Simultaneous multiplexed amplicon sequencing and transcriptome profiling in single cells.

Area of Science:

• Molecular Biology
• Genomics
• Virology

Background:

• Single-cell sequencing technologies are crucial for understanding cellular heterogeneity.
• Analyzing viral RNA and host responses simultaneously presents technical challenges.
• Characterizing B cell receptor (BCR) repertoires alongside transcriptomes requires integrated methods.

Purpose of the Study:

• To introduce and validate droplet-assisted RNA targeting by single-cell sequencing (DART-seq).
• To demonstrate DART-seq's capability for multiplexed analysis of viral and host nucleic acids.
• To showcase DART-seq for simultaneous profiling of B lymphocyte antibody genes and transcriptomes.

Main Methods:

• Development of DART-seq, a droplet-based microfluidic platform.
• Application of DART-seq for simultaneous transcriptome profiling and amplicon sequencing.
• Targeting non-A-tailed viral transcripts and host cell mRNA in infected cells.
• Simultaneous determination of natively paired heavy and light chain variable region amplicons in B lymphocytes.

Main Results:

• DART-seq successfully enabled multiplexed amplicon sequencing and transcriptome profiling in single cells.
• Simultaneous characterization of segmented dsRNA virus non-A-tailed transcripts and host cell transcriptome was achieved.
• Natively paired variable region heavy and light chain amplicons and the transcriptome of B lymphocytes were simultaneously determined.

Conclusions:

• DART-seq is a versatile technology for comprehensive single-cell multi-omic analysis.
• The method facilitates simultaneous investigation of viral infections and host responses at the single-cell level.
• DART-seq provides a powerful tool for B cell repertoire analysis and immune profiling.