Extended Data Fig. 1: Workflow of HiPR-FISH experiments.
From: Highly multiplexed spatial mapping of microbial communities
Environmental microbial consortia are first split into two samples. One sample is used to generate full-length 16S amplicon sequences using PCR and PacBio sequencing. The resulting sequence file is used to generate a list of probes, which are purchased from a commercial vendor. The other sample is used for imaging experiments. Fixed samples are hybridized using an encoding hybridization buffer containing the amplified complex probes and read out using a readout hybridization buffer containing fluorescently labelled readout probes. Samples are then embedded and imaged on a standard confocal microscope in the spectral imaging mode. Resulting raw images are registered and segmented. The spectra of individual cells are measured using the raw image and the segmentation image and classified using a machine learning algorithm. Finally, classified images are used for downstream quantitative measurements of microbial spatial associations.
