Synthetic standards combined with error and bias correction improve the accuracy and quantitative resolution of antibody repertoire sequencing in human naïve and memory B cells

High-throughput sequencing of immunoglobulin (Ig) repertoires (Ig-seq) is a powerful method for quantitatively interrogating B cell receptor sequence diversity. When applied to human repertoires, Ig-seq provides insight into fundamental immunological questions, and can be implemented in diagnostic and drug discovery projects. However, a major challenge in Ig-seq is ensuring accuracy, as library preparation protocols and sequencing platforms can introduce substantial errors and bias that compromise immunological interpretation. Here, we have established an approach for performing highly accurate human Ig-seq by combining synthetic standards with a comprehensive error and bias correction pipeline. First, we designed a set of 85 synthetic antibody heavy-chain standards (in vitro transcribed RNA) to assess correction workflow fidelity. Next, we adapted a library preparation protocol that incorporates unique molecular identifiers (UIDs) for error and bias correction which, when applied to the synthetic standards, resulted in highly accurate data. Finally, we performed Ig-seq on purified human circulating B cell subsets (naïve and memory), combined with a cellular replicate sampling strategy. This strategy enabled robust and reliable estimation of key repertoire features such as clonotype diversity, germline segment, and isotype subclass usage, and somatic hypermutation. We anticipate that our standards and error and bias correction pipeline will become a valuable tool for researchers to validate and improve accuracy in human Ig-seq studies, thus leading to potentially new insights and applications in human antibody repertoire profiling.

Schlagwörter

Fachgebiet (DDC)

600 - Technik, Medizin, angewandte Wissenschaften

Projekt

Veranstaltung

Startdatum der Ausstellung

Enddatum der Ausstellung

Startdatum der Konferenz

Enddatum der Konferenz

Datum der letzten Prüfung

ISBN

ISSN

1664-3224

Sprache

Englisch

Während FHNW Zugehörigkeit erstellt

Nein

Zukunftsfelder FHNW

Publikationsstatus

Veröffentlicht

Begutachtung

Peer-Review der ganzen Publikation

Open Access-Status

Gold

Lizenz

Zitation

FRIEDENSOHN, Simon, John M. LINDNER, Vanessa CORNACCHIONE, Mariavittoria IAZEOLLA, Enkelejda MIHO, Andreas ZINGG, Simon MENG, Elisabetta TRAGGIAI und Sai T. REDDY, 2018. Synthetic standards combined with error and bias correction improve the accuracy and quantitative resolution of antibody repertoire sequencing in human naïve and memory B cells. Frontiers in Immunology. 20 Juni 2018. Bd. 9, S. 1401. DOI 10.3389/fimmu.2018.01401. Verfügbar unter: https://doi.org/10.26041/fhnw-9965

Komplettanzeige