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September 22, 2019  |  

Single molecule real-time (SMRT) sequencing comes of age: applications and utilities for medical diagnostics.

Short read massive parallel sequencing has emerged as a standard diagnostic tool in the medical setting. However, short read technologies have inherent limitations such as GC bias, difficulties mapping to repetitive elements, trouble discriminating paralogous sequences, and difficulties in phasing alleles. Long read single molecule sequencers resolve these obstacles. Moreover, they offer higher consensus accuracies and can detect epigenetic modifications from native DNA. The first commercially available long read single molecule platform was the RS system based on PacBio’s single molecule real-time (SMRT) sequencing technology, which has since evolved into their RSII and Sequel systems. Here we capsulize how SMRT sequencing is revolutionizing constitutional, reproductive, cancer, microbial and viral genetic testing.© The Author(s) 2018. Published by Oxford University Press on behalf of Nucleic Acids Research.


September 22, 2019  |  

Mapping and characterizing N6-methyladenine in eukaryotic genomes using single-molecule real-time sequencing.

N6-Methyladenine (m6dA) has been discovered as a novel form of DNA methylation prevalent in eukaryotes; however, methods for high-resolution mapping of m6dA events are still lacking. Single-molecule real-time (SMRT) sequencing has enabled the detection of m6dA events at single-nucleotide resolution in prokaryotic genomes, but its application to detecting m6dA in eukaryotic genomes has not been rigorously examined. Herein, we identified unique characteristics of eukaryotic m6dA methylomes that fundamentally differ from those of prokaryotes. Based on these differences, we describe the first approach for mapping m6dA events using SMRT sequencing specifically designed for the study of eukaryotic genomes and provide appropriate strategies for designing experiments and carrying out sequencing in future studies. We apply the novel approach to study two eukaryotic genomes. For green algae, we construct the first complete genome-wide map of m6dA at single-nucleotide and single-molecule resolution. For human lymphoblastoid cells (hLCLs), it was necessary to integrate SMRT sequencing data with independent sequencing data. The joint analyses suggest putative m6dA events are enriched in the promoters of young full-length LINE-1 elements (L1s), but call for validation by additional methods. These analyses demonstrate a general method for rigorous mapping and characterization of m6dA events in eukaryotic genomes.© 2018 Zhu et al.; Published by Cold Spring Harbor Laboratory Press.


September 22, 2019  |  

Real-time assembly of ribonucleoprotein complexes on nascent RNA transcripts.

Cellular protein-RNA complexes assemble on nascent transcripts, but methods to observe transcription and protein binding in real time and at physiological concentrations are not available. Here, we report a single-molecule approach based on zero-mode waveguides that simultaneously tracks transcription progress and the binding of ribosomal protein S15 to nascent RNA transcripts during early ribosome biogenesis. We observe stable binding of S15 to single RNAs immediately after transcription for the majority of the transcripts at 35?°C but for less than half at 20?°C. The remaining transcripts exhibit either rapid and transient binding or are unable to bind S15, likely due to RNA misfolding. Our work establishes the foundation for studying transcription and its coupled co-transcriptional processes, including RNA folding, ligand binding, and enzymatic activity such as in coupling of transcription to splicing, ribosome assembly or translation.


September 21, 2019  |  

DNA-guided delivery of single molecules into zero-mode waveguides.

