Multi-seq for Advanced Sample Multiplexing in Single-Cell RNA

Single-cell RNA sequencing (scRNA-seq) has transformed our understanding of cellular diversity, but traditional methods can be costly and inefficient when processing multiple samples. Multi-seq enables sample multiplexing by labeling cells with unique nucleotide barcodes before sequencing, reducing costs while preserving data integrity.

This method enhances scalability and minimizes batch effects, making it valuable for large-scale studies. Understanding barcode synthesis, attachment to cells, and incorporation into sequencing libraries is essential for accurate data interpretation.

Nucleotide Barcodes And Their Synthesis

Multi-seq relies on precisely designed nucleotide barcodes that serve as unique molecular identifiers. These short, synthetic oligonucleotides must balance specificity and diversity to prevent misassignment while remaining compatible with high-throughput sequencing platforms. Barcode sequences are curated to minimize similarity, reducing the risk of cross-contamination.

Synthesis typically employs solid-phase oligonucleotide synthesis, allowing precise control over sequence composition. This process sequentially adds nucleotide monomers using phosphoramidite chemistry for high fidelity. Advances such as microarray-based oligonucleotide production and enzymatic DNA synthesis have improved scalability and cost-effectiveness, generating large barcode libraries with minimal sequence errors.

Chemical modifications enhance barcode stability and functionality. Phosphorothioate linkages increase resistance to nuclease degradation, while unique molecular identifiers (UMIs) help correct amplification biases, improving cell identification accuracy. Computational modeling and empirical validation ensure barcode sequences perform optimally in complex biological samples.

Barcode Attachment To Cells

Effective barcode integration ensures accurate sample identification. One widely used approach involves lipid-modified oligonucleotides, which use hydrophobic anchors like cholesterol or palmitic acid to insert into the cell membrane without compromising viability. This non-invasive strategy maintains high labeling efficiency.

Barcode stability is critical, particularly during washing and processing. Cholesterol-modified barcodes exhibit superior retention due to their preferential partitioning into lipid rafts, specialized membrane microdomains that enhance stability. Polyethylene glycol (PEG) spacers reduce steric hindrance, improving barcode incorporation without interfering with cellular processes.

Covalent attachment methods further enhance barcode retention. Click chemistry, specifically strain-promoted azide-alkyne cycloaddition (SPAAC), creates stable covalent bonds between barcodes and cell surface proteins, ensuring irreversible labeling. Another strategy employs enzymatic ligation, leveraging natural glycosylation pathways to attach barcodes to cell-surface glycans, though efficiency varies by cell type.

Single-Cell Sequencing Library Generation

Generating a high-quality single-cell sequencing library requires precise molecular handling to capture each cell’s transcriptome while preserving sample multiplexing. After labeling, cells are lysed to release RNA, which serves as the template for library preparation. Specialized reverse transcription primers anneal to polyadenylated mRNA, incorporating UMIs and sample-specific barcodes for accurate gene expression quantification.

During polymerase chain reaction (PCR) amplification, excessive cycles can introduce artifacts and distort gene expression profiles. High-fidelity enzymes enhance efficiency while minimizing errors. UMIs enable computational correction of amplification biases, ensuring sequencing libraries accurately reflect the original transcriptome.

Library complexity is refined through fragmentation and adapter ligation, preparing cDNA fragments for high-throughput sequencing platforms like Illumina or 10x Genomics. Controlled fragmentation generates optimal insert lengths, improving sequencing efficiency. Enzymatic fragmentation, such as tagmentation using transposases, streamlines library preparation by simultaneously fragmenting and tagging DNA. Adapter ligation incorporates platform-specific sequencing adapters, facilitating cluster generation and read assignment. Unique dual indexes enhance error correction, reducing sample misassignment risks.

Barcoded Data Interpretation

Accurate interpretation of barcoded data requires systematic demultiplexing, assigning sequencing reads to their respective samples based on unique barcodes. Computational pipelines like Cell Ranger and STARsolo efficiently match barcode sequences to corresponding cells. Error-correction models address sequencing errors and PCR artifacts by leveraging known barcode structures.

Quality control filters out low-confidence reads and barcode collisions, where distinct cells are mistakenly assigned the same barcode due to sequencing artifacts. Statistical models analyze barcode frequency distributions, flagging anomalies. Doublet detection algorithms, such as DoubletFinder and Scrublet, identify instances where two cells are mistakenly merged, refining the dataset before analysis.