Efficient in situ barcode sequencing using padlock probe-based BaristaSeq

Abstract

Cellular DNA/RNA tags (barcodes) allow for multiplexed cell lineage tracing and neuronal projection mapping with cellular resolution. Conventional approaches to reading out cellular barcodes trade off spatial resolution with throughput. Bulk sequencing achieves high throughput but sacrifices spatial resolution, whereas manual cell picking has low throughput. In situ sequencing could potentially achieve both high spatial resolution and high throughput, but current in situ sequencing techniques are inefficient at reading out cellular barcodes. Here we describe BaristaSeq, an optimization of a targeted, padlock probe-based technique for in situ barcode sequencing compatible with Illumina sequencing chemistry. BaristaSeq results in a five-fold increase in amplification efficiency, with a sequencing accuracy of at least 97%. BaristaSeq could be used for barcode-assisted lineage tracing, and to map long-range neuronal projections.\n\nKey PointsO_LIIn situ sequencing by gap-filling padlock probes is limited by the strand displacement of DNA polymerases\nC_LIO_LIIllumina sequencing chemistry offers superior signal-to-noise ratio in situ compared to sequencing by ligation\nC_LIO_LIBaristaSeq as an accurate method for barcode sequencing in situ with improved gap-filling efficiency\nC_LI