Extended Data Fig. 4: Probe design pipeline and amplification strategy.
From: Highly multiplexed spatial mapping of microbial communities
a, Full-length 16S sequences are first grouped by taxa. The consensus sequence for each taxon is used to design probes. Each probe within each taxon is then blasted against the database of full-length 16S sequences. Several probe quality metrics are calculated on the basis of the blast results and are used to select probes. All selected probes are conjugated to the appropriate readout sequences and blasted against the database of full-length 16S sequences to remove probes with any potential mis-hybridization sites owing to the conjugation of the readout sequences. b, Schematic for probe synthesis. Complex oligo pools are amplified using limited-cycle PCR. The T7 promoter introduced during PCR allows the templates to be in-vitro-transcribed. Reverse transcription then converts RNA to cDNA. Finally, alkaline hydrolysis removes the RNA strand to generate the final single-stranded DNA probe pool.
