Simultaneous profiling of chromatin accessibility and methylation on human cell lines with nanopore sequencing.

Probing epigenetic features on DNA has tremendous potential to advance our understanding of the phased epigenome. In this study, we use nanopore sequencing to evaluate CpG methylation and chromatin accessibility simultaneously on long strands of DNA by applying GpC methyltransferase to exogenously label open chromatin. We performed nanopore sequencing of nucleosome occupancy and methylome (nanoNOMe) on four human cell lines (GM12878, MCF-10A, MCF-7 and MDA-MB-231). The single-molecule resolution allows footprinting of protein and nucleosome binding, and determination of the combinatorial promoter epigenetic signature on individual molecules. Long-read sequencing makes it possible to robustly assign reads to haplotypes, allowing us to generate a fully phased human epigenome, consisting of chromosome-level allele-specific profiles of CpG methylation and chromatin accessibility. We further apply this to a breast cancer model to evaluate differential methylation and accessibility between cancerous and noncancerous cells.

Effects of Polyamine Binding on the Stability of DNA i-Motif Structures.

B-form DNA can adopt alternative structures under conditions such as superhelical duress. Alternative DNA structures are favored when there is asymmetric distribution of guanosine and cytosine on complimentary DNA strands. A guanosine-rich strand can form a four-stranded structure known as a quadruplex (G4). The complimentary cytosine-rich strand can utilize intercalating cytosine-cytosine base pairing to form a four-stranded structure known as the i-motif (iM). Both secondary structures are energetically uphill from double-strand DNA (dsDNA), meaning that additional factors are needed for their formation. Most iMs require slightly acidic conditions for structure stabilization. However, crowding agents such as polyethylene glycols and dextrans can shift the pK a of the iM to near-physiological pH ≈ 7. Nucleic acids have long been known to be bound and stabilized by polyamines such as putrescine, spermidine, and spermine. Polyamines have very high concentrations in cells (0.1-30 mM), and their binding to DNA is driven by electrostatic interactions. Polyamines typically bind in the minor groove of DNA. However, because of the unusual structure of iMs, it was unknown whether polyamines might also bind and stabilize iMs. The study described here was undertaken to analyze polyamine-iM interactions. The thermal stability and pH dependence of iM structures were determined in the presence of polyamines. In contrast to dsDNA, our results suggest that polyamines have considerably weaker interactions with iMs, as demonstrated by the minimal change in iM pH dependence and thermal stability. Our results suggest that polyamines are unlikely to provide a significant source of iM stabilization in vivo.

Effects of 5-Hydroxymethylcytosine Epigenetic Modification on the Stability and Molecular Recognition of VEGF i-Motif and G-Quadruplex Structures.

Promoters often contain asymmetric G- and C-rich strands, in which the cytosines are prone to epigenetic modification via methylation (5-mC) and 5-hydroxymethylation (5-hmC). These sequences can also form four-stranded G-quadruplex (G4) or i-motif (iM) secondary structures. Although the requisite sequences for epigenetic modulation and iM/G4 formation are similar and can overlap, they are unlikely to coexist. Despite 5-hmC being an oxidization product of 5-mC, the two modified bases cluster at distinct loci. This study focuses on the intersection of G4/iM formation and 5-hmC modification using the vascular endothelial growth factor (VEGF) gene promoter's CpG sites and examines whether incorporation of 5-hmC into iM/G4 structures had any physicochemical effect on formation, stability, or recognition by nucleolin or the cationic porphyrin, TMPyP4. No marked changes were found in the formation or stability of iM and G4 structures; however, changes in recognition by nucleolin or TMPyP4 occurred with 5-hmC modification wherein protein and compound binding to 5-hmC modified G4s was notably reduced. G4/iM structures in the VEGF promoter are promising therapeutic targets for antiangiogenic therapy, and this work contributes to a comprehensive understanding of their governing principles related to potential transcriptional control and targeting.

SAGE2: parallel human genome assembly.

