Single-molecule real-time sequencing

Last updated

Single-molecule real-time (SMRT) sequencing is a parallelized single molecule DNA sequencing method. Single-molecule real-time sequencing utilizes a zero-mode waveguide (ZMW). [1] 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 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. [2]

Contents

Technology

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.

Template preparation

To prepare the library, DNA fragments are put into a circular form using hairpin adapter ligations. [3]

Phospholinked nucleotide

For each of the nucleotide bases, there is a corresponding fluorescent dye molecule that enables 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. [4]

Zero-Mode Waveguide

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. [5]

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. [6] [7]

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. [4] [8]

Sequencing Performance

Sequencing performance can be measured in read length, accuracy, and total throughput per experiment. PacBio sequencing systems using ZMWs have the advantage of long read lengths, although error rates are on the order of 5-15% and sample throughput is lower than Illumina sequencing platforms. [9]

On 19 Sep 2018, Pacific Biosciences [PacBio] released the Sequel 6.0 chemistry, synchronizing the chemistry version with the software version. Performance is contrasted for large-insert libraries with high molecular weight DNA versus shorter-insert libraries below ~15,000 bases in length. For larger templates average read lengths are up to 30,000 bases. For shorter-insert libraries, average read length are up to 100,000 bases while reading the same molecule in a circle several times. The latter shorter-insert libraries then yield up to 50 billion bases from a single SMRT Cell. [10]

History

Pacific Biosciences (PacBio) commercialized SMRT sequencing in 2011, [11] after releasing a beta version of its RS instrument in late 2010. [12]

RS and RS II

SMRT Cell for a RS or RS II Sequencer RSSmrtCell.jpg
SMRT Cell for a RS or RS II Sequencer

At commercialization, read length had a normal distribution with a mean of about 1100 bases. 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 2500 to 2900 bases. [13]

The XL chemistry kit released in late 2012 increased average read length to more than 4300 bases. [14] [15]

On August 21, 2013, PacBio released a new DNA polymerase Binding Kit P4. This P4 enzyme has average read lengths of more than 4,300 bases when paired with the C2 sequencing chemistry and more than 5,000 bases when paired with the XL chemistry. [16] The enzyme’s accuracy is similar to C2, reaching QV50 between 30X and 40X coverage. The resulting P4 attributes provided higher-quality assemblies using fewer SMRT Cells and with improved variant calling. [16] When coupled with input DNA size selection (using an electrophoresis instrument such as BluePippin) yields average read length over 7 kilobases. [17]

On October 3, 2013, PacBio released new reagent combination for PacBio RS II, the P5 DNA polymerase with C3 chemistry (P5-C3). Together, they extend sequencing read lengths to an average of approximately 8,500 bases, with the longest reads exceeding 30,000 bases. [18] Throughput per SMRT cell is around 500 million bases demonstrated by sequencing results from the CHM1 cell line. [19]

On October 15, 2014, PacBio announced the release of new chemistry P6-C4 for the RS II system, which represents the company's 6th generation of polymerase and 4th generation chemistry--further extending the average read length to 10,000 - 15,000 bases, with the longest reads exceeding 40,000 bases. The throughput with the new chemistry was estimated between 500 million to 1 billion bases per SMRT Cell, depending on the sample being sequenced. [20] [21] This was the final version of chemistry released for the RS instrument.

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. [22] 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. [23] [24] The highest throughput mode in November 2013 used P5 binding, C3 chemistry, BluePippin size selection, and a PacBio RS II officially yielded 350 million bases per SMRT Cell though a human de novo data set released with the chemistry averaging 500 million bases per SMRT Cell. Throughput varies based on the type of sample being sequenced. [25] With the introduction of P6-C4 chemistry typical throughput per SMRT Cell increased to 500 million bases to 1 billion bases.

RS Performance
C1C2P4-XLP5-C3P6-C4
Average read length bases11002500 - 29004300 - 5000850010,000 - 15,000
Throughput per SMRT Cell30M - 40M60M - 100M250M - 300M350M - 500M500M - 1B

Sequel

SMRT Cell for a Sequel Sequencer SequelSmrtCell.jpg
SMRT Cell for a Sequel Sequencer

In September 2015, the company announced the launch of a new sequencing instrument, the Sequel System, that increased capacity to 1 million ZMW holes. [26] [27]

With the Sequel instrument initial read lengths were comparable to the RS, then later chemistry releases increased read length.

