The human genome reference sequence remains incomplete owing to the challenge of assembling long tracts of near-identical tandem repeats in centromeres. We implemented a nanopore sequencing strategy ...to generate high-quality reads that span hundreds of kilobases of highly repetitive DNA in a human Y chromosome centromere. Combining these data with short-read variant validation, we assembled and characterized the centromeric region of a human Y chromosome.
The sequencing of individual DNA strands with nanopores is under investigation as a rapid, low-cost platform in which bases are identified in order as the DNA strand is transported through a pore ...under an electrical potential. Although the preparation of solid-state nanopores is improving, biological nanopores, such as α-hemolysin (αHL), are advantageous because they can be precisely manipulated by genetic modification. Here, we show that the transmembrane β-barrel of an engineered αHL pore contains 3 recognition sites that can be used to identify all 4 DNA bases in an immobilized single-stranded DNA molecule, whether they are located in an otherwise homopolymeric DNA strand or in a heteropolymeric strand. The additional steps required to enable nanopore DNA sequencing are outlined.
Functional truncated membrane pores Stoddart, David; Ayu, Mariam; Höfler, Lajos ...
Proceedings of the National Academy of Sciences - PNAS,
02/2014, Letnik:
111, Številka:
7
Journal Article
Recenzirano
Odprti dostop
Membrane proteins are generally divided into two classes. Integral proteins span the lipid bilayer, and peripheral proteins are located at the membrane surface. Here, we provide evidence for membrane ...proteins of a third class that stabilize lipid pores, most probably as toroidal structures. We examined mutants of the staphylococcal α-hemolysin pore so severely truncated that the protein cannot span a bilayer. Nonetheless, the doughnut-like structures elicited well-defined transmembrane ionic currents by inducing pore formation in the underlying lipids. The formation of lipid pores, produced here by a structurally defined protein, is supported by the lipid and voltage dependences of pore formation, and by molecular dynamics simulations. We discuss the role of stabilized lipid pores in amyloid disease, the action of antimicrobial peptides, and the assembly of the membrane-attack complexes of the immune system.
The MinION sequencer has made in situ sequencing feasible in remote locations. Following our initial demonstration of its high performance off planet with Earth-prepared samples, we developed and ...tested an end-to-end, sample-to-sequencer process that could be conducted entirely aboard the International Space Station (ISS). Initial experiments demonstrated the process with a microbial mock community standard. The DNA was successfully amplified, primers were degraded, and libraries prepared and sequenced. The median percent identities for both datasets were 84%, as assessed from alignment of the mock community. The ability to correctly identify the organisms in the mock community standard was comparable for the sequencing data obtained in flight and on the ground. To validate the process on microbes collected from and cultured aboard the ISS, bacterial cells were selected from a NASA Environmental Health Systems Surface Sample Kit contact slide. The locations of bacterial colonies chosen for identification were labeled, and a small number of cells were directly added as input into the sequencing workflow. Prepared DNA was sequenced, and the data were downlinked to Earth. Return of the contact slide to the ground allowed for standard laboratory processing for bacterial identification. The identifications obtained aboard the ISS,
and
, matched those determined on the ground down to the species level. This marks the first ever identification of microbes entirely off Earth, and this validated process could be used for in-flight microbial identification, diagnosis of infectious disease in a crewmember, and as a research platform for investigators around the world.
For the past two decades, microbial monitoring of the International Space Station (ISS) has relied on culture-dependent methods that require return to Earth for analysis. This has a number of ...limitations, with the most significant being bias towards the detection of culturable organisms and the inherent delay between sample collection and ground-based analysis. In recent years, portable and easy-to-use molecular-based tools, such as Oxford Nanopore Technologies’ MinION™ sequencer and miniPCR bio’s miniPCR™ thermal cycler, have been validated onboard the ISS. Here, we report on the development, validation, and implementation of a swab-to-sequencer method that provides a culture-independent solution to real-time microbial profiling onboard the ISS. Method development focused on analysis of swabs collected in a low-biomass environment with limited facility resources and stringent controls on allowed processes and reagents. ISS-optimized procedures included enzymatic DNA extraction from a swab tip, bead-based purifications, altered buffers, and the use of miniPCR and the MinION. Validation was conducted through extensive ground-based assessments comparing current standard culture-dependent and newly developed culture-independent methods. Similar microbial distributions were observed between the two methods; however, as expected, the culture-independent data revealed microbial profiles with greater diversity. Protocol optimization and verification was established during NASA Extreme Environment Mission Operations (NEEMO) analog missions 21 and 22, respectively. Unique microbial profiles obtained from analog testing validated the swab-to-sequencer method in an extreme environment. Finally, four independent swab-to-sequencer experiments were conducted onboard the ISS by two crewmembers. Microorganisms identified from ISS swabs were consistent with historical culture-based data, and primarily consisted of commonly observed human-associated microbes. This simplified method has been streamlined for high ease-of-use for a non-trained crew to complete in an extreme environment, thereby enabling environmental and human health diagnostics in real-time as future missions take us beyond low-Earth orbit.