Summary: De novo genome assembly of next-generation sequencing data is a fundamental problem in bioinformatics. There are many programs that assemble small genomes, but very few can assemble whole human genomes. We present a new algorithm for parallel overlap graph construction, which is capable of assembling human genomes and improves upon the current state-of-the-art in genome assembly. Availability and implementation: SAGE2 is written in C ++ and OpenMP and is freely available (under the GPL 3.0 license) at github.com/lucian-ilie/SAGE2. Contact: ilie@uwo.ca. Supplementary information: Supplementary data are available at Bioinformatics online.

Correcting Illumina data.

Next-generation sequencing technologies revolutionized the ways in which genetic information is obtained and have opened the door for many essential applications in biomedical sciences. Hundreds of gigabytes of data are being produced, and all applications are affected by the errors in the data. Many programs have been designed to correct these errors, most of them targeting the data produced by the dominant technology of Illumina. We present a thorough comparison of these programs. Both HiSeq and MiSeq types of Illumina data are analyzed, and correcting performance is evaluated as the gain in depth and breadth of coverage, as given by correct reads and k-mers. Time and memory requirements, scalability and parallelism are considered as well. Practical guidelines are provided for the effective use of these tools. We also evaluate the efficiency of the current state-of-the-art programs for correcting Illumina data and provide research directions for further improvement.

SAGE: String-overlap Assembly of GEnomes.

BACKGROUND: De novo genome assembly of next-generation sequencing data is one of the most important current problems in bioinformatics, essential in many biological applications. In spite of significant amount of work in this area, better solutions are still very much needed. RESULTS: We present a new program, SAGE, for de novo genome assembly. As opposed to most assemblers, which are de Bruijn graph based, SAGE uses the string-overlap graph. SAGE builds upon great existing work on string-overlap graph and maximum likelihood assembly, bringing an important number of new ideas, such as the efficient computation of the transitive reduction of the string overlap graph, the use of (generalized) edge multiplicity statistics for more accurate estimation of read copy counts, and the improved use of mate pairs and min-cost flow for supporting edge merging. The assemblies produced by SAGE for several short and medium-size genomes compared favourably with those of existing leading assemblers. CONCLUSIONS: SAGE benefits from innovations in almost every aspect of the assembly process: error correction of input reads, string-overlap graph construction, read copy counts estimation, overlap graph analysis and reduction, contig extraction, and scaffolding. We hope that these new ideas will help advance the current state-of-the-art in an essential area of research in genomics.

[Pleasure and confusion. A footnote to Freud's translations of Mill]. / Lust und Verwirrung. Eine Fuessnote zu Freuds Mill-Ubersetzungen.

In 1863 Theodor Gomperz came to England to propose to Helen Taylor Mill, step-daughter of J. S. Mill. For several months he delayed the proposal while studying transcripts of the Philodemus papyri in the Bodleian Library. There a threatening note, supposedly left on his desk, triggered an attack of paranoia. My study of this incident, initially a mere footnote, expanded into an examination of the obscure causes of this attack. The philosophical question of the nature of desire and the researcher's passion to reconstruct a fragmented classical text are related to Gomperz's unfocussed relationship with both Mill and his step-daughter, and his ensuing confusion between reality and fantasy. The incident is considered paradigmatic of the perils of scholarly research, when the desire to possess knowledge becomes entangled with transferential relationships.

RACER: Rapid and accurate correction of errors in reads.

MOTIVATION: High-throughput next-generation sequencing technologies enable increasingly fast and affordable sequencing of genomes and transcriptomes, with a broad range of applications. The quality of the sequencing data is crucial for all applications. A significant portion of the data produced contains errors, and ever more efficient error correction programs are needed. RESULTS: We propose RACER (Rapid and Accurate Correction of Errors in Reads), a new software program for correcting errors in sequencing data. RACER has better error-correcting performance than existing programs, is faster and requires less memory. To support our claims, we performed extensive comparison with the existing leading programs on a variety of real datasets. AVAILABILITY: RACER is freely available for non-commercial use at www.csd.uwo.ca/∼ilie/RACER/.

Mysteries of nature.

