Sequel 2 sequencing 2.0 kit

Plasma DNA was extracted from 1.2–4 mL of human plasma and 1.1–1.4 mL of pooled mice plasma with the QIAamp circulating nucleic acid kit (Qiagen) according to the manufacturer's instructions. The SMRTbell express template prep kit 2.0 (PacBio) was used for the library preparation of plasma DNA. Briefly, DNA molecules were ligated with hairpin adaptors to form a circularized template. Sequencing primer v4 was annealed to the sequencing template, followed by binding of polymerase to templates using a Sequel II binding kit 2.1 and internal control kit 1.0 (PacBio). SMRT cell 8M was used for sequencing with a Sequel II sequencing 2.0 kit (PacBio), and sequencing movies were collected for 30 h. Nanopore library preparation and sequencing of pregnant samples are as described previously (Yu et al. 2023b (link)). For healthy samples, nanopore libraries were prepared using the native barcoding kit (ONT SQK-NBD114.96) according to the manufacturer's instructions except that a bead-to-sample ratio of three was used in all clean-up steps using AMPure XP beads with prolonged incubation time for DNA repair, end-prep, barcode, and adapter ligation. Short fragment buffer, which retained DNA fragments of all sizes, was used in adapter ligation. Each library was loaded onto a PromethION flow cell R10.4.1 and sequenced on a PromethION device for 72 h.

For CCS library preparation, ≥3 ug of high molecular weight genomic DNA (more than 50% of fragments ≥40 kb) was sheared to ~15 kb using the Megaruptor 3 (Diagenode B06010003), followed by DNA repair and ligation of PacBio adapters using the PacBio SMRTbell Express Template Prep Kit 2.0 (100-938-900) and removal of incomplete ligation products with the SMRTbell Enzyme Clean Up Kit 2.0 (PacBio 101-938-500). Libraries were then size-selected for 15 kb +/− 20% using the PippinHT with 0.75% agarose cassettes (Sage Science). Following quantification with the Qubit dsDNA High Sensitivity assay (Thermo Q32854), libraries were diluted to 60 pM per SMRT cell, hybridized with PacBio V5 sequencing primer, and bound with SMRT seq polymerase using Sequel II Binding Kit 2.2 (PacBio 101-908-100). CCS sequencing was performed on the Sequel iIe instrument using 8M SMRT Cells (101-389-001) and Sequel II Sequencing 2.0 Kit (101-820-200), utilizing PacBio’s adaptive loading feature with a 2 hour pre-extension time and 30 hour movie time per SMRT cell. Initial quality filtering, basecalling, adapter marking, and CCS error correction was done automatically on board the Sequel iIe.