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SPRINTseq Technology

Overview

SPRINTseq (SPatially Resolved and sIgnal-diluted Next-generation Targeted sequencing) is a rapid and signal crowdedness-robust in-situ sequencing strategy developed by the Huang Lab. It combines hybrid block coding with molecular dilution to achieve fast, high-sensitivity, high-resolution spatial transcriptomics.

Key Innovations

Hybrid Block Coding

Traditional in-situ sequencing uses a one-hot or composite color code per sequencing cycle. In dense tissues, overlapping signals ("crowdedness") corrupt base calling. SPRINTseq introduces a hybrid block code that is:

  • Crowdedness-robust: Tolerates signal overlap from neighboring rolonies
  • Error-correcting: Detects and corrects sequencing errors
  • Efficient: High decoding rate with fewer wasted code words

Signal Dilution

Physical dilution of molecular signals further counteracts crowdedness. By randomly subsampling which rolonies are read in each imaging field, SPRINTseq reduces local signal density without sacrificing global transcript recovery.

Performance

Metric Value
Gene panel 108 genes (mouse brain)
Transcripts recovered > 142 million
Cells profiled 453,843 (4 coronal slices)
Sequencing time < 9.5 hours per coronal slice
Resolution Near optical diffraction limit
Application demonstrated Cellular and subcellular molecular architecture of Alzheimer's disease

Workflow

SPRINTseq shares the same library construction workflow as PRISM (sample pre-treatment → RCA). The key difference is in the readout step:

  1. Library Construction: Standard padlock probe hybridization, ligation, and RCA (see RCA Protocol)
  2. Flow Cell Assembly: Mount slide into a microfluidic flow cell for automated reagent delivery
  3. In-situ Sequencing: Multi-cycle sequencing-by-synthesis (SBS) using two-color reversible terminator chemistry
  4. Base Calling & Decoding: Hybrid block code decoding to assign gene identity to each rolony
  5. Morphology: DAPI / WGA / H&E staining

Comparison to PRISM

Feature PRISM SPRINTseq
Readout Single-round fluorescence imaging Multi-cycle in-situ sequencing
Encoding Color-intensity barcodes (radius vector) Hybrid block coding
Multiplexing Up to 64-plex Scales with sequencing cycles
Equipment Conventional fluorescence microscope Microscope + fluidics system
Speed Fastest (one imaging round) Fast (< 9.5 h per slice)
Best for Rapid spatial profiling Deep targeted transcriptome, variant detection

Code & Data

Publication

Chang T, Han W, Jiang M, et al. Rapid and signal crowdedness-robust in situ sequencing through hybrid block coding. PNAS 120(47), e2309227120 (2023). DOI: 10.1073/pnas.2309227120

Documentation Status

Detailed SPRINTseq sequencing protocols are being digitized. The library construction steps (pre-treatment through RCA) are shared with PRISM and fully documented in the Protocols section.