Detection and Characterization of Clonal Hematopoiesis.

Clonal hematopoiesis (CH) is the age-related expansion of hematopoietic stem cell clones resulting from the acquisition of somatic point mutations or mosaic chromosomal alterations (mCAs). It is linked to adverse systemic effects, including hematologic malignancies, cardiovascular diseases, metabolic disorders, as well as liver and kidney ailments, ultimately contributing to elevated overall mortality.Given its diverse biological and clinical implications, the identification of clonal hematopoiesis holds significance in various contexts. While traditionally centered on mutations associated with myeloid malignancies, stem/progenitor cell involvement has been documented for various lymphoid malignancies, including T-cell lymphoma, chronic lymphocytic leukemia (CLL), and follicular lymphoma (FL). Lymphoid CH (L-CH) involves a broader spectrum of genes and occurs at a lower prevalence, resulting in reduced mutation prevalences per gene. This characteristic poses challenges for efficient CH detection.The major strategies to identify CH are whole exome sequencing (WES), whole genome sequencing (WGS), or targeted sequencing. Targeted sequencing allows for much higher sequencing depth compared to WES and WGS because of the focus on genes known to be associated with CH and therefore allows detecting potential variants at low frequencies with high precision. Here, we describe an error-corrected targeted sequencing approach for detection of CH in bone marrow (BM) or peripheral blood (PB) samples, which we have successfully established and used in various cohorts. This protocol includes the process of DNA isolation from PB and BM samples, library preparation with molecular tags including quality control steps and computational analysis including variant filtering.