Single molecule real time sequencing

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Single molecule real time sequencing (also known as SMRT) is a parallelized single molecule DNA sequencing by synthesis technology developed by Pacific Biosciences. Single molecule real time sequencing utilizes the zero-mode waveguide (ZMW), developed in the laboratories of Harold G. Craighead and Watt W. Webb[1] at Cornell University. A single DNA polymerase enzyme is affixed at the bottom of a ZMW with a single molecule of DNA as a template. The ZMW is a structure that creates an illuminated observation volume that is small enough to observe only a single nucleotide of DNA (also known as a base) being incorporated by DNA polymerase. Each of the four DNA bases is attached to one of four different fluorescent dyes. When a nucleotide is incorporated by the DNA polymerase, the fluorescent tag is cleaved off and diffuses out of the observation area of the ZMW where its fluorescence is no longer observable. A detector detects the fluorescent signal of the nucleotide incorporation, and the base call is made according to the corresponding fluorescence of the dye. Sequence data generated from single molecule real time sequencing was first published in January 2009 in the journal Science.[2]

Technology[edit]

The DNA sequencing is done on a chip that contains many ZMWs. Inside each ZMW, a single active DNA polymerase with a single molecule of single stranded DNA template is immobilized to the bottom through which light can penetrate and create a visualization chamber that allows monitoring of the activity of the DNA polymerase at a single molecule level. The signal from a phospho-linked nucleotide incorporated by the DNA polymerase is detected as the DNA synthesis proceeds which results in the DNA sequencing in real time.

Phospholinked nucleotide[edit]

For each of the nucleotide bases, there are four corresponding fluorescent dye molecules that enable the detector to identify the base being incorporated by the DNA polymerase as it performs the DNA synthesis. The fluorescent dye molecule is attached to the phosphate chain of the nucleotide. When the nucleotide is incorporated by the DNA polymerase, the fluorescent dye is cleaved off with the phosphate chain as a part of a natural DNA synthesis process during which a phosphodiester bond is created to elongate the DNA chain. The cleaved fluorescent dye molecule then diffuses out of the detection volume so that the fluorescent signal is no longer detected.

Zero-mode waveguide[edit]

The zero-mode waveguide (ZMW) is a nanophotonic confinement structure that consists of a circular hole in an aluminum cladding film deposited on a clear silica substrate.[3] The ZMW holes are ~70 nm in diameter and ~100 nm in depth. Due to the behavior of light when it travels through a small aperture, the optical field decays exponentially inside the chamber.[4] The observation volume within an illuminated ZMW is ~20 zeptoliters (20 X 10−21 liters). Within this volume, the activity of DNA polymerase incorporating a single nucleotide can be readily detected.

Sequencing performance[edit]

Sequencing performance for the technology can be measured in read length and total throughput per experiment.

Pacific Biosciences commercialized SMRT sequencing in 2011,[5] after releasing a beta version of its instrument in late 2010.[6] At commercialization read length had a normal distribution with a mean of about 1.1 kilobases. A new chemistry kit released in early 2012 increased the sequencer's read length; an early customer of the chemistry cited mean read lengths of 2.5 to 2.9 kilobases.[7] The XL chemistry kit released in late 2012 increased average read length to more than 4.3 kilobases.[8][9] The P4 binding kit released in August 2013 combined with the XL chemistry kit yielded average read lengths to more than 5 kilobases[10] and when coupled with input DNA size selection (using an electrophoresis instrument such as BluePippin) yields average read length over 7 kilobases.[11]

Throughput per experiment for the technology is both influenced by the read length of DNA molecules sequenced as well as total multiplex of a SMRT Cell. The prototype of the SMRT Cell contained about 3000 ZMW holes that allowed parallelized DNA sequencing. At commercialization, the SMRT Cells were each patterned with 150,000 ZMW holes that were read in two sets of 75,000.[12] In April 2013, the company released a new version of the sequencer called the "PacBio RS II" that uses all 150,000 ZMW holes concurrently, doubling the throughput per experiment.[13][14] The highest throughput mode in November 2013 uses P5 binding, C3 chemistry, BluePippin size selection, and a PacBio RS II officially yields 350 megabases per SMRT Cell though a Human de novo data set released with the chemistry averaged 500 megabases per SMRT Cell. Throughput varies based on the type of sample being sequenced.[15]

Application[edit]

Single molecule real time sequencing may be applicable for a broad range of genomics research, namely:

  • De novo genome sequencing: Read lengths from the single molecule real time sequencing are comparable to or greater than that from the Sanger sequencing method based on dideoxynucleotide chain termination. The longer read length allows de novo genome sequencing and easier genome assemblies.[16] SMRT sequencing has been demonstrated for de novo genome sequencing in publications analyzing the E. coli outbreak in Germany in 2011 and in the cholera outbreak in Haiti in 2010, both in the New England Journal of Medicine.[17][18] Scientists are also using single molecule real time sequencing in hybrid assemblies for de novo genomes to combine short-read sequence data with long-read sequence data.[19][20] In 2012, several peer-reviewed publications were released demonstrating the automated finishing of bacterial genomes,[21][22] including one paper that updated the Celera Assembler with a pipeline for genome finishing using long SMRT sequencing reads.[23] In 2013, scientists estimated that long-read sequencing could be used to fully assemble and finish the majority of bacterial and archaeal genomes.[24]
  • Resequencing: A same DNA molecule can be resequenced independently by creating the circular DNA template and utilizing a strand displacing enzyme that separates the newly synthesized DNA strand from the template. Scientists from Pacific Biosciences, the University of California, and other institutes used this circular consensus approach with SMRT sequencing to prove the validity of activating internal tandem duplication mutations in FLT3 as a therapeutic target in acute myeloid leukemia. Their findings were published in the journal Nature in April 2012.[25] In August 2012, scientists from the Broad Institute published an evaluation of SMRT sequencing for SNP calling.[26]
  • Methylation detection: The dynamics of polymerase can indicate whether a base is methylated.[27] Scientists demonstrated the use of single molecule real time sequencing for detecting methylation and other base modifications in a series of peer-reviewed papers in 2011.[28][29][30] In 2012 a team of scientists used SMRT sequencing to generate the full methylomes of six bacteria, reporting their findings in the Nucleic Acids Research journal.[31] In November 2012, scientists published a report on genome-wide methylation of an outbreak strain of E. coli.[32]
  • Full isoform sequencing: Long reads make it possible to sequence full gene isoforms, including the 5' and 3' ends. This type of sequencing to capture isoforms and splice variants has been included in papers describing full human transcriptome sequencing and human embryonic stem cells.[33][34]
  • In Vitro Diagnostics: On 25 September 2013 a partnership was announced between Pacific Biosciences and Roche Diagnostics for the development of in vitro diagnostic products using the technology.[35]

