Light-Seq: Whole transcriptome sequencing of hard-to-access cells

A new spatial transcriptomics technique — a DNA nanotechnology-driven method known as "light-seq" — allows researchers to "geotag" entire RNA sequences with unique DNA barcodes unique to a handful of cells of interest
.
These target cells are selected
under the microscope through a photocrosslinking process.

With the help of DNA nanotechnology, barcoded RNA sequences are translated into coherent strands of DNA, which can then be collected from tissue samples and identified
using next-generation sequencing (NGS).
The Light-Seq process can be repeated with different barcodes for different cell populations in the same sample and left as is for subsequent analysis
.

The work was published in
the journal Nature Methods.

"Light-Seq's unique combination of features fills an unmet need: the ability to perform image information, spatial conditioning, deep sequencing analysis of hard-to-isolate cell populations or rare cell types in preserved tissues, and one-to-one correspondence of their highly refined gene expression states to spatial, morphological, and underlying disease-related features," said
Peng Yin, Ph.
D.
, a core faculty member at the Wyss Institute.

Previously, Yin's team had developed the spatial transcriptomics method SABER-FISH for direct imaging of gene expression
in intact tissue.
"With SABER-FISH, we are orders of magnitude away from capturing the complete gene expression program of the cell, because each cell has thousands of different RNA molecules
.
RNA molecules are too dense to capture them in their entirety with current imaging techniques," notes
technology developer Dr.
Jocelyn Kishi.
"Light-Seq solves this problem by combining high-resolution barcode tagging with whole transcriptome sequencing through NGS, giving us the best of both worlds and an additional key advantage
.
"

Dr.
Ninning Liu, a postdoc in Yin's team, said: "To perform specific sequencing of cells at custom-selected locations of intact tissue samples, we developed a new method to light-crosslink DNA barcoding to copies of RNA molecules, as well as a DNA nanotechnology-driven procedure to make them and their attached RNA sequences readable by NGS
.
"

First, DNA primers form "base pairs" with RNA molecules in cells and are expanded to generate cDNA
.
The DNA barcode strand containing the ultrafast photocrosslinker nucleotide is then base-paired
with the cDNA inside the cell.
When a target cell is illuminated under the microscope through a template-like optical device that places other non-target cells under the microscope in the dark, thus shielding them from the photocrosslinking
reaction.
After washing out barcoded DNA sequences that are not permanently attached in situ cells, the process can be repeated with different barcodes and light patterns to label more regions of
interest.

"To be able to integrate this barcode workflow with NGS, we designed a new splicing reaction
based on DNA nanotechnology.
This innovation allows us to convert our barcode cDNA into a continuous readout sequence
.
We can then extract the complete set of cDNA sequences containing barcodes from the sample and analyze them using standard NGS techniques," explains
Dr.
Sinem Saka.
"Ultimately, each barcode traces the complete transcriptome readout back to pre-selected cells in the tissue sample, which remain intact for subsequent analysis
.
This gives us a unique opportunity to revisit the exact same cells
after sequencing validation or further exploration.
" ”

Yin's team applied Light-Seq to a cross-section of the mouse retina to analyze three main layers
with different functions.
The researchers achieved sequence coverage comparable to single-cell sequencing methods and found that thousands of RNAs
were enriched between the three main layers of the retina.
They also showed that after sequence extraction, tissue samples remain intact and can be further imaged for proteins and other biomolecules
.
Finally, the team succeeded in isolating the intact transcriptome of retinal dopaminergic nonsecretory cells, a rare cell type that is difficult to isolate
.

"Our sequencing data clearly shows that Light-Seq can identify natural changes in
RNA structure.
Going forward, we are very interested in using Light-Seq to better understand the interactions between the immune system, disease-transmitting cells, and different therapeutic strategies such as gene and cell therapy," said Kishi, who is working with some of her co-authors to pursue a path
to commercialize Light-Seq.

Light-Seq: Light-directed in situ barcoding of biomolecules in fixed cells and tissues for spatially indexed sequencing