Whole Methylome Analysis by Ultra-Deep Sequencing Using Two-Base Encoding

Methylation, the addition of methyl groups to cytosine (C), plays an important role in the regulation of gene expression in both normal and dysfunctional cells. During bisulfite conversion and subsequent PCR amplification, unmethylated Cs are converted into thymine (T), while methylated Cs will not be converted. Sequencing of this bisulfite-treated DNA permits the detection of methylation at specific sites. Through the introduction of next-generation sequencing technologies (NGS) simultaneous analysis of methylation motifs in multiple regions provides the opportunity for hypothesis-free study of the entire methylome. Here we present a whole methylome sequencing study that compares two different bisulfite conversion methods (in solution versus in gel), utilizing the high throughput of the SOLiD™ System. Advantages and disadvantages of the two different bisulfite conversion methods for constructing sequencing libraries are discussed. Furthermore, the application of the SOLiD™ bisulfite sequencing to larger and more complex genomes is shown with preliminary in silico created bisulfite converted reads

The Near Infrared Imager and Slitless Spectrograph for the James Webb Space Telescope -- IV. Aperture Masking Interferometry

The James Webb Space Telescope's Near Infrared Imager and Slitless Spectrograph (JWST-NIRISS) flies a 7-hole non-redundant mask (NRM), the first such interferometer in space, operating at 3-5 \micron~wavelengths, and a bright limit of $\simeq 4$ magnitudes in W2. We describe the NIRISS Aperture Masking Interferometry (AMI) mode to help potential observers understand its underlying principles, present some sample science cases, explain its operational observing strategies, indicate how AMI proposals can be developed with data simulations, and how AMI data can be analyzed. We also present key results from commissioning AMI. Since the allied Kernel Phase Imaging (KPI) technique benefits from AMI operational strategies, we also cover NIRISS KPI methods and analysis techniques, including a new user-friendly KPI pipeline. The NIRISS KPI bright limit is $\simeq 8$ W2 magnitudes. AMI (and KPI) achieve an inner working angle of $\sim 70$ mas that is well inside the $\sim 400$ mas NIRCam inner working angle for its circular occulter coronagraphs at comparable wavelengths.Comment: 30 pages, 10 figure

The Near Infrared Imager and Slitless Spectrograph for the James Webb Space Telescope. IV. Aperture Masking Interferometry

The James Webb Space Telescope’s Near Infrared Imager and Slitless Spectrograph (JWST-NIRISS) flies a 7-hole non-redundant mask (NRM), the first such interferometer in space, operating at 3-5 μm wavelengths, and a bright limit of ≃4 mag in W2. We describe the NIRISS Aperture Masking Interferometry (AMI) mode to help potential observers understand its underlying principles, present some sample science cases, explain its operational observing strategies, indicate how AMI proposals can be developed with data simulations, and how AMI data can be analyzed. We also present key results from commissioning AMI. Since the allied Kernel Phase Imaging (KPI) technique benefits from AMI operational strategies, we also cover NIRISS KPI methods and analysis techniques, including a new user-friendly KPI pipeline. The NIRISS KPI bright limit is ≃8 W2 (4.6 μm) magnitudes. AMI NRM and KPI achieve an inner working angle of ∼70 mas, which is well inside the ∼400 mas NIRCam inner working angle for its circular occulter coronagraphs at comparable wavelengths.</p

A Platform-Independent Method for Detecting Errors in Metagenomic Sequencing Data: DRISEE

We provide a novel method, DRISEE (duplicate read inferred sequencing error estimation), to assess sequencing quality (alternatively referred to as “noise” or “error”) within and/or between sequencing samples. DRISEE provides positional error estimates that can be used to inform read trimming within a sample. It also provides global (whole sample) error estimates that can be used to identify samples with high or varying levels of sequencing error that may confound downstream analyses, particularly in the case of studies that utilize data from multiple sequencing samples. For shotgun metagenomic data, we believe that DRISEE provides estimates of sequencing error that are more accurate and less constrained by technical limitations than existing methods that rely on reference genomes or the use of scores (e.g. Phred). Here, DRISEE is applied to (non amplicon) data sets from both the 454 and Illumina platforms. The DRISEE error estimate is obtained by analyzing sets of artifactual duplicate reads (ADRs), a known by-product of both sequencing platforms. We present DRISEE as an open-source, platform-independent method to assess sequencing error in shotgun metagenomic data, and utilize it to discover previously uncharacterized error in de novo sequence data from the 454 and Illumina sequencing platforms

The Near Infrared Imager and Slitless Spectrograph for the James Webb Space Telescope. IV. Aperture Masking Interferometry

The James Webb Space Telescope’s Near Infrared Imager and Slitless Spectrograph (JWST-NIRISS) flies a 7-hole non-redundant mask (NRM), the first such interferometer in space, operating at 3-5 μm wavelengths, and a bright limit of ≃4 mag in W2. We describe the NIRISS Aperture Masking Interferometry (AMI) mode to help potential observers understand its underlying principles, present some sample science cases, explain its operational observing strategies, indicate how AMI proposals can be developed with data simulations, and how AMI data can be analyzed. We also present key results from commissioning AMI. Since the allied Kernel Phase Imaging (KPI) technique benefits from AMI operational strategies, we also cover NIRISS KPI methods and analysis techniques, including a new user-friendly KPI pipeline. The NIRISS KPI bright limit is ≃8 W2 (4.6 μm) magnitudes. AMI NRM and KPI achieve an inner working angle of ∼70 mas, which is well inside the ∼400 mas NIRCam inner working angle for its circular occulter coronagraphs at comparable wavelengths

