SOLiD Sequencing: Principle, Steps, Applications microbiologystudy

SOLiD (Sequencing by Oligonucleotide Ligation and Detection) is a second-generation sequencing platform that uses sequencing by ligation method to determine the sequence of nucleotides in target DNA.

It was developed and commercialized by Applied Biosystems in 2007. It is used in various fields including genome research, transcriptome studies, and clinical diagnostics. Instead of synthesizing DNA bases one at a time like in Illumina, SOLiD technology reads short DNA sequences using ligation which provides highly accurate base calling.

SOLiD sequencing is unique in using the ligation-based method with di-base probes which offers advantages in accuracy and data analysis. Unlike other next-generation sequencing platforms, SOLiD uses a special approach called color space analysis, which makes error detection more effective and helps identify complex genetic variations like single nucleotide polymorphisms (SNPs), insertions, and deletions.

Principle of SOLiD Sequencing

SOLiD sequencing is based on the principle of sequencing by ligation which involves the hybridization and ligation of fluorescent probes. It uses fluorescent signals to identify DNA sequences. While other methods like Sanger sequencing use one fluorophore for each nucleotide, SOLiD sequencing assigns a fluorophore to a combination of two nucleotides. Each fluorescence signal represents a pair of nucleotides. This means that the initial data doesn’t directly reveal the DNA sequence because each signal corresponds to one of four possible dinucleotide combinations. 

To determine the sequence of nucleotides, the SOLiD system uses the color space technique, which decodes the sequence by analyzing the signals emitted. The color space technique is unique to the SOLiD platform. In the color space approach, each pair of nucleotides is assigned a specific color and related pairs, such as reverse complements or complementary pairs, share the same color. To accurately identify the sequence, multiple readings are taken from different angles. 

In the SOLiD sequencing process, the DNA fragments to be sequenced are linked to tiny beads and amplified using emulsion PCR (emPCR). Then, the amplified fragments are paired with fluorescently labeled probes that contain two known bases. The ligase enzyme joins these probes to the DNA template, and the attached fluorescent labels are detected by imaging. After each ligation, the fluorescent dye is removed, and a new probe is added in the next cycle. The cycle restarts at a slightly shifted position. This ensures that the same DNA base is detected by two different ligation events which improves the accuracy of each base call.

Process/Steps of SOLiD Sequencing

1. Library Preparation

The first step in SOLiD sequencing is preparing the DNA library. Library preparation includes fragmenting DNA or RNA and ligating short adapters to the ends of the fragments. For the SOLiD system, two types of libraries can be created: fragment libraries or mate-paired libraries. The choice depends on the kind of experiment conducted and the information we want. 

2. Template Amplification

The next step is to prepare the DNA templates. In this process, DNA templates are amplified using emPCR on tiny beads inside microreactors, which contain all the necessary components for PCR. Each bead carries a cloned population of DNA fragments. After the templates are amplified on the beads, they are enriched and prepared for attachment to the sequencing surface. A special chemical modification is applied to the 3′ end of the DNA on these beads allowing them to bind to a glass surface or FlowChip for sequencing. 

3. Sequencing by Ligation

Primers bind to specific adapter sequences on the DNA templates, and the process of ligation begins. Four fluorescently labeled probes compete to bind to the template DNA strand in each cycle. Each probe is designed to read two bases at a time so they are called di-base probes. The fluorescence emitted by the bound probes allows the system to identify which base pairs are present. The sequencing process involves multiple rounds of ligation, detection, and cleavage. After each set of ligation cycles, the primers are reset to cover different positions on the DNA.

4. Primer Reset

The system performs five rounds of primer resets to ensure every base is accurately identified. Each round shifts the position of the primer slightly and allows the system to read each base twice with two different primers. 

5. Exact Call Chemistry (ECC)

In ECC, additional primers are used to encode the DNA in a way that enhances accuracy. When paired with a reference genome, this process can achieve up to 99.99% accuracy. Even without a reference, ECC allows data to be output in a base-space format which provides a reliable read of the DNA sequence.

6. Color Space System

SOLiD uses a unique color space coding system where each pair of nucleotides is represented by one of four colors. This two-base encoding system helps to detect sequencing errors or mutations. The color signals gathered during each cycle don’t immediately correspond to specific nucleotides. Multiple cycles are required to resolve the DNA sequence fully.