On January 23, 2017, the V2 chemistry was released. It increased average read lengths to between 10,000 and 18,000 bases. [28]

On March 8, 2018, the 2.1 chemistry was released. It increased average read length to 20,000 bases and half of all reads above 30,000 bases in length. Yield per SMRT Cell increased to 10 or 20 billion bases, for either large-insert libraries or shorter-insert (e.g. amplicon) libraries respectively. [29]

Pipette tip in an 8M SMRT Cell TipIn8MCell.jpg
Pipette tip in an 8M SMRT Cell

On 19 September 2018, the company announced the Sequel 6.0 chemistry with average read lengths increased to 100,000 bases for shorter-insert libraries and 30,000 for longer-insert libraries. SMRT Cell yield increased up to 50 billion bases for shorter-insert libraries. [10]

Sequel Performance
V22.16.0
Average read length bases10,000 - 18,00020,000 - 30,00030,000 - 100,000
Throughput per SMRT Cell5B - 8B10B - 20B20B - 50B

8M Chip

In April 2019 the company released a new SMRT Cell with eight million ZMWs, [30] increasing the expected throughput per SMRT Cell by a factor of eight. [31] Early access customers in March 2019 reported throughput over 58 customer run cells of 250 GB of raw yield per cell with templates about 15 kb in length, and 67.4 GB yield per cell with templates in higher weight molecules. [32] System performance is now reported in either high-molecular-weight continuous long reads or in pre-corrected HiFi (also known as Circular Consensus Sequence (CCS)) reads. For high-molecular-weight reads roughly half of all reads are longer than 50 kb in length.

Sequel II High-Molecular-Weight Performance
Early Access1.02.0
Throughput per SMRT Cell~67.4 GBUp to 160 GBUp to 200 GB

The HiFi performance includes corrected bases with quality above Phred score Q20, using repeated amplicon passes for correction. These take amplicons up to 20kb in length.

Sequel II HiFi Corrected Read Performance
Early Access1.02.0
Raw reads per SMRT Cell~250 GBUp to 360 GBUp to 500 GB
Corrected reads per SMRT Cell (>Q20)~25 GBUp to 36 GBUp to 50 GB

Application

Single-molecule real-time sequencing may be applicable for a broad range of genomics research.

For 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. [2] [33] [34] 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. [35] [36] In 2012, several peer-reviewed publications were released demonstrating the automated finishing of bacterial genomes, [37] [38] including one paper that updated the Celera Assembler with a pipeline for genome finishing using long SMRT sequencing reads. [39] In 2013, scientists estimated that long-read sequencing could be used to fully assemble and finish the majority of bacterial and archaeal genomes. [40]

The 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. [41] In August 2012, scientists from the Broad Institute published an evaluation of SMRT sequencing for SNP calling. [42]

The dynamics of polymerase can indicate whether a base is methylated. [43] Scientists demonstrated the use of single-molecule real-time sequencing for detecting methylation and other base modifications. [44] [45] [46] In 2012 a team of scientists used SMRT sequencing to generate the full methylomes of six bacteria. [47] In November 2012, scientists published a report on genome-wide methylation of an outbreak strain of E. coli. [48]

Long reads make it possible to sequence full gene isoforms, including the 5' and 3' ends. This type of sequencing is useful to capture isoforms and splice variants. [49] [50]

SMRT sequencing has several applications in reproductive medical genetics research when investigating families with suspected parental gonadal mosaicism. Long reads enable haplotype phasing in patients to investigate parent-of-origin of mutations. Deep sequencing enables determination of allele frequencies in sperm cells, of relevance for estimation of recurrence risk for future affected offspring. [51] [52]

Related Research Articles

<span class="mw-page-title-main">Genomics</span> Discipline in genetics

Genomics is an interdisciplinary field of biology focusing on the structure, function, evolution, mapping, and editing of genomes. A genome is an organism's complete set of DNA, including all of its genes as well as its hierarchical, three-dimensional structural configuration. In contrast to genetics, which refers to the study of individual genes and their roles in inheritance, genomics aims at the collective characterization and quantification of all of an organism's genes, their interrelations and influence on the organism. Genes may direct the production of proteins with the assistance of enzymes and messenger molecules. In turn, proteins make up body structures such as organs and tissues as well as control chemical reactions and carry signals between cells. Genomics also involves the sequencing and analysis of genomes through uses of high throughput DNA sequencing and bioinformatics to assemble and analyze the function and structure of entire genomes. Advances in genomics have triggered a revolution in discovery-based research and systems biology to facilitate understanding of even the most complex biological systems such as the brain.

