NextSeq 500 paired end sequencing; DNA sequencing of human blood sample for Clonal Hematopoiesis of Indeterminate Potential (CHIP) detection.
Blood samplings were performed from an antecubital vein using a 21-gauge needle or from the central venous line. Blood was collected in the early morning at least 12 hours after the last administration of anticoagulant drugs. The first 2 to 4 mL of blood were discarded, and the remaining was collected in EDTA tubes aliquoted and immediately stored at -80 Â°C. DNA was extracted from whole blood samples with DNeasy Blood & Tissue Kit (Qiagen), according to the manufacturer's instructions. Libraries were prepared using SureSelectXT Low Input Target Enrichment System for Illumina Paired-End Multiplexed Sequencing Library protocol (Agilent technologies), starting from 200 ng of gDNA and according to manufacturers instructions. Hybridization capture of DNA libraries was performed through a custom RNA probes panel designed to enrich the 9 genes commonly mutated in CHIP. To improve the identification of low allele frequency variants and exclude PCR duplicates, 10-bp unique molecular identifiers (UMIs) were incorporated into each DNA fragment during library preparation. Libraries quality and size distribution were assessed on Agilent 2200 TapeStation System (Agilent Technologies) with the DNA1000 Kit.