Complete characterization of the human immune cell transcriptome using accurate full-length cDNA sequencing

Area of Science:

• Genomics
• Immunology
• Molecular Biology

Background:

• The human immune system relies on diverse transcripts, including Human Leukocyte Antigen (HLA) and B cell/T cell receptors (BCR/TCR).
• Accurate HLA typing is crucial for transplant compatibility, while BCR/TCR repertoires offer health insights.
• Current sequencing methods for HLA typing and adaptive immune repertoire sequencing (AIRR-seq) are limited by incomplete knowledge of genetic diversity.

Purpose of the Study:

• To develop a deep and accurate full-length cDNA sequencing method.
• To improve high-resolution HLA typing and BCR/TCR repertoire analysis.
• To enable untargeted analysis of immune system genetic diversity.

Main Methods:

• Generated over 10,000,000 full-length cDNA sequences using nanopore sequencing and Rolling Circle Amplification to Concatemeric Consensus (R2C2) protocol.
• Achieved a median accuracy of 97.9% for the generated sequences.
• Applied the data for isoform-level transcriptome analysis, HLA allele sequencing, and AIRR data extraction.

Main Results:

• Demonstrated the utility of deep, accurate full-length cDNA sequencing for transcriptome analysis across thousands of loci.
• Generated accurate sequences for numerous HLA alleles, enhancing HLA typing capabilities.
• Extracted detailed adaptive immune repertoire sequencing (AIRR) data, facilitating adaptive immune system analysis.

Conclusions:

• The R2C2 protocol provides deep and accurate full-length cDNA sequencing, suitable for comprehensive transcriptome and immune repertoire analysis.
• The developed untargeted approaches for HLA and AIRR analysis overcome limitations of prior methods by not requiring prior knowledge of genetic diversity.
• This method significantly advances the study of immune system diversity and its clinical applications, such as transplantation and diagnostics.