<span class="mw-page-title-main">DNA sequencer</span> A scientific instrument used to automate the DNA sequencing process

A DNA sequencer is a scientific instrument used to automate the DNA sequencing process. Given a sample of DNA, a DNA sequencer is used to determine the order of the four bases: G (guanine), C (cytosine), A (adenine) and T (thymine). This is then reported as a text string, called a read. Some DNA sequencers can be also considered optical instruments as they analyze light signals originating from fluorochromes attached to nucleotides.

<span class="mw-page-title-main">DNA sequencing</span> Process of determining the nucleic acid sequence

DNA sequencing is the process of determining the nucleic acid sequence – the order of nucleotides in DNA. It includes any method or technology that is used to determine the order of the four bases: adenine, guanine, cytosine, and thymine. The advent of rapid DNA sequencing methods has greatly accelerated biological and medical research and discovery.

<span class="mw-page-title-main">Sanger sequencing</span> Method of DNA sequencing developed in 1977

Sanger sequencing is a method of DNA sequencing that involves electrophoresis and is based on the random incorporation of chain-terminating dideoxynucleotides by DNA polymerase during in vitro DNA replication. After first being developed by Frederick Sanger and colleagues in 1977, it became the most widely used sequencing method for approximately 40 years. It was first commercialized by Applied Biosystems in 1986. More recently, higher volume Sanger sequencing has been replaced by next generation sequencing methods, especially for large-scale, automated genome analyses. However, the Sanger method remains in wide use for smaller-scale projects and for validation of deep sequencing results. It still has the advantage over short-read sequencing technologies in that it can produce DNA sequence reads of > 500 nucleotides and maintains a very low error rate with accuracies around 99.99%. Sanger sequencing is still actively being used in efforts for public health initiatives such as sequencing the spike protein from SARS-CoV-2 as well as for the surveillance of norovirus outbreaks through the Center for Disease Control and Prevention's (CDC) CaliciNet surveillance network.

Epigenomics is the study of the complete set of epigenetic modifications on the genetic material of a cell, known as the epigenome. The field is analogous to genomics and proteomics, which are the study of the genome and proteome of a cell. Epigenetic modifications are reversible modifications on a cell's DNA or histones that affect gene expression without altering the DNA sequence. Epigenomic maintenance is a continuous process and plays an important role in stability of eukaryotic genomes by taking part in crucial biological mechanisms like DNA repair. Plant flavones are said to be inhibiting epigenomic marks that cause cancers. Two of the most characterized epigenetic modifications are DNA methylation and histone modification. Epigenetic modifications play an important role in gene expression and regulation, and are involved in numerous cellular processes such as in differentiation/development and tumorigenesis. The study of epigenetics on a global level has been made possible only recently through the adaptation of genomic high-throughput assays.

<span class="mw-page-title-main">RNA-Seq</span> Lab technique in cellular biology

RNA-Seq is a sequencing technique that uses next-generation sequencing (NGS) to reveal the presence and quantity of RNA in a biological sample, representing an aggregated snapshot of the cells' dynamic pool of RNAs, also known as transcriptome.

Optical mapping is a technique for constructing ordered, genome-wide, high-resolution restriction maps from single, stained molecules of DNA, called "optical maps". By mapping the location of restriction enzyme sites along the unknown DNA of an organism, the spectrum of resulting DNA fragments collectively serves as a unique "fingerprint" or "barcode" for that sequence. Originally developed by Dr. David C. Schwartz and his lab at NYU in the 1990s this method has since been integral to the assembly process of many large-scale sequencing projects for both microbial and eukaryotic genomes. Later technologies use DNA melting, DNA competitive binding or enzymatic labelling in order to create the optical mappings.

<span class="mw-page-title-main">Pacific Biosciences</span> American biotechnology company

<span class="mw-page-title-main">Transmission electron microscopy DNA sequencing</span> Single-molecule sequencing technology

Transmission electron microscopy DNA sequencing is a single-molecule sequencing technology that uses transmission electron microscopy techniques. The method was conceived and developed in the 1960s and 70s, but lost favor when the extent of damage to the sample was recognized.

