Helicos Single-Molecule Sequencing: Principle, Steps, Uses microbiologystudy

Helicos single-molecule sequencing, also called true single-molecule sequencing (tSMS), is a third-generation DNA sequencing technology that directly reads individual DNA or RNA molecules without requiring complex steps like PCR amplification.

It was the first commercially introduced single-molecule sequencing system launched by Helicos Biosciences in 2008. This system uses sequencing-by-synthesis (SBS) and requires minimal sample preparation.

Unlike traditional next-generation sequencing methods which require complex ligation and amplification steps, Helicos sequencing uses a simplified process. This amplification-free preparation eliminates potential biases and errors, providing accurate sequence data. 

Although Helicos pioneered single-molecule sequencing, the company faced financial difficulties and ultimately filed for bankruptcy in 2012. At present, Helicos Biosciences no longer operates and its technologies are no longer sold or broadly used in its original form. The sequencing technology was acquired and modified by other companies like SeqLL and Direct Genomics.

What is Single-Molecule Sequencing?

• The majority of sequencing data used to come from amplification-based methods, but single-molecule sequencing without amplification has now become a widely used technology.
• Single-molecule sequencing methods are third-generation sequencing technologies that use simplified preparation methods and directly sequence without amplification of DNA templates. 
• There are different single-molecule sequencing methods. They are currently divided into two main methods: DNA polymerase-based methods and direct inspection methods. 
• DNA polymerase-based methods include Helicos Biosciences’ tSMS and Pacific Biosciences’ SMRT sequencing. Direct detection methods include nanopore sequencing and microscopy-based methods. 
• All single-molecule sequencing systems use sequencing-by-synthesis techniques using laser excitation to detect fluorescently labeled nucleotides. However, they differ in how fluorescence is excited and detected, and in the methods for immobilizing DNA.

Principle of Helicos Single-Molecule Sequencing

The principle of Helicos single-molecule sequencing is based on sequencing-by-synthesis and directly sequencing individual nucleic acid molecules without amplification.

The workflow begins by fragmenting genomic DNA which is denatured into single strands, poly-A tailed, and hybridized to a glass flow cell surface coated with oligo-dT primers to initiate sequencing. The poly-A tails are filled using dTTP and polymerase. After filling, fluorescently-labeled nucleotides are added to lock the templates. This prevents further extension and ensures templates are ready for sequencing reactions. 

During sequencing, fluorescently-labeled virtual terminator nucleotides are added and complementary bases are incorporated one at a time into the immobilized DNA strands. Each incorporation is detected through laser-induced fluorescence. The fluorescent label is then removed and this cycle is repeated until the entire DNA fragment is sequenced. 

Steps of Helicos Single-Molecule Sequencing

1. Template Preparation

• Genomic DNA is first fragmented and denatured into single strands. Shorter DNA templates is beneficial for efficient sequencing due to the increased availability of 3′-hydroxyl ends required for sequencing.
• Fragmentation can be done using sonication or enzymatic shearing methods. Restriction enzymes can also be used for some applications rather than shearing.
• The single-stranded DNA (ssDNA) is modified with poly-A tails. These tails allow the DNA to bind to the flow cell surface coated with complementary poly(T) primers for sequencing. Terminal transferase is used to add unbiased poly-dA tails.
• After tailing, it is necessary to block the 3′ ends of the tailed DNA to prevent interference during sequencing.
• The poly-A-tailed DNA strands are then immobilized onto the glass surface of the sequencing flow cell.

2. Fill and Lock Step

• Polymerase and dTTP are added to fill any remaining complementary nucleotides at the poly-A tail. This step prevents the sequencing of any unpaired A residues in the template.
• After filling, the templates are locked for sequencing by adding fluorescently-labeled terminator nucleotides. This ensures that the templates are available for sequencing.

