So181

Extracted plasmids were amplified using random hexamer primers (Thermo Scientific SO181) and ϕ29 DNA polymerase (NEB M0269L), as previously described (47 (link)), with the exception of using the commercial ϕ29 reaction buffer from NEB (B0269L). Amplification products were cleaned and concentrated using the E.Z.N.A. Cycle-Pure kit from Omega BioTek (D6492-02) and quantified using Nanodrop.
Purified DNA was then sequenced using Oxford Nanopore’s ligation sequencing chemistry (SQK-LSK109) in conjunction with native barcoding expansions (EXP-NBD104/114) for high throughput, according to the manufacturer’s recommendations. All samples were sequenced on a MinION device using R9.4.1 flow cells. Samples were demultiplexed and base called either (i) in real time with MinKNOW v4.3.20 using the fast base-calling algorithm or (ii) asynchronously with Guppy GPU v5.0.11 on Ubuntu 20.04 LTS using the fast base-calling algorithm.

One hundred nanograms of pTXB1 plasmids was firstly linearized by 10 U XbaI (NEB, R0145S) at 37 °C for 1 h and purified by Zymo columns. Then, we took 0.5-ng linearized plasmids for multiple displacement amplification (MDA), with all dTTPs replaced by dUTPs. Specifically, the 1 μl DNA was mixed with 22 μl reaction buffer containing 1× phi29 reaction buffer (NEB, M0269S), 20 μM random primers (Thermo, SO181), and 1 mM dNTP (NEB, N0446S, and N0459S), then they were incubated at 98 °C for 3 min and immediately cooled down at 4 °C for 20 min. Two microliters of phi29 DNA polymerase was added, and the amplification was carried out at 30 °C for 5 h, terminated at 65 °C for 10 min. The products were purified by Zymo columns.