References[edit]

  1. ^ M.J. Levene, J. Korlach, S.W. Turner, M. Foquet, H.G. Craighead, W.W. Webb, Zero-Mode Waveguides for Single-Molecule Analysis at high concentrations. Science. 299 (2003) 682-686
  2. ^ Real-Time DNA Sequencing from Single Polymerase Molecules
  3. ^ J. Korlach, P.J. Marks, R.L. Cicero, J.J. Gray, D.L. Murphy, D.B. Roitman, T.T. Pham, G.A. otto, M. Foquet, S.W. Turner, Selective aluminum passivation for targeted immobilization of single DNA polymerase molecules in zero-mode waveguide nanostructures. PNAS. 105(2008) 1176-1181
  4. ^ M. Foquet, K.T. Samiee, X. Kong, B.P. Chauduri, P.M. Lundquist, S.W. Turner, J. Freudenthal, d.B. Roitman, Improved fabrication of zero-mode waveguides for single-molecule detection. Journal of Applied Physics. 103 (2008) 034301-1-034301-9
  5. ^ PacBio Ships First Two Commercial Systems; Order Backlog Grows to 44 | In Sequence | Sequencing | GenomeWeb
  6. ^ PacBio Reveals Beta System Specs for RS; Says Commercial Release is on Track for First Half of 2011 | In Sequence | Sequencing | GenomeWeb
  7. ^ After a Year of Testing, Two Early PacBio Customers Expect More Routine Use of RS Sequencer in 2012 | In Sequence | Sequencing | GenomeWeb
  8. ^ PacBio's XL Chemistry Increases Read Lengths and Throughput; CSHL Tests the Tech on Rice Genome | In Sequence | Sequencing | GenomeWeb
  9. ^ PacBio Users Report Progress in Long Reads for Plant Genome Assembly, Tricky Regions of Human Genome | In Sequence | Sequencing | GenomeWeb
  10. ^ PacBio Blog: New DNA Polymerase P4 Delivers Higher-Quality Assemblies Using Fewer SMRT Cells
  11. ^ Longing for the longest reads: PacBio and BluePippin | In between lines of code
  12. ^ Pacific Biosciences: Consumables
  13. ^ PacBio Launches PacBio RS II Sequencer
  14. ^ New Products: PacBio's RS II; Cufflinks | In Sequence | Sequencing | GenomeWeb
  15. ^ New PacBio RSII throughput record at Duke
  16. ^ Real-Time DNA Sequencing from Single Polymerase Molecules
  17. ^ Origins of the E. coli Strain Causing an Outbreak of Hemolytic-Uremic Syndrome in Germany
  18. ^ The Origin of the Haitian Cholera Outbreak Strain
  19. ^ GEN | Magazine Articles: Tech Tips: Next-Generation Sequencing
  20. ^ http://schatzlab.cshl.edu/presentations/2011-09-07.PacBio%20Users%20Meeting.pdf
  21. ^ http://genome.cshlp.org/content/early/2012/07/24/gr.141515.112.full.pdf
  22. ^ A hybrid approach for the automated finishing of bacterial genomes : Nature Biotechnology : Nature Publishing Group
  23. ^ Hybrid error correction and de novo assembly of single-molecule sequencing reads : Nature Biotechnology : Nature Publishing Group
  24. ^ Genome Biology | Abstract | Reducing assembly complexity of microbial genomes with single-molecule sequencing
  25. ^ http://www.nature.com/nature/journal/vaop/ncurrent/full/nature11016.html
  26. ^ BMC Genomics | Abstract | Pacific biosciences sequencing technology for genotyping and variation discovery in human data
  27. ^ http://www.nature.com/nmeth/journal/v7/n6/full/nmeth.1459.html
  28. ^ Characterization of DNA methyltransferase specificities using single-molecule, real-time DNA sequencing
  29. ^ http://www.nature.com/nmeth/journal/v9/n1/full/nmeth.1779.html
  30. ^ Genome Integrity | Full text | Direct Detection and Sequencing of Damaged DNA Bases
  31. ^ The methylomes of six bacteria
  32. ^ Genome-wide mapping of methylated adenine residues in pathogenic Escherichia coli using single-molecule real-time sequencing : Nature Biotechnology : Nature Publishing Group
  33. ^ http://www.nature.com/nbt/journal/v31/n11/full/nbt.2705.html
  34. ^ http://www.pnas.org/content/early/2013/11/25/1320101110.full.pdf
  35. ^ Pacific Biosciences to Partner With Roche on In Vitro Diagnostics Products

External links[edit]