Image search for the genome: the development and application of fluorescent single-molecule DNA barcodes

DNA sequence analysis is becoming increasingly affordable and accessible. Yet significant challenges remain in areas that sequencing can’t readily address. For example, the analysis of sequence-specific binding events (by drugs, transcription factors, etc) is not possible using the current DNA sequencing technologies. Imaging experiments, by contrast, can be readily multiplexed.
Here, I will describe the recent development of an approach to identify specific DNA molecules within large, complex mixtures of genomes, using fluorescence microscopy. We build on previous work showing that DNA can be fluorescently labelled at specific sequence motifs (e.g. 5’-TCGA-3’) using the DNA methyltransferase enzymes1. This chemistry can be applied as a tool to produce molecular barcodes of DNA sequence which can be rapidly read, compared and addressed to search for specific regions of genome within a mixed sample2.
In this talk, I will discuss the chemistry we have developed in order to produce these DNA maps and the development of analytical tools that allow us to search and query datasets containing gigabase pairs of genomic information. Imaging of these samples takes of the order of an hour to perform and the recorded dataset provides a permanent, memory-efficient record (~0.05 bits per basepair) of the total genomic content of a sample that can be stored and readily searched for a molecule or region of interest, retrospectively.

Left to right: Microscope image of deposited DNA barcodes; extracted barcodes; filtered and aligned barcodes (bottom traces show consensus barcode and reference barcode).

1 . M. H. Lauer, C. Vranken, J. Deen, W. Frederickx, W. Vanderlinden, N. Wand, V. Leen, M. H. Gehlen, J. Hofkens and R. K. Neely, Chem. Sci., 2017, 8, 3804–3811.
2 . N. Wand, D. A. Smith, A. Wilkinson, A. Rushton, S. J. W. Busby, I. Styles and R. K. Neely, bioRxiv, 2018, 450809.