This article examines a group photograph of the Psychiatry and Neurology section of the 66th Meeting of the Society of German Natural Scientists and Doctors in Vienna, 24-30 September 1894 which Sigmund Freud attended. The society's origins in Naturphilosophie are indicated and a number of the participants are identified on the photo. They and the events at the conference are related to Sigmund Freud's work at the time and to his gradual abandonment of anatomy and of heredity and degeneration as significant aetiological factors in the neuroses. Philosophical problems, such as how phenomena should be described and how 'nature' is conceptualized, are also considered in the light of their implications for Freud's life and thought at that period.

[Secrets of nature]. / Geheimnisse der Natur.

The article examines a group photograph of the Psychiatry and Neurology section of the 66th Meeting of the Society of German Natural Scientists and Doctors in Vienna, September 24-30, 1894 which Sigmund Freud attended. The society's origins in Naturphilosophie are indicated and a number of the participants are identified on the photo. They and the events at the conference are related to Sigmund Freud's work at the time and to his gradual abandonment of anatomy, and of heredity and degeneration as significant aetiological factors in the neuroses. Philosophical problems, such as how phenomena should be described and how "nature" is conceptualized, are also considered in the light of their implications for Freud's life and thought at that period.

[The Freud Museum in London as a research centre]. / Das Londoner Freud Museum als Forschungsstätte.

The paper presents a compact description of those resources of the Freud Museum most relevant for the Freud scholar: 1. the Archives with its collection of letters, documents, photos, and press cuttings from the 1920s and 1930s, as well as reproductions of paintings and photos of tourist features, compiled by Freud himself; 2. Freud's archaeological collection; 3. Freud's library.

["...that reflective gaze turned inward"]. / "... jener nach innen gekehrte nachdenkliche Blick".

The 1914 etching of Freud by Max Pollak differs from other portraits, firstly in that it shows him abstracted from the world and, secondly, because his antiquities are prominently featured. Links may be conjectured between his mood and such events of the time as the dispute with Jung and the Zurich group. Identification of the antiquities yields associations with aspects of Freud's working methods. An art work presents a different level of evidence from a photograph since intentionality plays a stronger role in it than structure. Consequently it throws more light on emotion than fact. The atmosphere of the etching prompts some reflections on representation and the uncanny. This article continues to argue the implicit case behind the author's preceding photograph studies: that iconography may provide a basis for writing history that can accomodate self-criticism and uncertainties.

[The moving sidewalk, 1900]. / Trottoir roulant, 1900.

Two photos of the same scene, but taken at an interval of a second or two, raise questions of the time lapse and what occurs between frozen frames. The subjects are Freud and a group of his relatives in the summer of 1900. As if waiting for a lecture which has not yet begun, and may never begin, they face their respective fates and futures. What will happen is implicit in what is happening and in what has already happened. The mechanisms of the Trottoir roulant or of dream scenes recalled are analogous to the experience of time reconstructed from these fragmented images.

Freud & Co.

A previously unknown photograph of the old Emanuel Freud with his youngest daughter Bertha inspires an attempt to salvage what can be rescued of these lost lives and reminds us that history is always incomplete. Emanuel's appearances in Freud's dreams exemplify the dream-like aspects of the photographic image, which momentarily highlights particular aspects of the sitter while neglecting others. Examining photographs for documentation involves a reappraisal of the language of historical evidence.

[Portrait of an alien enemy]. / Alien Enemy: Porträt eines Mädchens.

Official documents and photographs are forms of alienation yet they carry imprints of the inner life of their subjects. An identity paper and photograph of the eighteen-year-old Anna Freud (August 1914) serves as the basis for an investigation of various aspects of her identity at the time - firstly her position in England as an "alien enemy"; secondly her emotional and sexual predicament, brought to light primarily through her father's response to Ernest Jones' supposed advances; and finally her own sense of self as deduced from the expression in the portrait, backed up by the evidence of her poetry.

["... the child should know ..."]. / "... das Kind soll wissen ...".

Examination of an archive photograph of Angela Seidmann-Freud with her grandmother Maria (Mitzi) Freud leads to a consideraton of Tom Seidmann-Freud's work and fate. Circumstantial biographical events are related to the composition of the photograph. The events are a filter through which the photograph is viewed: the interaction between narrative and image has analogies in the educative strategies Tom developed in her children's books.