Spatiotemporal transcriptomic atlas of mouse organogenesis using DNA nanoball-patterned arrays.

Spatially resolved transcriptomic technologies are promising tools to study complex biological processes such as mammalian embryogenesis. However, the imbalance between resolution, gene capture, and field of view of current methodologies precludes their systematic application to analyze relatively large and three-dimensional mid- and late-gestation embryos. Here, we combined DNA nanoball (DNB)-patterned arrays and in situ RNA capture to create spatial enhanced resolution omics-sequencing (Stereo-seq). We applied Stereo-seq to generate the mouse organogenesis spatiotemporal transcriptomic atlas (MOSTA), which maps with single-cell resolution and high sensitivity the kinetics and directionality of transcriptional variation during mouse organogenesis. We used this information to gain insight into the molecular basis of spatial cell heterogeneity and cell fate specification in developing tissues such as the dorsal midbrain. Our panoramic atlas will facilitate in-depth investigation of longstanding questions concerning normal and abnormal mammalian development.

Spatiotemporal mapping of gene expression landscapes and developmental trajectories during zebrafish embryogenesis.

A major challenge in understanding vertebrate embryogenesis is the lack of topographical transcriptomic information that can help correlate microenvironmental cues within the hierarchy of cell-fate decisions. Here, we employed Stereo-seq to profile 91 zebrafish embryo sections covering six critical time points during the first 24 h of development, obtaining a total of 152,977 spots at a resolution of 10 × 10 × 15 µm3 (close to cellular size) with spatial coordinates. Meanwhile, we identified spatial modules and co-varying genes for specific tissue organizations. By performing the integrated analysis of the Stereo-seq and scRNA-seq data from each time point, we reconstructed the spatially resolved developmental trajectories of cell-fate transitions and molecular changes during zebrafish embryogenesis. We further investigated the spatial distribution of ligand-receptor pairs and identified potentially important interactions during zebrafish embryo development. Our study constitutes a fundamental reference for further studies aiming to understand vertebrate development.

Multiplex sample-to-answer detection of bacteria using a pipette-actuated capillary array comb with integrated DNA extraction, isothermal amplification, and smartphone detection.

A pipette-actuated capillary array comb (PAAC) system operated on a smartphone-based hand-held device has been successfully developed for the multiplex detection of bacteria in a "sample-to-answer" manner. The PAAC consists of eight open capillaries inserted into a cylindrical plastic base with a piece of chitosan-modified glass filter paper embedded in each capillary. During the sample preparation, a PAAC was mounted into a 1 mL pipette tip with an enlarged opening and was operated with a 1 mL pipette for liquid handling. The cell lysate was drawn and expelled through the capillaries three times to facilitate the DNA capture on the embedded filter discs. Following washes with water, the loop-mediated isothermal amplification (LAMP) reagents were aspirated into the capillaries, in which the primers were pre-fixed with chitosan. After that, the PAAC was loaded into the smartphone-based device for a one-hour amplification at 65 °C and end-point detection of calcein fluorescence in the capillaries. The DNA capture efficiency of a 1.1 mm-diameter filter disc was determined to be 97% of λ-DNA and the coefficient of variation among the eight capillaries in the PAAC was only 2.2%. The multiplex detection of genomic DNA extracted from Escherichia coli, Klebsiella pneumoniae, and Staphylococcus aureus provided limits of detection of 200, 500, and 500 copies, respectively, without any cross-contamination and cross reactions. "Sample-to-answer" detection of E. coli samples was successfully completed in 85 minutes, demonstrating a sensitivity of 200 cfu per capillary. The multiplex "sample-to-answer" detection, the streamlined operation, and the compact device should facilitate a broad range of applications of our PAAC system in point-of-care testing.

Visual Detection of Multiple Nucleic Acids in a Capillary Array.

Multi-target, short time, and resource-affordable methodologies for the detection of multiple nucleic acids in a single, easy to operate test are urgently needed in disease diagnosis, microbial monitoring, genetically modified organism (GMO) detection, and forensic analysis. We have previously described the platform called CALM (Capillary Array-based Loop-mediated isothermal amplification for Multiplex visual detection of nucleic acids). Herein, we describe improved fabrication and performance processes for this platform. Here, we apply a small, ready-to-use cassette assembled by capillary array for multiplex visual detection of nucleic acids. The capillary array is pre-treated into a hydrophobic and hydrophilic pattern before fixing loop-mediated isothermal amplification (LAMP) primer sets in capillaries. After assembly of the loading adaptor, LAMP reaction mixture is loaded and isolated into each capillary, due to capillary force by a single pipetting step. The LAMP reactions are performed in parallel in the capillaries. The results are visually read out by illumination with a hand-held UV flashlight. Using this platform, we demonstrate monitoring of 8 frequently appearing elements and genes in GMO samples with high specificity and sensitivity. In summary, the platform described herein is intended to facilitate the detection of multiple nucleic acids. We believe it will be widely applicable in fields where high-throughput nucleic acid analysis is required.

Visual detection of multiple genetically modified organisms in a capillary array.

There is an urgent need for rapid, low-cost multiplex methodologies for the monitoring of genetically modified organisms (GMOs). Here, we report a C[combining low line]apillary A[combining low line]rray-based L[combining low line]oop-mediated isothermal amplification for M[combining low line]ultiplex visual detection of nucleic acids (CALM) platform for the simple and rapid monitoring of GMOs. In CALM, loop-mediated isothermal amplification (LAMP) primer sets are pre-fixed to the inner surface of capillaries. The surface of the capillary array is hydrophobic while the capillaries are hydrophilic, enabling the simultaneous loading and separation of the LAMP reaction mixtures into each capillary by capillary forces. LAMP reactions in the capillaries are then performed in parallel, and the results are visually detected by illumination with a hand-held UV device. Using CALM, we successfully detected seven frequently used transgenic genes/elements and five plant endogenous reference genes with high specificity and sensitivity. Moreover, we found that measurements of real-world blind samples by CALM are consistent with results obtained by independent real-time PCRs. Thus, with an ability to detect multiple nucleic acids in a single easy-to-operate test, we believe that CALM will become a widely applied technology in GMO monitoring.