Counting absolute number of molecules using unique molecular identifiers

Advances in molecular biology have made it easy to identify different DNA or RNA species and to copy them. Identification of nucleic acid species can be accomplished by reading the DNA sequence; currently millions of molecules can be sequenced in a single day using massively parallel sequencing. Efficient copying of DNA-molecules of arbitrary sequence was made possible by molecular cloning, and the polymerase chain reaction. Differences in the relative abundance of a large number of different sequences between two or more samples can in turn be measured using microarray hybridization and/or tag sequencing. However, determining the relative abundance of two different species and/or the absolute number of molecules present in a single sample has proven much more challenging. This is because it is hard to detect individual molecules without copying them, and even harder to make defined number of copies of molecules. We show here that this limitation can be overcome by using unique molecular identifiers (umis), which make each molecule in the sample distinct

Counting molecules in cell-free DNA and single cells RNA

The field of Molecular Biology got started in earnest with the discovery of the molecular structure of DNA. This lead to a surge of interest into the relationships between DNA, RNA and proteins, and to the development of fundamental tools for manipulating those substances, such as cutting, ligating, amplifying, visualizing and size-selecting DNA. With these tools at hand it was possible to begin sequencing DNA, a process that took a leap forward in 2005 with the advent of Next Generation Sequencing (NGS). An inherent problem with NGS is that both the sequencing process and the library preparation introduce errors and biases. The massive amount of data generated by NGS, and the use of NGS in clinical settings, has created a demand for methods that can account for this and thereby making the sequencing data correct and reproducible. Part 1 of this thesis briefly describes the development of Molecular Biology from the discovery of the molecular structure of DNA until today. Part 2 describes the development of error correcting and molecule counting methods, and part 3 describes the results of the papers of the thesis. Paper I introduces the concept of Unique Molecular Identifiers (UMI) for reducing noise in molecular karyotyping and RNA sequencing data. Paper II compares the use of UMIs, a novel amplification-free method, and standard library preparation in Non-Invasive Prenatal Testing (NIPT) of fetal karyotype. Paper III uses the UMI concept together with single-cell tagged reverse transcription (STRT) to examine promoter preference in single cells. Finally paper IV use UMIs combined with PacBio sequencing to examine the full-length transcriptome in single cells

Towards improved cover glasses for photovoltaic devices

For the solar energy industry to increase its competitiveness there is a global drive to lower the cost of solar generated electricity. Photovoltaic (PV) module assembly is material-demanding and the cover glass constitutes a significant proportion of the cost. Currently, 3 mm thick glass is the predominant cover material for PV modules, accounting for 10-25% of the total cost. Here we review the state-of-the-art of cover glasses for PV modules and present our recent results for improvement of the glass. These improvements were demonstrated in terms of mechanical, chemical and optical properties by optimizing the glass composition, including addition of novel dopants, to produce cover glasses that can provide: (i) enhanced UV protection of polymeric PV module components, potentially increasing module service lifetimes; (ii) re-emission of a proportion of the absorbed UV photon energy as visible photons capable of being absorbed by the solar cells, thereby increasing PV module efficiencies; (iii) Successful laboratory-scale demonstration of proof-of-concept, with increases of 1-6% in Isc and 1-8% Ipm. Improvements in both chemical and crack resistance of the cover glass were also achieved through modest chemical reformulation, highlighting what may be achievable within existing manufacturing technology constraints

Deterministic evolution and stringent selection during preneoplasia

The earliest events during human tumour initiation, although poorly characterized, may hold clues to malignancy detection and prevention1. Here we model occult preneoplasia by biallelic inactivation of TP53, a common early event in gastric cancer, in human gastric organoids. Causal relationships between this initiating genetic lesion and resulting phenotypes were established using experimental evolution in multiple clonally derived cultures over 2 years. TP53 loss elicited progressive aneuploidy, including copy number alterations and structural variants prevalent in gastric cancers, with evident preferred orders. Longitudinal single-cell sequencing of TP53-deficient gastric organoids similarly indicates progression towards malignant transcriptional programmes. Moreover, high-throughput lineage tracing with expressed cellular barcodes demonstrates reproducible dynamics whereby initially rare subclones with shared transcriptional programmes repeatedly attain clonal dominance. This powerful platform for experimental evolution exposes stringent selection, clonal interference and a marked degree of phenotypic convergence in premalignant epithelial organoids. These data imply predictability in the earliest stages of tumorigenesis and show evolutionary constraints and barriers to malignant transformation, with implications for earlier detection and interception of aggressive, genome-instable tumours

The FIELDS Instrument Suite for Solar Probe Plus: Measuring the Coronal Plasma and Magnetic Field, Plasma Waves and Turbulence, and Radio Signatures of Solar Transients.

