microDNA-seq of B-ALL patient : Plasma sample at relapse, sequencing with high coverage
The eluted microDNA was used for rolling-circle amplification using degenerate 6-mer primers and Phi29 DNA polymerase. We employed an in-house protocol using oligonucleotides containing internal C3 spacers to minimize the production of self-priming by-products [17, 18]. Amplified microDNA concatemers were purified using Amicon Ultra-0.5 centrifugal filter devices (MilliporeSigma, Burlington, MA, USA) and quantified using a Qubit dsDNA BR Assay Kit (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA). Five hundred nanograms of Amplified microDNA (500 ng) was fragmented using the Illumina DNA Prep kit to generate 5/ 21 dual-indexed paired-end libraries following the Illumina DNA protocol with IDT for Illumina Unique Dual Indexes (Set D, Cat. No. 20027216). High-throughput sequencing was performed on an Illumina NextSeq 550 system using a 300-cycle mid-output kit (~100130 million paired-end 2150 bp reads)