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What is High Throughput Sequencing?
High-throughput sequencing (HTS) technologies allow researchers to determine the order of nucleotides in DNA and discover new parts of the genome faster and easier than previous methods. In 2001, scientists completed the first draft of the human genome using Sanger DNA sequencing, an expensive and laborious method when sequencing large amounts of DNA. To uncover more details about the genome, researchers rushed to design and implement HTS technologies.1 While early HTS technologies were faster than earlier methods, they were also less accurate, and most researchers continued to use Sanger-based methods to confirm genetic variants. Today, HTS methods are much more accurate and reliable and normally no longer require secondary confirmation.
Popular high-throughput sequencing methods
Illumina sequencing currently dominates the HTS market. This methodology involves ligating adapter sequences to smaller pieces of DNA which are then attached to a glass slide. During the sequencing reaction, DNA polymerases add fluorescently labeled nucleotides to each nascent DNA strand. The sequencer images the resulting color flashes and records the corresponding nucleotide. Then each fluorophore is enzymatically removed and the process is repeated, amplifying each strand of DNA. This method generates short DNA sequence reads that are aligned to a known reference genome. Illumina sequencing is a versatile method that can be optimized for a researcher’s needs. For example, slower sequencing sequences may increase accuracy, while faster sequencing sequences may reduce accuracy.1.2
Ion-Torrent technology uses emulsion PCR to amplify adapter-ligated DNA sequences on the surface of beads that are dispensed into microwells. When nucleotides bind to a growing DNA strand, hydrogen ions are released into the microwell, changing the pH. A sensor detects these pH changes after each nucleotide addition, and the sequence is determined based on this information. Since Ion-Torrent sequencing does not use imaging, this technique increases sequencing speeds and reduces costs. Ion-Torrent sequencing also generates short reads similar to Illumina sequencing, which makes alignment difficult if the shortest read is located in a repetitive sequence. Ion-Torrent is also dependent on emulsion PCR which, like any PCR method, can introduce amplification biases as some sequences are easier to amplify than others.1.3
Single Molecule Real-Time Sequencing (SMRT)
In SMRT sequencing, scientists cap DNA molecules with single-stranded hairpin adapters, and sequencing ensues without multiple amplification steps. Similar to Illumina sequencing, DNA synthesis occurs with the addition of fluorescent nucleotides. In SMRT sequencing, however, the reaction occurs at the bottom of a very small chamber. As the nucleotides bind to the growing DNA strand, the chamber prevents background noise and the fluorescent signal is imaged in real time by recorded video and analyzed to reveal the DNA sequence. This method allows researchers to sequence longer pieces of DNA and allows for greater overlaps between the ends of DNA molecules, thus avoiding the need for a reference genome. Longer DNA sequences can also cover repetitive DNA, allowing researchers to cover more of the genome.1.4
High throughput sequencing applications
Whole genome sequencing
With their large size and numerous repeated sequences, whole genomes continue to present the greatest sequencing challenges. The Illumina and Ion-Torrent technologies are examples of short-read sequencing and require researchers to use template genomes during post-sequencing assembly. If the sequence of a genome is already known, these technologies quickly and inexpensively detect variations at the genome level. Meanwhile, SMRT technology can perform long-read sequencing, giving it the ability to generate overlapping sequences, making it easier to align them. Without the need for a template, researchers use SMRT sequencing to assemble new genomes of understudied species.5
Exome sequencing is the fastest and most cost-effective variant detection method, and it provides a rapid means of detecting inherited mutations in a clinical setting. Scientists can only isolate exome DNA strands and sequence them with HTS. Therefore, only the parts of the genome coding for proteins are sequenced, which represents approximately 1% of human DNA. Although it may seem like a tiny fraction of the genome, eighty percent of mutations in Mendelian disease are in protein-coding sequences.6.7
RNA sequencing (RNA-seq)
By converting RNA molecules into DNA using reverse transcriptase, scientists use HTS technologies to quantify gene expression throughout the genome. Many studies have focused on bulk RNA-seq analysis, where RNA is extracted, sequenced, and quantified from whole tissues. Recently, single-cell RNA-seq, where RNA is extracted and quantified from each individual cell in a tissue, has provided new insights into tissue heterogeneity and cell-cell communication.8
DNA-protein/DNA-DNA interactions (ChIP and Hi-C sequencing)
Scientists also use HTS technologies to determine the DNA binding sites of proteins. In chromatin immunoprecipitation (ChIP), proteins are bound to their DNA binding sites via fixation. The DNA is then cut into smaller pieces and an antibody lowers the protein-DNA complex. After sequencing the DNA fragments, researchers can identify where a protein binds in the genome.9 Researchers use a similar technique called Hi-C to determine the locations of DNA-DNA interactions bound by specific proteins. Once antibodies extract these proteins, researchers sequence the attached DNA molecules and gain insight into the 3D structure of the genome.ten
- JA Reuter et al., “High-Throughput Sequencing Technologies”, Mol Cell58:586-97, 2015.
2. J. Guo et al., “Four-color DNA sequencing with reversible 3′-o-modified nucleotide terminators and chemically cleavable fluorescent dideoxynucleotides”, PNAS105:9145-50, 2008.
3. JM Rothberg et al., “An integrated semiconductor device enabling non-optical genome sequencing”, Nature475:348-52, 2011.
4. J. Eid et al., “Real-time DNA sequencing from single polymerase molecules”, Science323:133-38, 2009.
5. SL Amarasinghe et al., “Opportunities and challenges in analyzing long-read sequencing data”, Genome Biol9:30 p.m., 2020.
6. G. Lightbody et al., “Review of High-Throughput Sequencing Applications in Personalized Medicine: Barriers and Facilitators to Future Advances in Research and Clinical Application,” Brief Bioinform20:1795-1811, 2019.
7. SM Rego et al., “High-Throughput Sequencing and Disease Risk Assessment,” Perspect Med by Cold Spring Harb9, 2019.
8. S. Chen et al., “High-throughput sequencing of transcriptome and chromatin accessibility in the same cell”, Nat Biotechnol37:1452-57, 2019.
9. R. Mundade et al., “Role of chip-seq in the discovery of transcription factor binding sites, the mechanism of differential gene regulation, epigenetic marks and beyond”, Cell cycle13:2847-52, 2014.
10. K. Pal et al., “Hi-c analysis: from data generation to integration”, Biophys Rev11:67-78, 2019.
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