Full-Length Clonal Immunoglobulin Rearrangements in cfDNA: Improved Recovery and Sequencing

Abstract

Background: Circulating tumor DNA present in cell-free DNA (cfDNA) serves as diagnostic, predictive, and prognostic markers in cancer patients. In lymphoma patients, clonal immunoglobulin (cIg) sequences enable accurate identification of disease states. Despite its importance, there is sparse literature surrounding the preanalytical and analytical conditions that affect cIg detection in cfDNA. This study evaluates the effects of cfDNA isolation and next-generation sequencing (NGS) library preparation on IGH sequencing.

Methods: Pooled plasma obtained from healthy donors and plasma collected from study participants (N = 118) enrolled in cross-sectional studies were analyzed. Two cfDNA isolation methods, paramagnetic bead and spin-column, were evaluated with plasma volume being variable. Two NGS library preparation procedures, single and dual library purification, were evaluated with cfDNA input being variable.

Results: Paramagnetic bead-based cfDNA isolation improved recovery of cfDNA when compared to the column-based method. Higher purity of cfDNA resulted in more reproducible detection of cIg sequences. NGS library conditions affected IGH sequencing read depth, read diversity, and enrichment for full-length immunoglobulin sequences. A modified library procedure consisting of repeat library purifications improved the read depth of short and intermediate length targets independent of cfDNA input.

Conclusions: This is the first study to evaluate preanalytical and analytical factors that impact immunoglobulin sequencing in cfDNA. Our findings show optimal cfDNA isolation and library preparation procedures can improve the detection of clonal immunoglobulin rearrangements.