7. Data Analysis

The sequencing reads are decoded from color space to nucleotide sequence for downstream analysis after aligning the sequence to a reference genome. The raw color space data is converted into nucleotide sequences and processed for further applications.

Advantages of SOLiD Sequencing

• SOLiD sequencing offers high accuracy due to the two-base encoding and dual-reading approach. The two-base encoding system ensures that each base is read twice, reducing errors and increasing confidence in base calling.
• It is cost-effective compared to other next-generation sequencing platforms, especially for applications that require high accuracy.
• The color space system allows built-in error checking that ensures even small errors or mutations can be detected. This helps to distinguish between true genetic variants and measurement errors.
• This method allows the easy identification of errors, improving the overall quality of the sequencing output.
• It helps minimize the misinterpretation of sequencing results and reduces the likelihood of errors.

Limitations of SOLiD Sequencing

• SOLiD sequencing has shorter read lengths compared to other platforms. This limits its ability to sequence and map long, contiguous regions of DNA and makes it less suitable for de novo genome assembly.
• The emPCR and ligation-based methods in SOLiD sequencing make the process complex and time-consuming.
• The color space coding system requires specialized software and more complex computational analysis to interpret.
• The color space data must be converted back to bae space after analysis which adds complexity to the workflow.
• The conversion of data from color space to base space often requires alignment to a reference genome that limits de novo applications.
• Errors during the emPCR amplification step can lead to biases and incorporation mistakes, affecting the overall quality of the sequencing data.
• The advent of simpler and faster sequencing technologies like Illumina and Oxford Nanopore has led to a decline in the use of SOLiD sequencing.

Applications of SOLiD Sequencing

• SOLiD Sequencing has applications in whole-genome sequencing (WGS) for sequencing entire genomes, especially for organisms with complex genomes.
• SOLiD Sequencing can be used for RNA-Seq to study gene expression patterns, alternative splicing events, and transcriptome variations across different tissues or conditions. It can detect lowly expressed genes and provides accurate gene expression profiling.
• Its accuracy allows the detection of methylation patterns, histone modifications, and other epigenetic changes that influence gene regulation. It has applications in methylation and ChIP-Seq.
• It can detect complex genomic rearrangements, such as insertions, deletions, and structural variations.
• SOLiD Sequencing has been used in the diagnosis of genetic disorders, pathogen detection, and precision medicine, where accuracy is essential for detecting rare variants.
• The high accuracy of the SOLiD technology is useful for targeted resequencing. This allows region-specific resequencing when aligned with a known reference sequence which is useful for focused genomic analysis, such as studying specific mutations or variants.

References

1. Garrido-Cardenas, J., Garcia-Maroto, F., Alvarez-Bermejo, J., & Manzano-Agugliaro, F. (2017). DNA Sequencing Sensors: An Overview. Sensors, 17(3), 588. https://doi.org/10.3390/s17030588
2. Goodwin, S., McPherson, J. D., & McCombie, W. R. (2016). Coming of age: ten years of next-generation sequencing technologies. Nature Reviews Genetics, 17(6), 333–351. https://doi.org/10.1038/nrg.2016.49
3. Heather, J. M., & Chain, B. (2015). The sequence of sequencers: The history of sequencing DNA. Genomics, 107(1), 1–8. https://doi.org/10.1016/j.ygeno.2015.11.003
4. Metzker, M. L. (2009). Sequencing technologies — the next generation. Nature Reviews Genetics, 11(1), 31–46. https://doi.org/10.1038/nrg2626
5. Satam, H., Joshi, K., Mangrolia, U., Waghoo, S., Zaidi, G., Rawool, S., Thakare, R. P., Banday, S., Mishra, A. K., Das, G., & Malonia, S. K. (2023). Next-Generation Sequencing Technology: Current Trends and Advancements. Biology, 12(7), 997. https://doi.org/10.3390/biology12070997
6. Solid sequencing – Genomics.org. (n.d.). Retrieved from https://genomics.org/Solid_sequencing
7. SOLID® Next-Generation Sequencing Chemistry | Thermo Fisher Scientific – NP. (n.d.). Retrieved from https://www.thermofisher.com/np/en/home/life-science/sequencing/next-generation-sequencing/solid-next-generation-sequencing/solid-next-generation-sequencing-systems-reagents-accessories/solid-next-generation-sequencing-chemistry.html