# Characterization of Chronic Lymphocytic Leukemia Immunoglobulin Rearrangements from Partial Read Sequencing

## TL;DR

This paper introduces a new, cost-effective sequencing method to analyze immunoglobulin rearrangements in chronic lymphocytic leukemia, enabling faster and accurate determination of mutational status and B-cell clones.

## Contribution

A novel, low-cost sequencing strategy for IGH locus characterization in CLL using shorter reads and a clone-centered analysis pipeline.

## Key findings

- The method enables accurate clonality and mutational status determination using MiSeq 150 × 2 sequencing.
- Validated on 319 CLL patients and 47 healthy donors, showing reliable results compared to Sanger sequencing.
- The clone-centered consensus strategy overcomes limitations of traditional Sanger sequencing for IGHV mutational status.

## Abstract

The determination of the mutational status in the immunoglobulin variable region is an established prognostic biomarker for chronic lymphocytic leukemia (CLL). The length and inner variability of the variable, diversity, and joining (VDJ) rearranged sequences compromise B-cell clone characterization using next-generation sequencing (NGS), and a standardization is needed to adapt the procedure to the current clinical guidelines. Here, we develop a complete strategy for sequencing the variable domain of the immunoglobulin heavy chain (IGH) locus with a simple, low-cost, and efficient method that enables sequencing using shorter reads (MiSeq 150 × 2), allowing for faster results. Clonality and mutational status determination are performed within the same analysis pipeline. We tested and validated the method using 319 CLL patients previously diagnosed with IGH locus characterized using Sanger sequencing, along with 47 healthy donor samples. The analysis method follows a clone-centered consensus sequence strategy to identify B-cell clones and establish a clonal threshold specific for each patient’s clonality profile, thereby overcoming the limitations of Sanger sequencing which is the gold standard used for determining immunoglobulin heavy variable (IGHV) mutational status.

Graphical Abstract