<span class="mw-page-title-main">Ion semiconductor sequencing</span>

Ion semiconductor sequencing is a method of DNA sequencing based on the detection of hydrogen ions that are released during the polymerization of DNA. This is a method of "sequencing by synthesis", during which a complementary strand is built based on the sequence of a template strand.

<span class="mw-page-title-main">DNA nanoball sequencing</span>

DNA nanoball sequencing is a high throughput sequencing technology that is used to determine the entire genomic sequence of an organism. The method uses rolling circle replication to amplify small fragments of genomic DNA into DNA nanoballs. Fluorescent nucleotides bind to complementary nucleotides and are then polymerized to anchor sequences bound to known sequences on the DNA template. The base order is determined via the fluorescence of the bound nucleotides This DNA sequencing method allows large numbers of DNA nanoballs to be sequenced per run at lower reagent costs compared to other next generation sequencing platforms. However, a limitation of this method is that it generates only short sequences of DNA, which presents challenges to mapping its reads to a reference genome. After purchasing Complete Genomics, the Beijing Genomics Institute (BGI) refined DNA nanoball sequencing to sequence nucleotide samples on their own platform.

Massive parallel sequencing or massively parallel sequencing is any of several high-throughput approaches to DNA sequencing using the concept of massively parallel processing; it is also called next-generation sequencing (NGS) or second-generation sequencing. Some of these technologies emerged between 1993 and 1998 and have been commercially available since 2005. These technologies use miniaturized and parallelized platforms for sequencing of 1 million to 43 billion short reads per instrument run.

<span class="mw-page-title-main">Illumina dye sequencing</span> DNA sequencing method

Illumina dye sequencing is a technique used to determine the series of base pairs in DNA, also known as DNA sequencing. The reversible terminated chemistry concept was invented by Bruno Canard and Simon Sarfati at the Pasteur Institute in Paris. It was developed by Shankar Balasubramanian and David Klenerman of Cambridge University, who subsequently founded Solexa, a company later acquired by Illumina. This sequencing method is based on reversible dye-terminators that enable the identification of single nucleotides as they are washed over DNA strands. It can also be used for whole-genome and region sequencing, transcriptome analysis, metagenomics, small RNA discovery, methylation profiling, and genome-wide protein-nucleic acid interaction analysis.

In DNA sequencing, a read is an inferred sequence of base pairs corresponding to all or part of a single DNA fragment. A typical sequencing experiment involves fragmentation of the genome into millions of molecules, which are size-selected and ligated to adapters. The set of fragments is referred to as a sequencing library, which is sequenced to produce a set of reads.

Magnetic sequencing is a single-molecule sequencing method in development. A DNA hairpin, containing the sequence of interest, is bound between a magnetic bead and a glass surface. A magnetic field is applied to stretch the hairpin open into single strands, and the hairpin refolds after decreasing of the magnetic field. The hairpin length can be determined by direct imaging of the diffraction rings of the magnetic beads using a simple microscope. The DNA sequences are determined by measuring the changes in the hairpin length following successful hybridization of complementary nucleotides.

<span class="mw-page-title-main">Scaffolding (bioinformatics)</span>

Scaffolding is a technique used in bioinformatics. It is defined as follows:

Link together a non-contiguous series of genomic sequences into a scaffold, consisting of sequences separated by gaps of known length. The sequences that are linked are typically contiguous sequences corresponding to read overlaps.

Single-cell sequencing examines the nucleic acid sequence information from individual cells with optimized next-generation sequencing technologies, providing a higher resolution of cellular differences and a better understanding of the function of an individual cell in the context of its microenvironment. For example, in cancer, sequencing the DNA of individual cells can give information about mutations carried by small populations of cells. In development, sequencing the RNAs expressed by individual cells can give insight into the existence and behavior of different cell types. In microbial systems, a population of the same species can appear genetically clonal. Still, single-cell sequencing of RNA or epigenetic modifications can reveal cell-to-cell variability that may help populations rapidly adapt to survive in changing environments.

<span class="mw-page-title-main">Epitranscriptomic sequencing</span>

In epitranscriptomic sequencing, most methods focus on either (1) enrichment and purification of the modified RNA molecules before running on the RNA sequencer, or (2) improving or modifying bioinformatics analysis pipelines to call the modification peaks. Most methods have been adapted and optimized for mRNA molecules, except for modified bisulfite sequencing for profiling 5-methylcytidine which was optimized for tRNAs and rRNAs.

Third-generation sequencing is a class of DNA sequencing methods currently under active development.

