A genome of an organism is the collection of all the genes in the organism’s complete set of chromosomes.  Genes, which are units of heredity, encode all the proteins responsible for controlling everything from organism’s shape and size down to the function of every organ, tissue and cell. On a molecular level, genes are ordered sequence of four letters: A, C, G and T, which represent four nitrogenous-rich chemicals, also called bases, held together by weak bonds such that A pairs only with T and G pairs only with C.  The long chain consisting of two strands that result from these paired bases form a double-helix structure, called deoxyribonucleic acid (DNA) (Fig: 1). A human genome consists of approximately 3 billion base pairs, which make up about 30,000 genes. Genome sequence, hence, is essentially a read-out of the entire sequence of the base pairs.

So how do you obtain the sequence of a genome? Genome sequencing technology has evolved considerably from the days of extremely low-throughput, high-cost and labor-intensive slab gel based sequencing to the current day state-of-the-art high-throughput, lower-cost and automated capillary-electrophoresis based sequencing done at Broad Institute, Cambridge, MA (www.broad.mit.edu). Our sequencing platform can currently sequence a human genome within 3 months at a cost of under 10 million dollars. The Human Genome Project, the first such effort to sequence a complete human genome completed in 2002 and took 11 years and 1 billion dollars.

The genome shotgun sequencing is based on the Sanger sequencing method (named after British scientist Fred Sanger, who invented the method in 1977). Due to the limitation of current technology, you can’t just put a whole genome through a sequencer and directly read the sequence of that genome. First you take the genome and cut it into hundreds of thousands of small unique fragments of overlapping sequence, each about a few thousands base pairs long. Next you need to make enough (on the order of tens of millions) copies of each fragment, attach fluorescent label through enzymatic reaction and run them through capillaries, where the labels from all the fragments collectively fluoresce well enough to be detected by a laser.

Before getting copied, DNA fragments are first attached to cloning vectors, which are circular pieces of DNA into which the fragment to be copied is introduced. This step is called template preparation. Next, these vectors with attached fragments are introduced into bacteria called E. Coli., which under the right conditions of temperature, time and nutrition multiply exponentially together with the vectors inside it. Thus, you end up with millions of copies of each fragment. In a high-throughput laboratory environment, each of the colonies of these fragments, also called clones, is picked into different wells of 384-well plate pre-filled with growth media, using a 3-axis robot ( Fig: 2) with 384 pins that are decontaminated between plates. For a single mammalian genome, there are about tens of thousands of these plates that need to be processed over few months. The plates are fed through automated stackers on the robot that processes hundreds of plates per day. After the bacterial growth step, the plates are through another robotic platform, where all the clones are transferred into another special 384-well plate capable of withstanding higher temperature for the enzymatic reaction to occur. Together with clones, an enzyme called phi29 is added into the plates. All the plates have unique barcodes which are tracked in real-time through a Laboratory Information Management System (LIMS). These robots are capable of processing up to 1200 plates/day (Fig: 3). The automated liquid dispenser unit with 384 disposable pipette tips mounted on this robot has the capacity to deliver sample volume as low as a few hundred nanoliters into each well. Low volume range is a critical requirement for the dispenser to accomplish lowest possible usage of the highly expensive enzymes and reagents used for the reactions in this and the sequencing step. The plates are then incubated at room temperature for about 16 hours during the amplification step where bacterial cells are lysed to expose the inserted genomic DNA, which is then exponentially amplified based on rolling circle amplification. This step results in enough DNA for the sequencing reaction to occur in the next step.

In the sequencing step, DNA is transferred into another plate with the addition of another enzyme called DNA polymerase together with four different colored fluorescent labels representing the four different bases. This most expensive reagent is added using a special non-contact dispensing robot that dispenses on-the-fly. This dispenser is capable of dispensing on the order of tens of nanoliters. The plates are then heat cycled resulting in sequencing products with fluorescent labels attached to them. Finally, these plates are put on the detectors (Fig: 4), where the products are electrokinetically injected into capillaries, which have been filled with the separation matrix. DNA, being negatively charged, moves toward the positive end of the capillaries upon the application of electrical voltage. The speed of the migration is a function of DNA size. So by taking into account the speed and the dye color (unique for each base), detected through the laser, the software identifies the sequence of bases on a given fragment. Repeating this process for all the fragments, you finally have the sequence (Fig: 5) of all the small fragments of DNA. Since all of these fragments have overlapping sequences, the next step is to assemble them into whole genome based on the overlapping ends.

Various other technologies are being developed at Broad and elsewhere with the goal of bringing the cost of sequencing one mammalian genome down,  first to $ 100,000 and then to $ 1000,  while cutting time down,  first to weeks and then to days. The sequencing community is actively developing other platforms (microfluidic chip with offline detection, integrated microfluidic and online optical detection etc) as well as pursuing other approaches (single molecule sequencing using zero-mode waveguides, solid-state nanopores etc) in the hope of reaping the benefits promised by these technologies. Only time will tell us which technology is best suited to advance our ability to sequence genome quickly, cheaply and effectively.

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