SOLiD is known for its speed, accuracy, and affordability. It produces millions of short sequence reads all at once, making for a speedy and high-throughput sequencing of short-read sequences. The way it works is through a clever 2-base encoding system that translates the raw data generated by the sequencing platform into sequence data. The SOLiD sequencing platform is composed of a combination of a DNA polymerase, a DNA ligase, fluorescently-labeled oligonucleotides, and magnetic beads.
The SOLiD sequencing method involves several steps, starting with the preparation of a library of DNA fragments. Each DNA fragment is attached to a magnetic bead with a unique sequence that is identical for all fragments. This ensures that the starting sequence for each fragment is known and the same. Next, two adapters are attached to the fragment library, and emulsion polymerase chain reaction (PCR) is performed, which produces clonal beads and amplifies the fragments. Finally, the beads are deposited on a glass slide and analyzed using digital imaging.
One of the most critical steps in SOLiD sequencing is the ligation of fluorescently-labeled oligonucleotides to the template DNA. These oligonucleotides, called sequencing probes, are designed to hybridize to the sequence adapter at the library template. Each base is queried by two labeled oligonucleotides, which have one of 16 specific dinucleotides. They use two bases encoding, to decode platform data, each base pair of the probe is one of four possible colors. There are four dyes and four oligonucleotides for every dye. The scientists use primers that attach to the special sequence to help a set of four fluorescently labeled probes compete for ligation to the sequencing primer. These probes work by analyzing the first and second bases in each ligation reaction to determine the specificity of the probe.
The ligation process involves the addition of four different fluorescently-labeled probes, each representing one of the four nucleotides: A (adenine), C (cytosine), G (guanine), and T (thymine). The scientists perform multiple cycles of ligation, detection, and cleavage to determine the length of the read. The sequencing involves detection and removal of fluorochrome. After several cycles of ligation, they remove the extension product and reset the template with a primer that’s complementary to the n-1 position for a second round of ligation cycles. Scientists complete five rounds of primer reset for each sequence tag, which allows them to interrogate each base in two independent ligation reactions by two different primers.
The SOLiD sequencing method uses a two-base encoding system that assigns each unique pair of bases on the 3′ end of the probe one of four possible colors. For example, « AA » might be assigned to blue and « AC » to green. During sequencing, each base in the template is sequenced twice, and the resulting data are decoded using this system. For example, if the scientists want to know the base at position 5, they would use primer number 2 in ligation cycle 2 and primer number 3 in ligation cycle 1 to get the information they need.