The confocal detection principle is extended to a highly parallel optical system that continuously analyzes thousands of concurrent sample locations. This is achieved through the use of a holographic laser illumination multiplexer combined with a confocal pinhole array before a prism dispersive element used to provide spectroscopic information from each confocal volume. The system is demonstrated to detect and identify single fluorescent molecules from each of several thousand independent confocal volumes in real time.
Pacific Biosciences has developed a method for real-time sequencing of single DNA molecules (Eid et al., 2009), with intrinsic sequencing rates of several bases per second and read lengths into the kilobase range. Conceptually, this sequencing approach is based on eavesdropping on the activity of DNA polymerase carrying out template-directed DNA polymerization. Performed in a highly parallel operational mode, sequential base additions catalyzed by each polymerase are detected with terminal phosphate-linked, fluorescence-labeled nucleotides. This chapter will first outline the principle of this single-molecule, real-time (SMRT) DNA sequencing method, followed by descriptions of its underlying components and typical sequencing run conditions. Two examples are provided which illustrate that, in addition to the DNA sequence, the dynamics of DNA polymerization from each enzyme molecules is directly accessible: the determination of base-specific kinetic parameters from single-molecule sequencing reads, and the characterization of DNA synthesis rate heterogeneities. Copyright 2010 Elsevier Inc. All rights reserved.
Since the introduction of next generation sequencing, plant genome assembly projects do not need to rely on dedicated research facilities or community-wide consortia anymore, even individual research groups can sequence and assemble the genomes they are interested in. However, such assemblies are typically not based on the entire breadth of genomic technologies including genetic and physical maps and their contiguities tend to be low compared to the full-length gold standard reference sequences. Recently emerging third generation genomic technologies like long-read sequencing or optical mapping promise to bridge this quality gap and enable simple and cost-effective solutions for chromosomal-level assemblies.
Selective aluminum passivation for targeted immobilization of single DNA polymerase molecules in zero-mode waveguide nanostructures.
Optical nanostructures have enabled the creation of subdiffraction detection volumes for single-molecule fluorescence microscopy. Their applicability is extended by the ability to place molecules in the confined observation volume without interfering with their biological function. Here, we demonstrate that processive DNA synthesis thousands of bases in length was carried out by individual DNA polymerase molecules immobilized in the observation volumes of zero-mode waveguides (ZMWs) in high-density arrays. Selective immobilization of polymerase to the fused silica floor of the ZMW was achieved by passivation of the metal cladding surface using polyphosphonate chemistry, producing enzyme density contrasts of glass over aluminum in excess of 400:1. Yields of single-molecule occupancies of approximately 30% were obtained for a range of ZMW diameters (70-100 nm). Results presented here support the application of immobilized single DNA polymerases in ZMW arrays for long-read-length DNA sequencing.
Long, processive enzymatic DNA synthesis using 100% dye-labeled terminal phosphate-linked nucleotides.
We demonstrate the efficient synthesis of DNA with complete replacement of the four deoxyribonucleoside triphosphate (dNTP) substrates with nucleotides carrying fluorescent labels. A different, spectrally separable fluorescent dye suitable for single molecule fluorescence detection was conjugated to each of the four dNTPs via linkage to the terminal phosphate. Using these modified nucleotides, DNA synthesis by phi 29 DNA polymerase was observed to be processive for products thousands of bases in length, with labeled nucleotide affinities and DNA polymerization rates approaching unmodified dNTP levels. Results presented here show the compatibility of these nucleotides for single-molecule, real-time DNA sequencing applications.
Aquaculture is the fastest-growing food production sector in agriculture, with great potential to meet projected protein needs of human beings. Aquaculture is facing several challenges, including lack of a sufficient number of genetically improved species, lack of species-specific feeds, high mortality due to diseases and pollution of ecosystems. The rapid development of sequencing technologies has revolutionized biological sciences, and supplied necessary tools to tackle these challenges in aquaculture and thus ensure its sustainability and profitability. So far, draft genomes have been published in over 24 aquaculture species, and used to address important issues related to aquaculture. We briefly review the advances of next generation sequencing technologies, and summarize the status of whole genome sequencing and its general applications (i.e. establishing reference genomes and discovering DNA markers) and specific applications in tackling some important issues (e.g. breeding, diseases, sex determination & maturation) related to aquaculture. For sequencing a new genome, we recommend the use of 100–200 × short reads using Illumina and 50–60 × long reads with PacBio sequencing technologies. For identification of a large number of SNPs, resequencing pooled DNA samples from different populations is the most cost-effective way. We also discuss the challenges and future directions of whole genome sequencing in aquaculture.
Microsatellite length scoring by Single Molecule Real Time Sequencing – Effects of sequence structure and PCR regime.
Microsatellites are DNA sequences consisting of repeated, short (1-6 bp) sequence motifs that are highly mutable by enzymatic slippage during replication. Due to their high intrinsic variability, microsatellites have important applications in population genetics, forensics, genome mapping, as well as cancer diagnostics and prognosis. The current analytical standard for microsatellites is based on length scoring by high precision electrophoresis, but due to increasing efficiency next-generation sequencing techniques may provide a viable alternative. Here, we evaluated single molecule real time (SMRT) sequencing, implemented in the PacBio series of sequencing apparatuses, as a means of microsatellite length scoring. To this end we carried out multiplexed SMRT sequencing of plasmid-carried artificial microsatellites of varying structure under different pre-sequencing PCR regimes. For each repeat structure, reads corresponding to the target length dominated. We found that pre-sequencing amplification had large effects on scoring accuracy and error distribution relative to controls, but that the effects of the number of amplification cycles were generally weak. In line with expectations enzymatic slippage decreased proportionally with microsatellite repeat unit length and increased with repetition number. Finally, we determined directional mutation trends, showing that PCR and SMRT sequencing introduced consistent but opposing error patterns in contraction and expansion of the microsatellites on the repeat motif and single nucleotide level.
Recent technological developments have revolutionized the way we perform genetic analyses. In particular whole-genome sequencing provides access to the entire genetic makeup of an individual, and it is now an affordable approach for many research groups. As a consequence genome sequencing is pervading many fields of biological research. Sequencing technologies are evolving rapidly and so do their applications. Here we provide a first primer on whole-genome sequencing, focusing on two of the most popular applications: (1) de novo genome sequencing, in which the objective is obtaining a high-quality genome assembly that can serve as a reference for a species or variety, and (2) genome resequencing, when there is an available reference genome and the objective is to map sequence variation of an individual or a set of individuals. It is not our intention to provide a comprehensive overview of current methodologies that will likely soon become obsolete, but rather focus on general principles that will have a more general applicability.