Qubit dsdna br assay system

We purified all the amplicons using the Agencourt AMPure XP PCR Purification systems (Beckman Coulter, Pasadena, CA) and quantified the starting DNA library using the Qubit dsDNA BR Assay system (Invitrogen, Carlsbad, CA). The sequencing library construction was performed according to the Nextera XT sample preparation guide (Illumina, San Diego, CA) that uses transposome to fragment and simultaneously adds adapter and barcoding sequences.
The pooled and barcoded libraries were subsequently sequenced using the MiSeq sequencer with v2 kits, which generated 250-base paired-end sequence reads.

After visualization on the agarose gel, the LR-PCR fragments obtained were purified with Exo-Sap (Affymetrix, Santa Clara, USA) before they were quantified using Qubit dsDNA BR Assay System (Invitrogen, Carlsbad, USA) on the Qubit® 2.0 Fluorometer (Thermo Fisher, Waltham, USA).
In the next step, 27 amplicons of each patient were pooled in equimolar ratios. According to the manufacturer’s protocol, 1 ng of the pooled DNA fragments was subjected to the Nextera XT procedure (Illumina) using transposome technology. Finally, using Nextera XT DNA Sample Preparation Kit (Illumina) and Nextera^® XT Index Kit (96) (Illumina), we obtained eighty-seven both-side indexed DNA libraries ready for high-throughput sequencing. Quality control of the libraries was performed on TapeStation 2200 Instrument (Agilent, Santa Clara, USA) using an HS D1000 ScreenTape (Agilent). Normalization of all libraries was carried out with magnetic beads, according to the Nextera XT procedure.

In the study, the use of the Human Myeloid Neoplasm QIAseq Targeted DNA Panel was preferred as the NGS panel test. This panel encompasses 141 genes commonly mutated in myeloid neoplasms. It focuses on the most relevant variants in myeloid neoplasms by incorporating compiled databases such as Cancer Gene Census and Catalogue of Somatic Mutations in Cancer (COSMIC), as well as scientific networks including the Cancer Genome Atlas (TCGA) and the latest whole genome/exome sequencing studies [8 (link)]. An NGS panel test (Myeloid Neoplasms Panel, DHS-003Z, Qiagen, Hilden, Germany) (Table 2) was performed following the manufacturer’s protocol. Following DNA isolation, the NGS workflow continued with target enrichment, library preparation, template preparation, sequencing, variant calling, variant classification, and interpretation. Quality control of the prepared libraries was performed with the Qubit dsDNA BR Assay system (Invitrogen, Carlsbad, CA, USA). Sequencing was performed using a MiniSEQ High Output Reagent Cartridge (Illumina, Inc., San Diego, CA, USA) on a MiniSEQ NGS platform (MiniSEQ, MN00676, Illumina, Singapore).