Circular Consensus SequencingEdit

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Circular Consensus Sequencing (CCS) is a sequencing approach used within single-molecule real-time sequencing to produce high-accuracy, long DNA reads by repeatedly sequencing around a circular DNA template. The method yields reads known as HiFi reads, which combine substantial length with very high per-read accuracy, enabling robust genomic analyses that rely on long, accurate sequences. CCS is a core feature of the technologies developed by PacBio in collaboration with SMRT sequencing platforms, and it contrasts with traditional single-pass, short-read sequencing by collapsing multiple passes of the same DNA molecule into a single, high-confidence consensus sequence.

CCS relies on transforming the input DNA into circular templates called SMRTbell molecules. A DNA polymerase synthesizes a strand on the circular template, traversing the circle multiple times in a single continuous sequencing reaction. Each traversal generates a subread, and the collection of subreads corresponding to the same molecule is used to compute a consensus sequence. This consensus, when it reaches a suitable number of passes, is reported as a HiFi read. The process leverages the real-time observation of nucleotide incorporation in single-molecule real-time sequencing to assemble a high-fidelity read from independent passes over the same insert. The high accuracy arises from statistical weighting across passes and from the random nature of errors, which tend to cancel out in the consensus.

Technology and workflow - Core principle: Multiple passes over a circular template yield redundant observations of the same DNA insert, enabling statistical consensus to correct random errors. HiFi reads are the practical fruit of this approach, offering long read lengths with accuracy sufficient for many downstream analyses. - Template construction: DNA is converted to circular molecules by attaching hairpin adapters, creating a continuous loop for the polymerase to traverse. This circularization is central to producing repetitive reads from a single molecule. - Data processing: Subreads produced by each pass are aligned and combined to produce a circular consensus sequence. The quality of the HiFi read improves with the number of passes, though beyond a practical threshold diminishing returns set in. - Quality metrics: HiFi reads commonly achieve very high per-base accuracy, often quoted in the range of Q20 to Q30 (roughly 99% to 99.9% accuracy), with read lengths typically spanning many kilobases. The exact accuracy depends on factors such as insert size, polymerase performance, and the number of passes. - Instrumentation: The approach is implemented on PacBio instruments such as the Sequel family and subsequent platforms, which are designed to maximize polymerase processivity and the number of passes that can be achieved on a given template.

Applications and impact - De novo genome assembly: The combination of long length and high accuracy reduces the complexity of assembling repetitive regions and improves contiguity for complex genomes. See also de novo genome assembly. - Haplotyping and structural variation: Long, precise reads facilitate phasing of variants across long spans and enable more reliable detection of structural variants, including insertions, deletions, and complex rearrangements. See also phasing (genetics) and structural variant. - Transcriptomics and isoforms: Long, accurate reads support full-length transcript sequencing in approaches such as Iso-Seq, enabling better resolution of alternative splicing and isoform structures. - Epigenetics and base modifications: The kinetic signals captured during SMRT sequencing can reveal certain native base modifications, providing information about methylation and other epigenetic marks alongside the sequence itself. See base modification and epigenetics. - Comparative genomics and microbiomes: The ability to assemble complete genomes from diverse organisms—including those with repetitive content—has broad implications for comparative studies and metagenomics.

Comparison with other technologies - Short-read sequencing: Traditional short-read technologies (e.g., Illumina sequencing) offer very high throughput at low per-base cost but produce short fragments that complicate assembly and phasing. CCS provides a complementary option when long-range accuracy is essential. - Long-read sequencing: Other long-read platforms (e.g., Oxford Nanopore sequencing) produce very long reads but historically faced higher per-read error rates; CCS seeks to combine long read length with substantially improved accuracy, reducing reliance on post hoc error correction. - Cost and throughput considerations: CCS data generation typically involves higher per-sample costs and different throughput dynamics than some short-read workflows. The choice between CCS and alternative sequencing strategies often depends on the requirements for read length, accuracy, and downstream analyses.

Limitations and challenges - DNA quality and input: Successful CCS requires high-murity, high-molecular-weight DNA to maximize read length and the number of passes; degraded or fragmented DNA can limit performance. - Cost and infrastructure: The per-sample cost and the need for specialized instrumentation can constrain adoption in some settings, particularly where large-scale short-read surveys already exist. - Data analysis: Although the consensus approach reduces error rates, specialized software and expertise are still needed to process and interpret HiFi data, integrate it with other data types, and manage the large data volumes generated. - Coverage considerations: While CCS excels at accuracy, achieving uniform coverage across all genomic regions may still require careful library preparation and sequencing design, particularly in GC-rich or structurally complex regions.

History and development - Concept and early development: The circular consensus approach emerged from efforts to improve per-molecule accuracy in real-time sequencing by exploiting multiple observations of the same insert. The underlying sequencing technology is built on the principles of single-molecule real-time sequencing. - Commercialization and evolution: PacBio introduced and iterated on SMRTbell libraries and CCS-based read calling, with instrument generations (e.g., Sequel II) that increased throughput and read length while maintaining or enhancing HiFi accuracy. The term HiFi reads has become widely used to describe CCS-derived high-accuracy long reads.

See also - PacBio - SMRT sequencing - SMRTbell - HiFi reads - de novo genome assembly - structural variant - phasing (genetics) - Iso-Seq - base modification - epigenetics - Illumina sequencing - Oxford Nanopore sequencing - genome sequencing