PacBio RS II

RS II is a sequencer released by Pacific Biosciences in January 2013. It is based on Single Molecule Real Time (SMRT) Sequencing.

PacBio RS II
Photo of RS II: by

RS II is available in the following models:
  • RS II
  • RS II (P4)
  • RS II (P5)
  • RS II (P6)


Manufacturer Pacific Biosciences
Technology Single Molecule Real Time (SMRT) Sequencing
Release date January 2013
Price [2] $695,000
MSRP as of release date
Type Industrial
Weight 1091 Kg
Supports real-time analysis No
Demultiplexing support Yes
File format BAM
Read length PacBio RS II: 1x10000bp
PacBio RS II (P4): 1x10000bp
PacBio RS II (P5): 1x10000bp
PacBio RS II (P6): 1x20000bp
Run time PacBio RS II: Up to 32 hours
PacBio RS II (P4): Up to 32 hours
PacBio RS II (P5): Up to 32 hours
PacBio RS II (P6): Up to 32 hours
Reagent cost per Gb [1] PacBio RS II: Unavailable
PacBio RS II (P4): Unavailable
PacBio RS II (P5): Unavailable
PacBio RS II (P6): Unavailable
Data quality [3] PacBio RS II: 99.999% accuracy
PacBio RS II (P4): 99.999% accuracy
PacBio RS II (P5): 99.999% accuracy
PacBio RS II (P6): 99.999% accuracy
Maximum output per run (Gb)[4] PacBio RS II: 1.00
PacBio RS II (P4): 1.00
PacBio RS II (P5): 1.00
PacBio RS II (P6): 1.00
Primary error type [5] Indels
Notes about the specifications

  1. Cost per Gb: The cost per Gb is calculated by dividing the costs of sequencing reagents by the manufacturer’s maximum output per run. For these runs, the manufacturer may have included their own controls and used specific library preparation kits. Actual performance may vary based on sample and library type and quality, loading concentration, and other experimental factors. The sequencing reagent costs used for calculations do not include any other costs associated with preparing the samples or running the instrument, including reagents used for extraction, quality control or library preparation, labor, electricity, lab consumables, storage, etc. Performance metrics and prices are subject to change.
  2. Price: The final price may be different depending on the supplier, the region and any discounts provided through bulk orders or negotiation. Other factors that may influence the final price are warranty options, maintenance contracts, possible trade-ins of older instruments, and any financing arrangements chosen.
  3. Data quality: This is the maximum data quality that the instrument can offer based on manufacturer’s specifications and optimizations. The quality of a specific run will depend on multiple factors, including sample quality, sample quantity, library prep protocol, run conditions, etc.
  4. Maximum output: This is the maximum output of a full instrument run. This number is based on manufacturer’s specifications and optimizations.
  5. Primary error type: This is the most common type of error that may occur due to sequencing. Other types of error may occur as well.

More about the RS II

The PacBio RS II, released in 2013, was a pioneering instrument in the single-molecule, real-time (SMRT) sequencing space. Aimed at researchers tackling de novo genome assemblies, complex regions with repetitive elements, and long-range haplotyping, the RS II laid the groundwork for future PacBio sequencers like the Sequel II. It has since been discontinued by Pacific Biosciences.

Key Innovations

The core innovation of the RS II is its ability to generate long reads of DNA sequences. Unlike traditional next-generation sequencing (NGS) technologies that break DNA into smaller fragments, Single Molecule, Real-Time (SMRT) sequencing allows for reading single DNA molecules in real time. This is achieved using zero-mode waveguides (ZMWs) - microscopic wells containing immobilized DNA/polymerase complexes. As fluorescently labeled nucleotides are incorporated during DNA synthesis, the instrument detects the release of the fluorophore, translating it into base calls.

Features and Technologies

The RS II boasts impressive read lengths, averaging over 10,000 base pairs (bp) with a maximum approaching 60,000 bp, significantly higher than the original RS. This enables researchers to assemble complex genomes with fewer gaps and characterize regions like transposable elements and repetitive sequences that are challenging for traditional short-read sequencing. However, the RS II has a lower data output compared to newer sequencers, typically generating ~50,000 reads per SMRT cell.

Intended Uses and Specific Applications

The RS II excels in:

  • De novo genome assembly: Long read lengths are crucial for assembling genomes from scratch, particularly for large or complex genomes with high repeat content.
  • Variant discovery in complex regions: The ability to span repetitive regions makes the RS II valuable for identifying variants in areas inaccessible to short-read sequencing.
  • Long RNA-Seq (Iso-Seq): Full-length transcript characterization for isoform discovery and alternative splicing analysis benefits from long reads.

As the RS II is discontinued, PacBio is not officially supporting it anymore with reagents or specific library prep kits. However, an example of a recent suitable library prep kit is the SMRTbell prep kit 3.0.

Comparison with Competing Instruments

  • PacBio Sequel II: The Sequel II is the successor to the RS II, offering significantly higher data output (up to 7x more reads) per run due to increased ZMW density in its SMRT Cells at similar read lengths and run times. It also can offer high fidelity reads, which are higher accuracy than the RS II.
  • Oxford Nanopore Flongle: The Flongle, from Oxford Nanopore, utilizes a different technology (nanopore sequencing) to achieve long reads. It boasts even longer average read lengths than the RS II at shorter run times with a higher max output, all at a much lower cost. However, the accuracy is lower than the RS II.

Special Considerations

The RS II, while groundbreaking for its time, has been surpassed by newer PacBio sequencers in terms of data output and is now discontinued. Researchers might still consider the RS II if:

  • High accuracy long reads are a priority: The RS II offers good accuracy for long reads compared to some competing platforms.
  • Lower upfront instrument cost is a major factor: Since the instrument is discontinued, it may be possible to find a used RS II at a significantly reduced price.
  • Access to a Sequel II is unavailable: The RS II remains a valuable tool for researchers who do not have access to the higher-throughput Sequel II or Revio.

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This information on this page is up-to-date as of April 26, 2024 and based on the spec sheets published by the manufacturer.