The Near Infrared Imager and Slitless Spectrograph for the James Webb Space Telescope. IV. Aperture Masking Interferometry

The James Webb Space Telescope’s Near Infrared Imager and Slitless Spectrograph (JWST-NIRISS) flies a 7-hole non-redundant mask (NRM), the first such interferometer in space, operating at 3-5 μm wavelengths, and a bright limit of ≃4 mag in W2. We describe the NIRISS Aperture Masking Interferometry (AMI) mode to help potential observers understand its underlying principles, present some sample science cases, explain its operational observing strategies, indicate how AMI proposals can be developed with data simulations, and how AMI data can be analyzed. We also present key results from commissioning AMI. Since the allied Kernel Phase Imaging (KPI) technique benefits from AMI operational strategies, we also cover NIRISS KPI methods and analysis techniques, including a new user-friendly KPI pipeline. The NIRISS KPI bright limit is ≃8 W2 (4.6 μm) magnitudes. AMI NRM and KPI achieve an inner working angle of ∼70 mas, which is well inside the ∼400 mas NIRCam inner working angle for its circular occulter coronagraphs at comparable wavelengths.</p

The Near Infrared Imager and Slitless Spectrograph for the James Webb Space Telescope. IV. Aperture Masking Interferometry

The James Webb Space Telescope’s Near Infrared Imager and Slitless Spectrograph (JWST-NIRISS) flies a 7-hole non-redundant mask (NRM), the first such interferometer in space, operating at 3-5 μm wavelengths, and a bright limit of ≃4 mag in W2. We describe the NIRISS Aperture Masking Interferometry (AMI) mode to help potential observers understand its underlying principles, present some sample science cases, explain its operational observing strategies, indicate how AMI proposals can be developed with data simulations, and how AMI data can be analyzed. We also present key results from commissioning AMI. Since the allied Kernel Phase Imaging (KPI) technique benefits from AMI operational strategies, we also cover NIRISS KPI methods and analysis techniques, including a new user-friendly KPI pipeline. The NIRISS KPI bright limit is ≃8 W2 (4.6 μm) magnitudes. AMI NRM and KPI achieve an inner working angle of ∼70 mas, which is well inside the ∼400 mas NIRCam inner working angle for its circular occulter coronagraphs at comparable wavelengths

Rapid establishment of the European Bank for induced Pluripotent Stem Cells (EBiSC):The Hot Start experience

A fast track “Hot Start” process was implemented to launch the European Bank for Induced Pluripotent Stem Cells (EBiSC) to provide early release of a range of established control and disease linked human induced pluripotent stem cell (hiPSC) lines. Established practice amongst consortium members was surveyed to arrive at harmonised and publically accessible Standard Operations Procedures (SOPs) for tissue procurement, bio-sample tracking, iPSC expansion, cryopreservation, qualification and distribution to the research community. These were implemented to create a quality managed foundational collection of lines and associated data made available for distribution. Here we report on the successful outcome of this experience and work flow for banking and facilitating access to an otherwise disparate European resource, with lessons to benefit the international research community. eTOC: The report focuses on the EBiSC experience of rapidly establishing an operational capacity to procure, bank and distribute a foundational collection of established hiPSC lines. It validates the feasibility and defines the challenges of harnessing and integrating the capability and productivity of centres across Europe using commonly available resources currently in the field

Ribosomal frameshifting and misreading of mRNA in COVID-19 vaccines produces “off-target” proteins and immune responses eliciting safety concerns: Comment on UK study by Mulroney et al.

We comment on the study by Mulroney et al.(1) entitled: “N1-methylpseudouridylation of mRNA causes +1 ribosomal frameshifting.” The study found evidence in mice and humans for the misreading of the modRNA contained within the Pfizer COVID-19 vaccine to inadvertently produce “off-target” proteins capable of eliciting “off-target” immune responses. The authors propose that these novel proteins are the result of ribosomal frameshifting occasioned by the substitution of N1-methyl pseudouridine. The authors state that the “error prone” code is a safety concern with a “huge potential to be harmful” and that “it is essential that these therapeutics are designed to be free from unintended side-effects.” The findings reveal a developmental and regulatory failure to ask fundamental questions that could affect the safety and effectiveness of these products. According to WHO guidelines for mRNA vaccines, (2) manufacturers should provide details of “unexpected ORFs”(Open Reading Frames). The formation of these off-target proteins is not disclosed in the package insert for COMIRNATY. The finding that unintended proteins may be produced as a result of vaccination is sufficient cause for regulators to conduct full risk assessments of past or future harms that may have ensued. Given that this study was conducted under the auspices of the United Kingdom Government, we must assume UK regulators, manufacturers, and international regulatory agencies, including FDA, were apprised of the data many months ago. We await their account of what steps they have taken to investigate why the formation of off-target proteins was not discovered sooner, what toxic effects they may have caused and what steps they are taken to prevent harm in the future and to inform the public of these findings

Expanded genetic codes in next generation sequencing enable decontamination and mitochondrial enrichment.

We have developed a PCR method, coined Déjà vu PCR, that utilizes six nucleotides in PCR with two methyl specific restriction enzymes that respectively digest these additional nucleotides. Use of this enzyme-and-nucleotide combination enables what we term a "DNA diode", where DNA can advance in a laboratory in only one direction and cannot feedback into upstream assays. Here we describe aspects of this method that enable consecutive amplification with the introduction of a 5th and 6th base while simultaneously providing methylation dependent mitochondrial DNA enrichment. These additional nucleotides enable a novel DNA decontamination technique that generates ephemeral and easy to decontaminate DNA