Genome sequencing has gone through a precipitous drop in price since the first human genome was sequenced at a cost of billions of dollars. In fact, last year James Watson’s genome was sequenced for about $2 million. While this is still pricey, it represents a three orders of magnitude decrease on the way to the $1,000 price tag that would bring large-scale sequencing into the realm of feasibility.

This price schedule still has a way to go to fulfill the dream of the $100 genome that would make personalized medicine available for all. At Cambridge Healthtech’s “Next Generation Sequencing” meeting held last month, companies presented their technologies designed to move the field ever closer to that goal.

“Our company’s approach to sequencing is built on a three-tiered library strategy,” said Radoje Drmanac, Ph.D., CSO at Complete Genomics. The first consists of preparing DNA fragments of about 500 bp in length with 35 base paired-end reads, long enough to span the repetitive sequences. The second tier is composed of longer (5–10 kilo base pair) fragments with 35 base paired-end reads to cover most long repeats and rearrangements. Finally, the third tier consists of much longer fragments in the 100 kilo base pair size for much longer reads.

Dr. Drmanac described his company’s technology for obtaining independent sequence data from both homologues, in which the DNA from a small number (approximately 20) of human cells is divided between the wells of a 384-well plate. This means that the actual number of molecules diminishes to a handful per well. With such small numbers it is unlikely that any well will receive DNA from both homologous chromosomes.

Another feature of the Complete Genomics platform is the use of amplified DNA clusters referred to as DNA nano-balls. This technology uses a small circular DNA template consisting of approximately 80 bases of genomic DNA and four synthetic adaptors producing a head-to-tail concatemer containing more than 200 copies formed into a ball. One mL of reaction volume generates over 10 billion DNA nanoballs, sufficient for sequencing an entire human genome. The amplification takes place in solution and avoids the cost and challenges of relying on single fluorophore measurements used by single-molecule sequencing systems, Dr. Drmanac explained.

Unlike alternative approaches, clonal DNA amplification is not performed in emulsions or on surfaces. The amplification process occurs in solution and in a single reaction chamber, allowing for higher density and lower reagent usage. Additionally, the DNA-nanoball production process inherently produces clonal amplicons; it is not subject to the stochastic variation from limiting dilution.

“Our platform can be scaled up to eventually accommodate millions of sequences per year at an affordable cost,” said Dr. Drmanac. The company’s present commercial plan starts in June with the sequencing of hundreds of genomes with the aim of moving this number into the millions in five to six years. “With this many analyzed genomes scientists and doctors will have in-depth insight in causes of the 1,000 most frequent genetic disorders.

According to Dr. Drmanac, one of the major accomplishments has been the sequencing of a Caucasian HapMap sample generating 91x average read coverage (that is, a repetition of the sequencing 91 times to ensure maximum accuracy) of the genome using Complete Genomics third-generation genome sequencing technology. The sequencing revealed 3.3 million SNPs, 12% of them novel, and 40,000 insertions and deletions.

Squeezing through the Channels

“We are looking at the genome holistically, taking into account changes all the way from individual base pair mutations to large chromosomal rearrangements on the haplotype level,” explained Michael Boyce-Jacino, Ph.D., president and CEO of BioNanomatrix. “In fact, we believe major structural changes, combined with single base pair mutations, are the basis of human genetic variability.”

The hardware of the company’s technology are chips fabricated from fused silica with 100 nm wide channels etched in them to form a highly organized surface providing maximum density. The channels are covered with a roof of sprayed silicon dioxide or a glass coverslip to form tiny tunnels that can be as narrow as 10 nanometers. The molecules are driven through this nanochannel array with low voltage electrical pulses.

BioNanomatrix is applying semi- conducter nanofabrication technology to linearize long strands of DNA for massively parallel single-molecule genome analysis.

The BioNanomatrix sorting strategy ensures maximum efficiency of space by driving the DNA molecules through the nanochannels, which are designed with a narrowing configuration reminiscent of a highway with the lanes merging together at a point where toll booths are placed. In such a landscape the cars are sorted and forced into narrower and narrower lanes. In a similar fashion the balled up DNA is forced to unravel as it flows through the device until it ends up as long, linear DNA molecules. To identify landmarks along the molecule they are tagged with fluorescent molecules that identify individual bases visually in order to arrive at a sequence.

The DNA-filled nanochannels have a number of applications such as genome mapping in which the molecules are digested with nicking enzymes and labeled, with the pattern of labels allowing identification of deletions, mutations, and translocations.

