DNA detection: This illustration shows a single well, 70 to 100 nanometers in diameter, one of thousands on Pacific Biosciences’ chip. A DNA polymerase enzyme (shown in white) is immobilized at the bottom, with a piece of single-stranded DNA (shown in purple). Fluorescently labeled bases, each marked with a different color (shown here as gray shapes with red, orange, blue, or green circles), randomly diffuse into the well. When the polymerase enzyme detects that the correct base is present, it attaches the fluorescent molecule to the growing strand of DNA. Fluorescent light shines from below, illuminating that reaction, which is captured by a camera. The polymerase then moves to the next position on the strand.
Both Roche and Illumina have ramped up speed by running a massive number of sequencing reactions in parallel. However, these methods generate relatively short lengths of sequence, about 50 to 200 bases pairs, which must then be computationally assembled into a complete sequence. The shorter the piece, the more computationally difficult it is to sew them together. “With a 35 base read, you can’t assemble 25 percent of the genome,” says Turner.
Pacific Biosciences has already generated what Turner believes to be “the longest sequencing trace ever made.” In a proof-of-principle experiment, scientists continuously sequenced the same 135 base circle of DNA 12 times–it was like repeatedly driving around a rotary. While the company hasn’t yet repeated the feat with natural DNA, the ability to generate long reads could be especially important when sequencing unknown DNA or stretches with a highly repetitive series of bases. “Many of the dynamic regions of the genome that are associated with disease consist of duplicated and complex repetitive sequences that cannot be accurately assessed at present,” says Evan Eichler, a geneticist at the University of Washington, in Seattle. “Long-sequence reads are necessary to comprehensively understand human genetic variation.”
Pacific Biosciences’ machine may also enable scientists to generate more accurate sequences. Existing methods generate a consensus sequence by reading the same section of multiple copies of DNA, which may have some copying errors, and pooling the results. “All short-read technologies are less accurate than traditional methods,” says Nusbaum. With Pacific Biosciences technology, scientists would theoretically be able to sequence the same piece of DNA multiple times. “That means we’ll be able to detect rare mutations with unprecedented accuracy, orders of magnitude better than others,” says Turner.
He adds that the long reads, high accuracy, and quick turnaround time make the technology ideal for diagnostics, such as those for cancer, which involves long stretches of repetitive sequences, and infectious disease, in which small sequence changes may drastically change the infectiousness and virulence of a pathogen.
Perhaps the biggest excitement comes from Turner’s predictions for the future. He says that it will be easy to further improve the company’s technology with higher-resolution cameras, faster DNA polymerases, and more densely packed chips. Currently, each chip has thousands of wells, only about one-third of which are used. (The remaining two-thirds house either no enzyme molecules or more than one, and thus fail to generate useful information.) But the chips, which are created using semiconductor fabrication methods, have the capacity to hold 10 million wells.
Turner calculates that with a camera that can track one million wells, a polymerase that operates at about 50 bases per second (the current rate is 10), and full use of all the wells on the plate, Pacific Biosciences technology could read 100 gigabases an hour. That translates to full coverage of a human genome–the same genome sequenced 15 times–in just 15 minutes.
“I’m pretty excited about the possibilities of this technology, but I remain to be convinced,” says Nusbaum. “They still have some significant technical hurdles ahead of them.”