The α-hemolysin (αHL) protein nanopore has been investigated previously as a base detector for the strand sequencing of DNA and RNA. Recent findings have suggested that shorter pores might provide ...improved base discrimination. New work has also shown that truncated-barrel mutants (TBM) of αHL form functional pores in lipid bilayers. Therefore, we tested TBM pores for the ability to recognize bases in DNA strands immobilized within them. In the case of TBMΔ6, in which the barrel is shortened by ∼16 Å, one of the three recognition sites found in the wild-type pore, R1, was almost eliminated. With further mutagenesis (Met113 → Gly), R1 was completely removed, demonstrating that TBM pores can mediate sharpened recognition. Remarkably, a second mutant of TBMΔ6 (Met113 → Phe) was able to bind the positively charged β-cyclodextrin, am7βCD, unusually tightly, permitting the continuous recognition of individual nucleoside monophosphates, which would be required for exonuclease sequencing mediated by nanopore base identification.
Registration of range images of surfaces is a fundamental problem in three-dimensional modelling. This process is performed by finding a rotation matrix and translation vector between two sets of ...data points requiring registration. Many techniques have been developed to solve the registration problem. Therefore, it is important to understand the accuracy of various registration techniques when we decide which technique will be selected to perform registration task. This paper presents a new approach to test and compare registration techniques in terms of accuracy. Among various registration methods, iterative closest point-based algorithms and reference marker methods are two types of commonly applied methods which are used to accomplish this task because they are easy to implement and relatively low cost. These two methods have been selected to perform a comprehensively quantitative evaluation by using the proposed method and the registration results are verified using the calibrated NPL freeform standard.
At the end of the sixteenth century, Queen Elizabeth I forced the Irish Franciscans into exile. Of the four continental provinces to which the Irish Franciscans fled, the Prague Franciscan College of ...the Immaculate Conceptionof the Virgin Mary was the largest in its time. This monograph documents this intense point of contact betweentwo small European lands, Ireland and Bohemia. The Irish exiles changed the course of Bohemian history in significantways, both positive — the Irish students and teachers of medicine who contributed to Bohemia’s culture and sciences— and negative — the Irish officers who participated in the murder of Albrecht of Valdštejn and their successors who served in the Imperial forces. Dealing with a hitherto largely neglected theme, Parez and Kucharová attemptto place the Franciscan College within Bohemian history and to document the activities of its members. This wealth of historical material from the Czech archives, presented in English for the first time, will be of great aid for international researchers, particularly those interested in Bohemia or the Irish diaspora.
High-order three-dimensional (3D) interactions between more than two genomic loci are common in human chromatin, but their role in gene regulation is unclear. Previous high-order 3D chromatin assays ...either measure distant interactions across the genome or proximal interactions at selected targets. To address this gap, we developed Pore-C, which combines chromatin conformation capture with nanopore sequencing of concatemers to profile proximal high-order chromatin contacts at the genome scale. We also developed the statistical method Chromunity to identify sets of genomic loci with frequencies of high-order contacts significantly higher than background ('synergies'). Applying these methods to human cell lines, we found that synergies were enriched in enhancers and promoters in active chromatin and in highly transcribed and lineage-defining genes. In prostate cancer cells, these included binding sites of androgen-driven transcription factors and the promoters of androgen-regulated genes. Concatemers of high-order contacts in highly expressed genes were demethylated relative to pairwise contacts at the same loci. Synergies in breast cancer cells were associated with tyfonas, a class of complex DNA amplicons. These results rigorously link genome-wide high-order 3D interactions to lineage-defining transcriptional programs and establish Pore-C and Chromunity as scalable approaches to assess high-order genome structure.
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