High throughput sequencing technologies play major role in studies on genomics, epigenomics, and transcriptomics. This technology is capable of sequencing multiple DNA molecules in parallel, enabling hundreds of millions of DNA molecules to be sequenced at a time. This advantage allows high throughput sequencing to be used to create large data sets, generating more comprehensive insights into the cellular genomic and transcriptomic signatures of various diseases and developmental stages. Within high throughput sequencing technologies, whole exome sequencing can be used to identify novel variants and other mutations that may underlie many genetic cardiac disorders, whereas RNA sequencing can be used to analyze how the transcriptome changes
High throughput sequencing or next-generation sequencing technologies no longer relied on this labor-intensive and time-intensive procedure.
The High-throughput sequencing method is fast, cheap ways to sequence and analyze large genomes. The method involves the amplification of DNA templates by the polymerase chain reaction and the physical binding of template DNA to a solid surface or to tiny beads called microbeads. These techniques are often referred to as massively parallel DNA sequencing, because thousands or millions of sequencing reactions are run at once to greatly speed up the process. This animation describes one such high-throughput method, in which a single 10-hour run can produce 400 million or more base pairs of DNA sequence information.
- For massively parallel sequencing using microbeads, the genomic DNA is first cut into fragments of 300–800 base pairs.
- Small DNA adaptors are added to each end of a fragment, and the double-stranded DNA is denatured to single-stranded DNA.
- One and a half million tiny microbeads, each less than 20 microns in diameter, are coated with DNA primers complementary to one of the adaptors. The single-stranded DNA molecules attach to the primers by complementary base pairing under conditions that favor just one DNA fragment attachment per bead.
- For the next step, each bead must occupy its own tiny reaction chamber. A fine emulsion of oil and the reaction mixture is created by homogenization, such that each bead is isolated in its own reaction bubble. Inside the bubbles, an amplification procedure called PCR (for polymerase chain reaction) will amplify the DNA so that enough identical DNA strands are available to analyze.
- In PCR, DNA polymerase synthesizes a second strand of DNA starting from a primer. The two long strands are separated by heat, and then new primers are allowed to bind. DNA synthesis continues from the new primers. With each cycle, heat separates the strands that are not permanently affixed to the bead by the attached primer. The PCR cycles continue until each bead has about 2 million identical copies of DNA.
- Beads are loaded into tiny wells with room for a single bead each, resulting in an array of a million beads. Each well consists of a different amplified DNA fragment.
- Along with a single bead, the well contains beads covered with two types of enzymes, sulfurylase and luciferase, that create light signals during the sequencing reaction.
- Some sequencing reactions employ fluorescently labeled dNTPs. However, the technique depicted here, called pyrosequencing, uses a different strategy. First, a primer is allowed to attach to the DNA, and then DNA polymerase begins to add nucleotides. A single type of dNTP, in this case dTTP is flowed across the wells and becomes part of the reaction. The thymine can form a base pair with the adenine in the DNA strand, allowing the polymerase to attach the incoming nucleotide to the primer. In the process, phosphate groups are snipped off the dNTP in the form of a pyrophosphate ion.
- On the enzyme bead, the enzymes use the pyrophosphate and other substrates to perform a series of reactions. One enzyme converts the pyrophosphate into ATP and the other uses the energy in ATP to produce a flash of light. The light is detected by a camera and recorded.
- The nucleotides are washed out of the wells, and a new nucleotide is added. Because an adenine does not pair with a guanine, no nucleotides are incorporated, and no light is emitted from the well.
- The next set of nucleotides bear a cytosine base, and one is incorporated into the sequence, emitting light through the action of the enzyme beads.
- The next nucleotides bear a guanine base. Three nucleotides are added to the growing chain, and each pyrophosphate ion results in the emission of a photon. Three times as much light is emitted from the reaction with the guanine-bearing nucleotides, indicating that three cytosine bases appear consecutively in the template strand.
- The four nucleotides are flowed sequentially into the wells, and the set of four flows is repeated for 100 cycles. This grid represents a moment in time in which a single type of nucleotide has been flowed across the wells. In the dark wells, no nucleotide is incorporated. In the dimly lit wells, a single nucleotide is incorporated. In the brighter wells, multiple nucleotides are incorporated. Data from 100 cycles are processed.
- The data from a single well can be depicted on a chart. A calibration sequence is built into each sequencing reaction, showing the level of light emitted for the addition of single nucleotides. For the sequencing reaction in this well, a thymine was incorporated first. Adenine could not be incorporated next, but the following flow with cytosine worked. The light emitted from the next reaction with guanine is three times as intense as for the single incorporations of nucleotides, indicating that three guanine-bearing nucleotides were incorporated in a row. One flow of nucleotides after another reveals the sequence of the DNA.
- With 1,000,000 reads occurring simultaneously and an average read length of 400 bases, a single 10-hour run time can produce 400 million bases of sequence information.
- Software packages assemble the sequence fragments into longer pieces, and in this way determine the overall sequence of a genome.
There is no separation between the elongation and detection steps. The bases are identified as the sequencing reaction proceeds. While HTS decreased cost and time, their ‘reads’ were relatively short. That is, in order to assemble an entire genome, intensive computation was necessary, that put together millions of short stretches of sequenced DNA to create the overall nucleotide sequence of a chromosome or genome.