A plant genome assembly represents the complete genomic sequence of a plant species, which is assembled into chromosomes and other organelles by using DNA fragments that are obtained from different types of sequencing technology.

References

  1. ↑ Levene MJ, Korlach J, Turner SW, et al. (2003). "Zero-Mode Waveguides for Single-Molecule Analysis at High Concentrations". Science . 299 (5607): 682–6. Bibcode:2003Sci...299..682L. doi:10.1126/science.1079700. PMID   12560545. S2CID   6060239.
  2. 1 2 Eid J, Fehr A, Gray J, et al. (2009). "Real-Time DNA Sequencing from Single Polymerase Molecules". Science . 323 (5910): 133–8. Bibcode:2009Sci...323..133E. doi:10.1126/science.1162986. PMID   19023044. S2CID   54488479.
  3. ↑ Friedmann, Theodore (2012). Advances in genetics (in Dutch). Oxford: Academic. ISBN   978-0-12-394395-8. OCLC   813987819.
  4. 1 2 "Pacific Biosciences Develops Transformative DNA Sequencing Technology" (PDF). Pacific Biosciences Technology Backgrounder. 2008.
  5. ↑ Korlach J, Marks PJ, Cicero RL, et al. (2008). "Selective aluminum passivation for targeted immobilization of single DNA polymerase molecules in zero-mode waveguide nanostructures". PNAS . 105 (4): 1176–81. Bibcode:2008PNAS..105.1176K. doi: 10.1073/pnas.0710982105 . PMC   2234111 . PMID   18216253.
  6. ↑ Foquet M, Samiee KT, Kong X, et al. (2008). "Improved fabrication of zero-mode waveguides for single-molecule detection". J. Appl. Phys. 103 (3): 034301–034301–9. Bibcode:2008JAP...103c4301F. doi:10.1063/1.2831366. S2CID   38892226.
  7. ↑ Zhu, Paul; Craighead, Harold G. (2012-06-09). "Zero-Mode Waveguides for Single-Molecule Analysis". Annual Review of Biophysics. Annual Reviews. 41 (1): 269–293. doi:10.1146/annurev-biophys-050511-102338. ISSN   1936-122X. PMID   22577821.
  8. ↑ Baibakov, Mikhail; Barulin, Aleksandr; Roy, Prithu; Claude, Jean-Benoît; Patra, Satyajit; Wenger, Jérôme (1999-02-22). "Zero-mode waveguides can be made better: fluorescence enhancement with rectangular aluminum nanoapertures from the visible to the deep ultraviolet". Nanoscale Advances. 2 (9): 4153–4160. doi:10.1039/D0NA00366B. PMC   9417158 . PMID   36132755.
  9. ↑ Pollock, Jolinda; Glendinning, Laura; Wisedchanwet, Trong; Watson, Mick (2018). "The Madness of Microbiome: Attempting To Find Consensus "Best Practice" for 16S Microbiome Studies". Applied and Environmental Microbiology. 84 (7): e02627-17. Bibcode:2018ApEnM..84E2627P. doi: 10.1128/AEM.02627-17 . PMC   5861821 . PMID   29427429.
  10. 1 2 "PacBio Post". Twitter. 19 Sep 2018.
  11. ↑ Karow J (3 May 2011). "PacBio Ships First Two Commercial Systems; Order Backlog Grows to 44" . GenomeWeb.
  12. ↑ Karow J (7 Dec 2010). "PacBio Reveals Beta System Specs for RS; Says Commercial Release is on Track for First Half of 2011" . GenomeWeb.
  13. ↑ Karow J (10 Jan 2012). "After a Year of Testing, Two Early PacBio Customers Expect More Routine Use of RS Sequencer in 2012" . GenomeWeb.
  14. ↑ Heger M (13 Nov 2012). "PacBio's XL Chemistry Increases Read Lengths and Throughput; CSHL Tests the Tech on Rice Genome" . GenomeWeb.
  15. ↑ Heger M (5 Mar 2013). "PacBio Users Report Progress in Long Reads for Plant Genome Assembly, Tricky Regions of Human Genome" . GenomeWeb.
  16. 1 2 "New DNA Polymerase P4 Delivers Higher-Quality Assemblies Using Fewer SMRT Cells". PacBio Blog. 21 Aug 2013.
