Ultrasensitive DNA Immune Repertoire Sequencing Using Unique Molecular Identifiers

Abstract

Background Immune repertoire sequencing of the T-cell receptor can identify clonotypes that have expanded as a result of antigen recognition or hematological malignancies. However, current sequencing protocols display limitations with nonuniform amplification and polymerase-induced errors during sequencing. Here, we developed a sequencing method that overcame these issues and applied it to γδ T cells, a cell type that plays a unique role in immunity, autoimmunity, homeostasis of intestine, skin, adipose tissue, and cancer biology. Methods The ultrasensitive immune repertoire sequencing method used PCR-introduced unique molecular identifiers. We constructed a 32-panel assay that captured the full diversity of the recombined T-cell receptor delta loci in γδ T cells. The protocol was validated on synthetic reference molecules and blood samples of healthy individuals. Results The 32-panel assay displayed wide dynamic range, high reproducibility, and analytical sensitivity with single-nucleotide resolution. The method corrected for sequencing-depended quantification bias and polymerase-induced errors and could be applied to both enriched and nonenriched cells. Healthy donors displayed oligoclonal expansion of γδ T cells and similar frequencies of clonotypes were detected in both enrichment and nonenriched samples. Conclusions Ultrasensitive immune repertoire sequencing strategy enables quantification of individual and specific clonotypes in a background that can be applied to clinical as well as basic application areas. Our approach is simple, flexible, and can easily be implemented in any molecular laboratory.

Counting unique molecular identifiers in sequencing using a multitype branching process with immigration

“…The number of approaches and applications that use UMIs in sequencing is rapidly increasing. In cancer diagnostics, the use of UMIs is crucial since it allows to correct for both polymerase induced errors and amplification biases [10]. Many sample types and matrices are challenging to analyse due to limited amounts of DNA and enzymatic inhibitors.…”

Ultrasensitive circulating tumor DNA analysis enables precision medicine: experimental workflow considerations

“…These molecular barcodes are random sequences, usually between four and fourteen nucleotides long, that enable tracking of all sequencing reads back to the original DNA molecule. Subsequently, unique molecular identifiers allow for correction of both DNA polymerase-induced errors [133] and uneven amplification [134] when used in combination with deep sequencing [135,136]. The requirement for deep sequencing limits ultrasensitive sequencing to targeted approaches due to costs.…”

Dual UMIs and Dual Barcodes With Minimal PCR Amplification Removes Artifacts and Acquires Accurate Antibody Repertoire

“…In addition to inter-and intra-sample chimeras, another important factor that impedes Rep-seq applications is base errors introduced by PCR and HTS (28,34,(36)(37)(38). These base errors can be eliminated by taking consensus sequences (34,36).…”

“…It's reported that the substitution errors for amplicon sequencing have been greatly corrected by using quality score combined with Hamming graph and read overlapping (31). Moreover, amplification biases have been largely addressed by the introduction of unique molecular identifiers (UMIs), the random-tandem sequences with huge diversity, during reverse transcription (RT), thus subsequent PCR amplification of each cDNA molecule can be quantified and corrected by grouping antibodies based on UMIs or UMI pairs and subsequent consensus sequence building (28,29,(32)(33)(34)(35)(36)(37)(38). Besides, with the capability of tracking individual RNA molecules throughout PCR amplification and sequencing (33,35,39), UMIs also possess the potential for identifying and removing intra-sample chimeras.…”