Zero-mode waveguides (ZMWs) are powerful analytical tools corresponding to optical nanostructures fabricated in a thin metallic film capable of confining an excitation volume to the range of attoliters. This small volume of confinement allows single-molecule fluorescence experiments to be performed at physiologically relevant concentrations of fluorescently labeled biomolecules. Exactly one molecule to be studied must be attached at the floor of the ZMW for signal detection and analysis; however, the massive parallelism of these nanoarrays suffers from a Poissonian-limited distribution of these biomolecules. To date, there is no method available that provides full single-molecule occupancy of massively arrayed ZMWs. Here we report the performance of a DNA-guided method that uses steric exclusion properties of large DNA molecules to bias the Poissonian-limited delivery of single molecules. Non-Poissonian statistics were obtained with DNA molecules that contain a free-biotinylated extremity for efficient binding to the floor of the ZMW, which resulted in a decrease of accessibility for a second molecule. Both random-coiled and condensed DNA conformations drove non-Poissonian single-molecule delivery into ZMW arrays. The results suggest that an optimal balance between the rigidity and flexibility of the macromolecule is critical for favorable accessibility and single occupancy. The optimized method provides a means for full exploitation of these massively parallelized analytical tools.


September 21, 2019  |  

A flexible and efficient template format for circular consensus sequencing and SNP detection.

A novel template design for single-molecule sequencing is introduced, a structure we refer to as a SMRTbell template. This structure consists of a double-stranded portion, containing the insert of interest, and a single-stranded hairpin loop on either end, which provides a site for primer binding. Structurally, this format resembles a linear double-stranded molecule, and yet it is topologically circular. When placed into a single-molecule sequencing reaction, the SMRTbell template format enables a consensus sequence to be obtained from multiple passes on a single molecule. Furthermore, this consensus sequence is obtained from both the sense and antisense strands of the insert region. In this article, we present a universal method for constructing these templates, as well as an application of their use. We demonstrate the generation of high-quality consensus accuracy from single molecules, as well as the use of SMRTbell templates in the identification of rare sequence variants.


September 21, 2019  |  

A Sequel to Sanger: amplicon sequencing that scales.

Although high-throughput sequencers (HTS) have largely displaced their Sanger counterparts, the short read lengths and high error rates of most platforms constrain their utility for amplicon sequencing. The present study tests the capacity of single molecule, real-time (SMRT) sequencing implemented on the SEQUEL platform to overcome these limitations, employing 658 bp amplicons of the mitochondrial cytochrome c oxidase I gene as a model system.By examining templates from more than 5000 species and 20,000 specimens, the performance of SMRT sequencing was tested with amplicons showing wide variation in GC composition and varied sequence attributes. SMRT and Sanger sequences were very similar, but SMRT sequencing provided more complete coverage, especially for amplicons with homopolymer tracts. Because it can characterize amplicon pools from 10,000 DNA extracts in a single run, the SEQUEL can reduce greatly reduce sequencing costs in comparison to first (Sanger) and second generation platforms (Illumina, Ion).SMRT analysis generates high-fidelity sequences from amplicons with varying GC content and is resilient to homopolymer tracts. Analytical costs are low, substantially less than those for first or second generation sequencers. When implemented on the SEQUEL platform, SMRT analysis enables massive amplicon characterization because each instrument can recover sequences from more than 5 million DNA extracts a year.


September 21, 2019  |  

Detecting AGG interruptions in females with a FMR1 premutation by long-read Single-Molecule Sequencing: A 1 year clinical experience.

The fragile X syndrome arises from the FMR1 CGG expansion of a premutation (55-200 repeats) to a full mutation allele (>200 repeats) and is the most frequent cause of inherited X-linked intellectual disability. The risk for a premutation to expand to a full mutation allele depends on the repeat length and AGG triplets interrupting this repeat. In genetic counseling it is important to have information on both these parameters to provide an accurate risk estimate to women carrying a premutation allele and weighing up having children. For example, in case of a small risk a woman might opt for a natural pregnancy followed up by prenatal diagnosis while she might choose for preimplantation genetic diagnosis (PGD) if the risk is high. Unfortunately, the detection of AGG interruptions was previously hampered by technical difficulties complicating their use in diagnostics. Therefore we recently developed, validated and implemented a new methodology which uses long-read single-molecule sequencing to identify AGG interruptions in females with a FMR1 premutation. Here we report on the assets of AGG interruption detection by sequencing and the impact of implementing the assay on genetic counseling.


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