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

Rena R Xian1,2, Prisca Mbonu1, Lisa M Haley1

  • 1Department of Pathology, Johns Hopkins School of Medicine, Baltimore, MD, United States.

Clinical Chemistry
|May 26, 2025
PubMed
Summary
This summary is machine-generated.

Optimizing cell-free DNA (cfDNA) isolation and library preparation enhances the detection of clonal immunoglobulin (cIg) sequences in lymphoma patients. This study identifies key preanalytical and analytical factors for improved IGH sequencing in cfDNA.

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Area of Science:

  • Molecular Oncology
  • Genomics
  • Biotechnology

Background:

  • Circulating tumor DNA (ctDNA) in cell-free DNA (cfDNA) are vital biomarkers in oncology.
  • Clonal immunoglobulin (cIg) sequences in cfDNA are crucial for lymphoma diagnosis and monitoring.
  • Limited data exists on preanalytical and analytical variables impacting cIg detection in cfDNA.

Purpose of the Study:

  • To investigate the impact of cfDNA isolation and next-generation sequencing (NGS) library preparation on IGH sequencing.
  • To identify optimal protocols for enhanced cIg detection in cfDNA.

Main Methods:

  • Evaluated two cfDNA isolation methods (paramagnetic bead vs. spin-column) using variable plasma volumes.
  • Assessed two NGS library preparation procedures (single vs. dual purification) with variable cfDNA input.
  • Analyzed plasma from 118 healthy donors and lymphoma patients.

Main Results:

  • Paramagnetic bead isolation yielded higher cfDNA recovery than spin-column.
  • Increased cfDNA purity correlated with reproducible cIg sequence detection.
  • NGS library preparation influenced IGH sequencing metrics, including read depth and diversity.
  • A modified dual library purification improved target read depth.

Conclusions:

  • This study is the first to systematically evaluate factors affecting immunoglobulin sequencing in cfDNA.
  • Optimized cfDNA isolation and library preparation significantly improve the detection of clonal immunoglobulin rearrangements.