It suggests a deluge of charged particles that are here to drastically change the way we do science. Whether or not the technology ends up being as disruptive as surrounding press would suggest, there is no question that their core sequencing technology represents a huge deviation from the techniques used for sequencing in the past decades.

Sanger Sequencing: The Chain Termination Method

*Most of the following information was adapted from the Wikipedia entry on DNA Seqnencing.

Developed by Ferderick Sanger in 1975, it would contain a DNA synthesis reaction mixture of normal deoxynucleotide triphosphates (dNTPs) along with dideoxynucleotide triphosphates (ddNTPs). These ddNTPs would be incorporated into a chain of nucleic acids much like the normal dNTPs. However, the ddNTPs lack the oxygen from the 3′-hydroxyl (-OH) group necessary to form a phosphodiester bond and continue the expansion of the DNA chain. Once a ddNTP is incorporated into a DNA strand, there is no further expansion of that oligonucleotide by DNA polymerase.

By labeling the four different types of ddNTPs (A, T, C and G) with different fluorescent signals, once a ddNTP is incorporated into the elongating DNA strand, the light signals emitted by the molecule allow for the interpretation of the base at that position. Originally, oligonucleotides would then be separated by gel electrophoresis to understand at exactly which position the base was incorporated, but eventually, more automated methods came about to help improve the process. The following video does a good job of explaining exactly how chain termination methods work.

Knowledge in Light: The Dominance of Fluorescence

Fluorescence signaling has been the primary and dominant method for sequencing for the past few decades. As seen in the picture to the left, nucleotide addition results in the release of a diphosphate group, the energy of which has been used to catalyze the fluorescence during Sanger sequencing. The ion torrent team cleverly realized that diphosphate is only one of the reaction products released upon incorporation of a nucleic acid. Formation of the phosphodiester bond between the new nitrogenous base and the previously incorporated base also results in the release of a H+ from the 3′ hydroxyl group of the chain backbone.

Instead of using the energy released from the diphosphate groups to catalyze fluorescence, the ion torrent team decided to try something radically different: an attempt to determine nucleotide addition by monitoring the release of hydrogen ions. This method requires no light, a huge deviation from the past decades of sequencing.

Sequencing in the Dark: A Semiconductor Platform

Described in 2006 by Nader Pourmand, Ronald Davis and their team at Stanford, ion torrent determines DNA sequence through electrical detection. As pictured here, the release of hydrogen ions induces a current that can be measured on their CMOS based platform. Rothberg and his team have built out a robust and disruptive platform that takes advantage of this principle.

From a practical standpoint, it seems as though the semiconductor chips have ample space to affix many different primers targeting specific regions of the genome. Once genomic DNA is added, sequencing essentially takes place by performing a series of washes. Each wash contains one of the four possible nitrogenous bases (A, T, C, or G). If a base is incorporated in a well, the release of hydrogen ions will be detected via current induction in that well. No light required.

I’m excited to see how this technology scales. For the time being, the limitations seem to be extremely similar to Illumina’s MiSeq. However, they are offering a much cheaper price point per sample, and they claim that scalability is going to be on par with what we’ve seen for the past decade of semiconductors. More on the difference between the two here.

While much of the talk is centered around the total number of base pairs that can be read in parallel, one of my main concerns is actually about the length of individual reads. In the quest to shrink and optimize whole genome sequencing, it is important that full genomic coverage is achieved. Shorter read lengths will be incapable of testing for certain parts of the genome where large repetitive elements or pseudo-genes prevent one from easily discerning the true sequence. Right now both companies claim that the max read lengths will be around 400bp for their latest platforms. For the time being, this is great. However, I predict that the future will look better for the company that is able to determine more bases per read.

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