LOTR-Seq: A protocol for large-scale simultaneous single-cell long-read genotyping of transcripts.

Area of Science:

• Genomics
• Molecular Biology
• Cancer Research

Background:

• Single-cell RNA sequencing (scRNA-seq) is crucial for understanding tumor heterogeneity.
• However, scRNA-seq has limitations in covering critical mutational hotspots.
• Accurate genotyping at the single-cell level is needed to complement transcriptomic data.

Purpose of the Study:

• To present LOTR-Seq, a new protocol for comprehensive single-cell analysis.
• To combine whole-transcriptome profiling with long-read genotyping.
• To enable detailed characterization of intra-tumoral heterogeneity, including mutations.

Main Methods:

• LOTR-Seq protocol development.
• Hematopoietic stem cell isolation from human bone marrow.
• Generation of full-length single-cell barcoded cDNA.
• Target enrichment and long-read sequencing for variant calling.

Main Results:

• Successful integration of short-read transcriptome analysis with long-read genotyping.
• Demonstration of the protocol's utility in analyzing transcriptional heterogeneity and mutations.
• Detailed steps provided for protocol execution.

Conclusions:

• LOTR-Seq offers a powerful approach to overcome limitations of standard scRNA-seq.
• This method provides a more complete picture of intra-tumoral heterogeneity.
• LOTR-Seq facilitates deeper insights into cancer biology and potential therapeutic targets.