  17. ↑ lexnederbragt (19 Jun 2013). "Longing for the longest reads: PacBio and BluePippin". In between lines of code.
  18. ↑ "New Chemistry for PacBio RS II Provides Average 8.5 kb Read Lengths for Complex Genome Studies". PacBio Blog. 3 Oct 2013.
  19. ↑ Chaisson MJ, Huddleston J, Dennis MY, et al. (2014). "Resolving the complexity of the human genome using single-molecule sequencing". Nature . 517 (7536): 608–11. Bibcode:2015Natur.517..608C. doi:10.1038/nature13907. PMC   4317254 . PMID   25383537.
  20. ↑ "Pacific Biosciences Releases New DNA Sequencing Chemistry to Enhance Read Length and Accuracy for the Study of Human and Other Complex Genomes". Pacific Biosciences (Press Release). 15 Oct 2014.
  21. ↑ "New Chemistry Boosts Average Read Length to 10 kb – 15 kb for PacBio RS II". PacBio Blog. 15 Oct 2014.
  22. ↑ "SMRT Cells, sequencing reagent kits, and accessories for the PacBio RS II". Pacific Biosciences. 2020. Archived from the original on 2013-04-21. Retrieved 2012-04-28.
  23. ↑ "PacBio Launches PacBio RS II Sequencer". Next Gen Seek. 11 Apr 2013. Archived from the original on 19 December 2019. Retrieved 18 April 2013.
  24. ↑ "New Products: PacBio's RS II; Cufflinks" . GenomeWeb. 16 Apr 2013.
  25. ↑ "Duke Sequencing Post". Twitter. 30 Aug 2013.
  26. ↑ "PacBio Announces Sequel Sequencing System". Bio-IT World. 30 Sep 2015. Archived from the original on 29 July 2020. Retrieved 16 November 2015.
  27. ↑ Heger M (1 Oct 2015). "PacBio Launches Higher-Throughput, Lower-Cost Single-Molecule Sequencing System" . GenomeWeb.
  28. ↑ "New Chemistry and Software for Sequel System Improve Read Length, Lower Project Costs". PacBio Blog. 9 Jan 2017.
  29. ↑ "New Software, Polymerase for Sequel System Boost Throughput and Affordability". PacBio Blog. 7 Mar 2018.
  30. ↑ "PacBio Launches Sequel II System". Bio-IT World. 26 Apr 2019.
  31. ↑ "Archived copy". Archived from the original on 2018-09-24. Retrieved 2018-09-24.{{cite web}}: CS1 maint: archived copy as title (link)
  32. ↑ Heger M (7 Mar 2019). "PacBio Shares Early-Access Customer Experiences, New Applications for Sequel II" . GenomeWeb.
  33. ↑ Rasko DA, Webster DR, Sahl JW, et al. (2011). "Origins of the E. coli Strain Causing an Outbreak of Hemolytic–Uremic Syndrome in Germany". N. Engl. J. Med. 365 (8): 709–17. doi:10.1056/NEJMoa1106920. PMC   3168948 . PMID   21793740.
  34. ↑ Chin CS, Sorenson J, Harris JB, et al. (2011). "The Origin of the Haitian Cholera Outbreak Strain". N. Engl. J. Med. 364 (1): 33–42. doi:10.1056/NEJMoa1012928. PMC   3030187 . PMID   21142692.
  35. ↑ Gao H, Green SJ, Jafari N, et al. (2012). "Tech Tips: Next-Generation Sequencing". Genetic Engineering & Biotechnology News. 32 (8).
  36. ↑ Schatz M (7 Sep 2011). "SMRT-assembly approaches" (PDF). schatzlab.cshl.edu (PacBio Users Meeting).
  37. ↑ Ribeiro FJ, Przybylski D, Yin S, et al. (2012). "Finished bacterial genomes from shotgun sequence data". Genome Res. 22 (11): 2270–7. doi:10.1101/gr.141515.112. PMC   3483556 . PMID   22829535.
  38. ↑ Bashir A, Klammer A, Robins WP, et al. (2012). "A hybrid approach for the automated finishing of bacterial genomes". Nat. Biotechnol. 30 (7): 701–7. doi:10.1038/nbt.2288. PMC   3731737 . PMID   22750883.
  39. ↑ Koren S, Schatz MC, Walenz BP, et al. (2012). "Hybrid error correction and de novo assembly of single-molecule sequencing reads". Nat. Biotechnol. 30 (7): 693–700. doi:10.1038/nbt.2280. PMC   3707490 . PMID   22750884.
