In Situ Hybridization, In Situ Transcription, and In Situ Polymerase Chain Reaction

In situ hybridization, in situ transcription, and in situ polymerase chain reaction (PCR) are techniques used to detect DNA and RNA sequences within a cell or tissue structure. These three in situ methodologies employ the principles of recombinant DNA to form double-stranded hybrids of DNA-DNA, DNA-RNA, or RNA-RNA. The essence of in situ hybridization (ISH) is the hybridization of a labeled probe to a complementary target sequence, whereas in situ transcription (IST) is the synthesis of complementary DNA incorporating a label directly on the target DNA or RNA within a cell or tissue. In the case of in situ PCR (ISPCR), it is the repeated in situ duplication of both the sense and antisense strands of DNA to increase the number of copies of the target sequence. ISH, IST, and ISPCR each have their advantages and disadvantages. The purpose of this chapter is to address in situ considerations requried of these techniques, emphasizing tissue fixation, pre-hybridization steps, DNA probes, RNA probes, oligoprobes, and probe labeling. Five successfully used protocols are presented as examples. Any given nucleotide target sequence may have its own unique set of optimum conditions, thus requiring some adjustment in the hands of the user

3D Correlations in the Lyman-α\alpha Forest from Early DESI Data

We present the first measurements of Lyman-$\alpha$ (Ly$\alpha$) forest correlations using early data from the Dark Energy Spectroscopic Instrument (DESI). We measure the auto-correlation of Ly$\alpha$ absorption using 88,509 quasars at $z>2$, and its cross-correlation with quasars using a further 147,899 tracer quasars at $z\gtrsim1.77$. Then, we fit these correlations using a 13-parameter model based on linear perturbation theory and find that it provides a good description of the data across a broad range of scales. We detect the BAO peak with a signal-to-noise ratio of $3.8\sigma$, and show that our measurements of the auto- and cross-correlations are fully-consistent with previous measurements by the Extended Baryon Oscillation Spectroscopic Survey (eBOSS). Even though we only use here a small fraction of the final DESI dataset, our uncertainties are only a factor of 1.7 larger than those from the final eBOSS measurement. We validate the existing analysis methods of Ly$\alpha$ correlations in preparation for making a robust measurement of the BAO scale with the first year of DESI data

Variability in the analysis of a single neuroimaging dataset by many teams

Data analysis workflows in many scientific domains have become increasingly complex and flexible. To assess the impact of this flexibility on functional magnetic resonance imaging (fMRI) results, the same dataset was independently analyzed by 70 teams, testing nine ex-ante hypotheses. The flexibility of analytic approaches is exemplified by the fact that no two teams chose identical workflows to analyze the data. This flexibility resulted in sizeable variation in hypothesis test results, even for teams whose statistical maps were highly correlated at intermediate stages of their analysis pipeline. Variation in reported results was related to several aspects of analysis methodology. Importantly, meta-analytic approaches that aggregated information across teams yielded significant consensus in activated regions across teams. Furthermore, prediction markets of researchers in the field revealed an overestimation of the likelihood of significant findings, even by researchers with direct knowledge of the dataset. Our findings show that analytic flexibility can have substantial effects on scientific conclusions, and demonstrate factors related to variability in fMRI. The results emphasize the importance of validating and sharing complex analysis workflows, and demonstrate the need for multiple analyses of the same data. Potential approaches to mitigate issues related to analytical variability are discussed

Variability in the analysis of a single neuroimaging dataset by many teams

Data analysis workflows in many scientific domains have become increasingly complex and flexible. To assess the impact of this flexibility on functional magnetic resonance imaging (fMRI) results, the same dataset was independently analyzed by 70 teams, testing nine ex-ante hypotheses. The flexibility of analytic approaches is exemplified by the fact that no two teams chose identical workflows to analyze the data. This flexibility resulted in sizeable variation in hypothesis test results, even for teams whose statistical maps were highly correlated at intermediate stages of their analysis pipeline. Variation in reported results was related to several aspects of analysis methodology. Importantly, meta-analytic approaches that aggregated information across teams yielded significant consensus in activated regions across teams. Furthermore, prediction markets of researchers in the field revealed an overestimation of the likelihood of significant findings, even by researchers with direct knowledge of the dataset. Our findings show that analytic flexibility can have substantial effects on scientific conclusions, and demonstrate factors related to variability in fMRI. The results emphasize the importance of validating and sharing complex analysis workflows, and demonstrate the need for multiple analyses of the same data. Potential approaches to mitigate issues related to analytical variability are discussed

Overview of the instrumentation for the Dark Energy Spectroscopic Instrument

The Dark Energy Spectroscopic Instrument (DESI) embarked on an ambitious 5 yr survey in 2021 May to explore the nature of dark energy with spectroscopic measurements of 40 million galaxies and quasars. DESI will determine precise redshifts and employ the baryon acoustic oscillation method to measure distances from the nearby universe to beyond redshift z > 3.5, and employ redshift space distortions to measure the growth of structure and probe potential modifications to general relativity. We describe the significant instrumentation we developed to conduct the DESI survey. This includes: a wide-field, 3.°2 diameter prime-focus corrector; a focal plane system with 5020 fiber positioners on the 0.812 m diameter, aspheric focal surface; 10 continuous, high-efficiency fiber cable bundles that connect the focal plane to the spectrographs; and 10 identical spectrographs. Each spectrograph employs a pair of dichroics to split the light into three channels that together record the light from 360–980 nm with a spectral resolution that ranges from 2000–5000. We describe the science requirements, their connection to the technical requirements, the management of the project, and interfaces between subsystems. DESI was installed at the 4 m Mayall Telescope at Kitt Peak National Observatory and has achieved all of its performance goals. Some performance highlights include an rms positioner accuracy of better than 0.″1 and a median signal-to-noise ratio of 7 of the [O ii] doublet at 8 × 10−17 erg s−1 cm−2 in 1000 s for galaxies at z = 1.4–1.6. We conclude with additional highlights from the on-sky validation and commissioning, key successes, and lessons learned