NASA's Solar Probe Plus (SPP) mission will make the first in situ measurements of the solar corona and the birthplace of the solar wind. The FIELDS instrument suite on SPP will make direct measurements of electric and magnetic fields, the properties of in situ plasma waves, electron density and temperature profiles, and interplanetary radio emissions, amongst other things. Here, we describe the scientific objectives targeted by the SPP/FIELDS instrument, the instrument design itself, and the instrument concept of operations and planned data products

Design, pointing control, and on-sky performance of the mid-infrared vortex coronagraph for the VLT/NEAR experiment

Vortex coronagraphs have been shown to be a promising avenue for high- contrast imaging in the close-in environment of stars at thermal infrared (IR) wavelengths. They are included in the baseline design of the mid-infrared extremely large telescope imager and spectrograph. To ensure good performance of these coronagraphs, a precise control of the centering of the star image in real time is needed. We previously developed and validated the quadrant analysis of coronagraphic images for tip-tilt sensing estimator (QACITS) pointing estimator to address this issue. While this approach is not wavelength-dependent in theory, it was never implemented for mid-IR observations, which leads to specific challenges and limitations. Here, we present the design of the mid-IR vortex coronagraph for the "new Earths in the α Cen Region (NEAR) experiment with the Very Large Telescope (VLT)/Very Large Telescope imager and spectrometer for the mid-infrared (VISIR) instrument and assess the performance of the QACITS estimator for the centering control of the star image onto the vortex coronagraph. We use simulated data and on-sky data obtained with VLT/VISIR, which was recently upgraded for observations assisted by adaptive optics in the context of the NEAR experiment. We demonstrate that the QACITS-based correction loop is able to control the centering of the star image onto the NEAR vortex coronagraph with a stability down to 0.015 λ / D rms over 4 h in good conditions. These results show that QACITS is a robust approach for precisely controlling in real time the centering of vortex coronagraphs for mid-IR observations.Peer reviewe

NEAR: New Earths in the Alpha Cen Region (bringing VISIR as a "visiting instrument" to ESO-VLT-UT4)

By adding a dedicated coronagraph, ESO in collaboration with the Breakthrough Initiatives, modifies the Very Large Telescope mid-IR imager (VISIR) to further boost the high dynamic range imaging capability this instru- ment has. After the VISIR upgrade in 2012, where coronagraphic masks were first added to VISIR, it became evident that coronagraphy at a ground-based 8m-class telescope critically needs adaptive optics, even at wavelengths as long as 10μm. For VISIR, a work-horse observatory facility instrument in normal operations, this is ”easiest” achieved by bringing VISIR as a visiting instrument to the ESO-VLT-UT4 having an adaptive M2. This “visit” enables a meaningful search for Earth-like planets in the habitable zone around both α-Cen1,2. Meaningful here means, achieving a contrast of ≈ 10^(-6) within ≈ 0.8arcsec from the star while maintaining basically the normal sensitivity of VISIR. This should allow to detect a planet twice the diameter of Earth. Key components will be a diffractive coronagraphic mask, the annular groove phase mask (AGPM), optimized for the most sensitive spectral band-pass in the N-band, complemented by a sophisticated apodizer at the level of the Lyot stop. For VISIR noise filtering based on fast chopping is required. A novel internal chopper system will be integrated into the cryostat. This chopper is based on the standard technique from early radio astronomy, conceived by the microwave pioneer Robert Dicke in 1946, which was instrumental for the discovery of the 3K radio background

Meta-analysis of five genome-wide association studies identifies multiple new loci associated with testicular germ cell tumor

The international Testicular Cancer Consortium (TECAC) combined five published genome-wide association studies of testicular germ cell tumor (TGCT; 3,558 cases and 13,970 controls) to identify new susceptibility loci. We conducted a fixed-effects meta-analysis, including, to our knowledge, the first analysis of the X chromosome. Eight new loci mapping to 2q14.2, 3q26.2, 4q35.2, 7q36.3, 10q26.13, 15q21.3, 15q22.31, and Xq28 achieved genome-wide significance (P < 5 × 10−8). Most loci harbor biologically plausible candidate genes. We refined previously reported associations at 9p24.3 and 19p12 by identifying one and three additional independent SNPs, respectively. In aggregate, the 39 independent markers identified to date explain 37% of father-to-son familial risk, 8% of which can be attributed to the 12 new signals reported here. Our findings substantially increase the number of known TGCT susceptibility alleles, move the field closer to a comprehensive understanding of the underlying genetic architecture of TGCT, and provide further clues to the etiology of TGCT

NIST interlaboratory study on glycosylation analysis of monoclonal antibodies : comparison of results from diverse analytical methods

Glycosylation is a topic of intense current interest in the development of biopharmaceuticals since it is related to drug safety and efficacy. This work describes results of an interlaboratory study on the glycosylation of the Primary Sample (PS) of NISTmAb, a monoclonal antibody reference material. Seventy‑six laboratories from industry, university, research, government, and hospital sectors in Europe, North America, Asia, and Australia submitted a total of 103 reports on glycan distributions. The principal objective of this study was to report and compare results for the full range of analytical methods presently used in the glycosylation  analysis of mAbs. Therefore, participation was unrestricted, with laboratories choosing their own measurement techniques. Protein glycosylation was determined in various ways, including at the level of intact mAb, protein fragments, glycopeptides, or released glycans, using a wide variety of methods for derivatization, separation, identification, and quantification. Consequently, the diversity of results was enormous, with the number of glycan compositions identified by each laboratory ranging from 4 to 48. In total, one hundred sixteen glycan compositions were reported, of which 57 compositions could be assigned consensus abundance values. These consensus medians provide community-derived values for NISTmAb PS. Agreement with the consensus medians did not depend on the specific method or laboratory type.. The study provides a view of the current state-of-the-art for biologic glycosylation measurement and suggests a clear need for harmonization of glycosylation analysis methods