Development of a sequencing system for spatial decoding of DNA barcode molecules at single-molecule resolution

Area of Science:

• Molecular Biology
• Genomics
• Biotechnology

Background:

• DNA barcodes revolutionize single-cell transcriptome analysis by enabling multiplexed cDNA library indexing.
• DNA barcodes are increasingly used in spatial transcriptomics, but single-molecule decoding remains a significant challenge for spatial resolution.

Purpose of the Study:

• To develop an in-house sequencing system capable of spatially decoding DNA barcode molecules at single-molecule resolution.
• To address the limitations in achieving high-resolution spatial identification of DNA barcodes for transcriptomic and proteomic applications.

Main Methods:

• An in-house sequencing system, inspired by the HeliScope single-molecule sequencing system, was engineered.
• The system was benchmarked using 30 types of DNA barcode molecules to assess read length and error rates.
• DNA barcode molecules conjugated to antibodies were spatially identified to demonstrate broader applicability.

Main Results:

• The developed system achieved an average read length of approximately 20 nucleotides with an error rate below 5% per nucleotide.
• The system successfully achieved single-molecule resolution for spatially identifying DNA barcode molecules.
• The method demonstrated the ability to spatially identify DNA barcode molecules bound to antibodies.

Conclusions:

• The novel sequencing system provides single-molecule resolution for spatially decoding DNA barcodes, overcoming a key challenge in spatial transcriptomics.
• The developed 'molecular foot printing' method shows significant potential for advancing both spatial transcriptomics and spatial proteomics.
• This technology offers a new tool for high-resolution molecular mapping in biological tissues.