3. Sequencing-by-Synthesis (SBS)

• After the fill and lock step, sequencing is initiated. Fluorescently-labeled virtual terminator nucleotides are added sequentially to incorporate the complementary base into the growing DNA strand using DNA polymerase. 
• Then the flow cell is illuminated with a laser which excites the fluorescent labels and the emitted signals are captured by a CCD camera. This captures the position of all labeled molecules.
• After each nucleotide incorporation, the fluorescent dyes and terminator groups are cleaved from the incorporated nucleotide, allowing the next cycle of nucleotide addition to proceed. 

4. Data Analysis

• Images captured during the sequencing run are processed to determine the nucleotide sequence information for each strand.
• The fluorescent signals are interpreted by base calling algorithms and translated into nucleotide sequences.
• The sequence information is then processed for downstream analysis including alignment to reference genome and variant detection.

Advantages of Helicos Single-Molecule Sequencing

• Helicos SMS allows direct sequencing by eliminating amplification. This preserves original DNA and RNA sequences.
• It eliminates biases related to GC content and molecule size, allowing for the sequencing of degraded or modified DNA and RNA directly. 
• This streamlined process involves direct hybridization of nucleic acids to a flow cell which reduces sample loss and bias.
• It can work with low amounts of starting material, making it suitable for rare or limited samples. 

Limitations of Helicos Single-Molecule Sequencing

• Helicos SMS produces short read lengths which can be a barrier for assembling complex genomes.
• Each run generates a massive amount of data which requires complex computational resources for data storage and analysis.
• The expensive instruments, operational expenses, and complex imaging system contribute to high overall costs which made it less accessible. 
• The Heliscope machine required a stable environment including heavy infrastructure like granite bases to minimize vibrations that could interfere with precise sequencing.
• Background noise during imaging can reduce accuracy, particularly in challenging regions like highly complex regions and homopolymers.
• Helicos technology struggled with limited throughput compared to competitors like Illumina.
• The inconsistent quality between runs makes it unreliable for research or clinical applications.

Applications of Helicos Single-Molecule Sequencing

• Helicos SMS can be used in RNA sequencing to directly sequence RNA without the need for reverse transcription into cDNA. It is useful for transcriptomic studies, capturing both coding and non-coding RNAs.
• It can be used in ChIP-seq which provides insights into regulatory and epigenetic processes by sequencing DNA fragments associated with specific proteins or epigenetic modifications.
• It is well-suited for applications like copy number variation (CNV) analysis. 
• It allows accurate detection of genetic variants and is useful in large-scale genomic studies. This is also useful in cancer research to identify rare mutations.
• It is also effective for studying genomes with high GC content, providing accurate representation compared to amplification-based methods.
• It is suitable for sequencing degraded or low-input DNA samples such as ancient and forensic samples.

The Fall of Helicos Single-Molecule Sequencing

• Despite being the first to commercialize single-molecule sequencing, Helicos Biosciences faced significant technical and economic challenges that led to its commercial failure. 
• The Heliscope instrument launched in 2008 offered unique technology that eliminated the need for DNA amplification and reduced errors. 
• However, the system was very expensive with high initial and operational costs. It also struggled with short read lengths, high error rates, environmental sensitivity, and limited scalability.
• When Helicos entered the market, competitors like Illumina had already dominated the market. Illumina’s platforms offered longer read lengths, faster processing, and significantly lower costs, making it difficult for Helicos to compete.
• Finally, Helicos stopped operations in 2011 after selling only a few machines worldwide. 
• Despite its failure, Helicos laid the foundation for developing newer single-molecule sequencing technologies like PacBio sequencing and Oxford Nanopore sequencing. 
• Following bankruptcy, the company’s patents were acquired by Direct Genomics, a company founded by Jiankui He, who was known for the controversial creation of the first germline genome-edited babies. Direct genomics worked on using the single-molecule sequencing approach for specific applications but the applications remained limited and the company faced controversies linked to its founder.
• The sequencing technology has been repurposed by SeqLL for niche targeted sequencing, particularly in areas like RNA sequencing.

References

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5. SEQLL – True Single Molecule Sequencing. (n.d.). Retrieved from https://seqll.com/
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