“To fill in the gaps we perform ultralong analysis, greater than 1 kb in length and up to the whole chromosome,” stated Dr. Boyce-Jacino. At this point we do not have a sequencing product, however, we are in the second year of a five-year NIST-ATP-funded collaboration with Complete Genomics to create a much lower cost sequencing solution.”

According to Dr. Boyce-Jacino, BioNanomatrix makes extensive use of DNA bar coding for identifying and classifying sequences. This is a technique for finding genes using short DNA sequences from standard and agreed-upon positions in the gene. Since DNA bar code sequences can be obtained quickly and cheaply they are of great value in ordering the sequencing information, especially in the case of large-scale analyses involving an entire genome.

The company’s initial focus on large-scale modifications of the genome at the cytogenetic level has been partly funded through SBIR grants from NIH, reflecting the fact that the commercial significance of cytogenetic changes is only now starting to be recognized.

BioNanomatrix and Complete Genomics have also combined their complementary technologies to collaborate under a five-year $8.8 million NIST-ATP grant with the aim of sequencing the entire human genome in eight hours at a cost of $100. Through this combination of public and private funding, the companies claim to be “on track to reach the goal of the $100 genome by 2012,” noted Dr. Boyce-Jacino.

Sequencing-by-Synthesis Chemistry

“We have obtained worldwide licensing for our exclusive technology,” commented Jerzy Olejnik, Ph.D., vp for process R&D at Intelligent Bio-Systems, referring to the sequencing-by-synthesis chemistry developed at Columbia University. The basis of the technology is the binding of PCR-amplified fragments of DNA to a solid surface, and then building of a complementary sequence using polymerase and specially modified reversible terminator fluorescent nucleotides in four colors. The reagents are unique and low-cost, as is the instrument itself, driving down the overall cost per base, said Dr. Olejnik.

“The system and sample-preparation kits associated with it allows high accuracy and sensitivity of sequencing as well as opening the possibility of specialized applications such as digital gene-expression profiling and methylation profiling,” Dr. Olejnik continued.

The instrumentation, which is referred to as PinPoint, is outfitted with a high-speed imaging system and an ordered array-based disposable chip.

Moving Sequencing Forward

“We have developed a method to rapidly sequence DNA known as HANS or hybridization-assisted nanopore sequencing,” stated John Oliver, Ph.D., vp for research at NABsys. It combines two sequencing strategies—direct nanopore sequencing and sequencing by hybridization. “In brief, the genome is fragmented into pieces of 100 kb or longer, which are made single stranded and then hybridized with a short oligonucleotide probe. When these fragments are driven through a nanopore by a potential, they create changes in the current-versus-time profile, which indicates the binding positions of the probes.”

nanopore array (NABsys)
Parallel analysis of hybridization reactions with a nanopore array (NABsys)

According to Dr. Oliver, nanopore sequencing differs radically from sequencing approaches that use fluorescent compounds. “With our platform, we don’t need to label the molecule,” Dr. Oliver explained. “When the single-stranded DNA enters the pore, it changes the resistance because the chip is mounted between two chambers with a buffer solution bathing the chip.”

The nanopores are constructed by perforating a silicon chip with a focused beam from a transmission electron microscope. Prototype wafers are built by hand, but in the future they will be printed robotically. With these devices the size of the DNA molecule or the distance between two probes can be determined with an error range of 7%. The process can now be expanded, providing a genome-length probe map for each genomic fragment, and this can be accomplished in parallel for the entire library of probes.

The second phase of the process is to reconstruct the sequence from the probe-hybridization data by a process known as “moving window sequencing by hybridization.” The result is a complete genome sequence. “We are still in the early stages of development,” Dr. Oliver added, “but we are moving ahead to perfect a rapid and inexpensive approach to genome sequencing.”

From the early days of the human genome project, a vast amount of sequence data has been generated, not only for humans, but for many other species as well. Yet the traditional Sanger method is too expensive, cumbersome, and slow to accommodate the demands of large-scale sequencing of many genomes. These barriers must be overcome to satisfy the demands of clinical medicine.

Several of the companies interviewed for this article are not yet offering their technology to the public, but will do so within the next few months. In addition, other radical technologies, including nanoknife edge sequencing are in the research stage. The technologies that are now being pursued will no doubt bring the $100 genome to reality within the next few years.

K. John John Morrow Jr. PhD, is president of Newport Biotech.



























K. John Morrow Jr., Ph.D. ([email protected]), is president of Newport Biotech and a contributing editor for GEN. Web: Phone: (513) 237-3303.

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