  40. ↑ Koren S, Harhay GP, Smith TP, et al. (2013). "Reducing assembly complexity of microbial genomes with single-molecule sequencing". Genome Biol. 14 (9): R101. arXiv: 1304.3752 . Bibcode:2013arXiv1304.3752K. doi: 10.1186/gb-2013-14-9-r101 . PMC   4053942 . PMID   24034426.
  41. ↑ Smith CC, Wang Q, Chin CS, et al. (2012). "Validation of ITD mutations in FLT3 as a therapeutic target in human acute myeloid leukaemia". Nature . 485 (7397): 260–3. Bibcode:2012Natur.485..260S. doi:10.1038/nature11016. PMC   3390926 . PMID   22504184.
  42. ↑ Carneiro MO, Russ C, Ross MG, et al. (2012). "Pacific Biosciences Sequencing Technology for Genotyping and Variation Discovery in Human Data". BMC Genom. 13 (1): 375. doi: 10.1186/1471-2164-13-375 . PMC   3443046 . PMID   22863213.
  43. ↑ Flusberg BA, Webster DR, Lee JH, et al. (2010). "Direct detection of DNA methylation during single-molecule, real-time sequencing". Nat. Methods . 7 (6): 461–5. doi:10.1038/nmeth.1459. PMC   2879396 . PMID   20453866.
  44. ↑ Clark TA, Murray IA, Morgan RD, et al. (2012). "Characterization of DNA Methyltransferase Specificities Using Single-Molecule, Real-Time DNA Sequencing". Nucleic Acids Res. 40 (4): e29. doi:10.1093/nar/gkr1146. PMC   3287169 . PMID   22156058.
  45. ↑ Song CX, Clark TA, Lu XY, et al. (2011). "Sensitive and Specific Single-Molecule Sequencing of 5-hydroxymethylcytosine". Nat Methods . 9 (1): 75–7. doi:10.1038/nmeth.1779. PMC   3646335 . PMID   22101853.
  46. ↑ Clark TA, Spittle KE, Turner SW, et al. (2011). "Direct Detection and Sequencing of Damaged DNA Bases". Genome Integr. 2 (1): 10. doi: 10.1186/2041-9414-2-10 . PMC   3264494 . PMID   22185597.
  47. ↑ Murray IA, Clark TA, Morgan RD, et al. (2012). "The Methylomes of Six Bacteria". Nucleic Acids Res. 40 (22): 11450–62. doi:10.1093/nar/gks891. PMC   3526280 . PMID   23034806.
  48. ↑ Fang G, Munera D, Friedman DI, et al. (2012). "Genome-wide Mapping of Methylated Adenine Residues in Pathogenic Escherichia Coli Using Single-Molecule Real-Time Sequencing". Nat. Biotechnol. 30 (12): 1232–9. doi:10.1038/nbt.2432. PMC   3879109 . PMID   23138224.
  49. ↑ Sharon D, Tilgner H, Grubert F, et al. (2013). "A Single-Molecule Long-Read Survey of the Human Transcriptome". Nat. Biotechnol. 31 (11): 1009–14. doi:10.1038/nbt.2705. PMC   4075632 . PMID   24108091.
  50. ↑ Au KF, Sebastiano V, Afshar PT, et al. (2013). "Characterization of the human ESC transcriptome by hybrid sequencing". PNAS . 110 (50): E4821–30. Bibcode:2013PNAS..110E4821A. doi: 10.1073/pnas.1320101110 . PMC   3864310 . PMID   24282307.
  51. ↑ Ardui S, Ameur A, Vermeesch JR, et al. (2018). "Single Molecule Real-Time (SMRT) Sequencing Comes of Age: Applications and Utilities for Medical Diagnostics". Nucleic Acids Res. 46 (5): 2159–68. doi:10.1093/nar/gky066. PMC   5861413 . PMID   29401301.
  52. ↑ Wilbe M, Gudmundsson S, Johansson J, et al. (2017). "A Novel Approach Using Long-Read Sequencing and ddPCR to Investigate Gonadal Mosaicism and Estimate Recurrence Risk in Two Families With Developmental Disorders". Prenatal Diagnosis. 37 (11): 1146–54. doi:10.1002/pd.5156. PMC   5725701 . PMID   28921562.
This page is based on this Wikipedia article
Text is available under the CC BY-SA 4.0 license; additional terms may apply.
Images, videos and audio are available under their respective licenses.