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Auto Tag: Kinetics

September 21, 2019

The kinetoplastid-infecting Bodo saltans virus (BsV), a window into the most abundant giant viruses in the sea.

Giant viruses are ecologically important players in aquatic ecosystems that have challenged concepts of what constitutes a virus. Herein, we present the giant Bodo saltans virus (BsV), the first characterized representative of the most abundant group of giant viruses in ocean metagenomes, and the first isolate of a klosneuvirus, a subgroup of the Mimiviridae proposed from metagenomic data. BsV infects an ecologically important microzooplankton, the kinetoplastid Bodo saltans. Its 1.39 Mb genome encodes 1227 predicted ORFs, including a complex replication machinery. Yet, much of its translational apparatus has been lost, including all tRNAs. Essential genes are invaded by homing endonuclease-encoding self-splicing introns that may defend against competing viruses. Putative anti-host factors show extensive gene duplication via a genomic accordion indicating an ongoing evolutionary arms race and highlighting the rapid evolution and genomic plasticity that has led to genome gigantism and the enigma that is giant viruses.© 2018, Deeg et al.

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September 21, 2019

Real-time DNA sequencing from single polymerase molecules.

We present single-molecule, real-time sequencing data obtained from a DNA polymerase performing uninterrupted template-directed synthesis using four distinguishable fluorescently labeled deoxyribonucleoside triphosphates (dNTPs). We detected the temporal order of their enzymatic incorporation into a growing DNA strand with zero-mode waveguide nanostructure arrays, which provide optical observation volume confinement and enable parallel, simultaneous detection of thousands of single-molecule sequencing reactions. Conjugation of fluorophores to the terminal phosphate moiety of the dNTPs allows continuous observation of DNA synthesis over thousands of bases without steric hindrance. The data report directly on polymerase dynamics, revealing distinct polymerization states and pause sites corresponding to DNA secondary structure. Sequence data were aligned with the known reference sequence to assay biophysical parameters of polymerization for each template position. Consensus sequences were generated from the single-molecule reads at 15-fold coverage, showing a median accuracy of 99.3%, with no systematic error beyond fluorophore-dependent error rates.

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September 21, 2019

Direct detection of DNA methylation during single-molecule, real-time sequencing.

We describe the direct detection of DNA methylation, without bisulfite conversion, through single-molecule, real-time (SMRT) sequencing. In SMRT sequencing, DNA polymerases catalyze the incorporation of fluorescently labeled nucleotides into complementary nucleic acid strands. The arrival times and durations of the resulting fluorescence pulses yield information about polymerase kinetics and allow direct detection of modified nucleotides in the DNA template, including N6-methyladenine, 5-methylcytosine and 5-hydroxymethylcytosine. Measurement of polymerase kinetics is an intrinsic part of SMRT sequencing and does not adversely affect determination of primary DNA sequence. The various modifications affect polymerase kinetics differently, allowing discrimination between them. We used these kinetic signatures to identify adenine methylation in genomic samples and found that, in combination with circular consensus sequencing, they can enable single-molecule identification of epigenetic modifications with base-pair resolution. This method is amenable to long read lengths and will likely enable mapping of methylation patterns in even highly repetitive genomic regions.

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September 21, 2019

The advantages of SMRT sequencing.

Of the current next-generation sequencing technologies, SMRT sequencing is sometimes overlooked. However, attributes such as long reads, modified base detection and high accuracy make SMRT a useful technology and an ideal approach to the complete sequencing of small genomes.

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