Promethion sequencer

Small eccDNAs were added to 5 μL of 100 μM random hexamer primers. The samples were denatured at 95°C for 5 min, followed by annealing at 50°C for 15 s, 30°C for 15 s and 20°C for 10 min, and then hold on ice for 5 min. A reaction mix was added so that the final concentrations were 1 × phi29 DNA polymerase reaction buffer, 0.2 mg/mL BSA, 2 mM dNTP and 5 U of phi29 DNA polymerase (NEB). RCA was performed at 30°C for 24 h. The products were incubated with 10 U of T7 endonuclease I (NEB) at 37°C for 30 min. The small eccDNA sequencing library was prepared using the Ligation Sequencing Kit (SQK‐LSK109) and sequenced on the PromethION sequencer (R9.4.1, all Oxford Nanopore Technologies) (Supplementary Figure S3A–C). Raw reads with quality values of ≥10 were selected to map the human genome (hg38) using the minimap2 software. Small eccDNAs were generated from the raw reads (the number of tandem repeat sequences ≥ 2) using the eccDNA_RCA_nanopore software. Briefly, the workflow of the eccDNA_RCA_nanopore software was to map the raw reads to the human genome and then call the tandem repeat sequences not present in the human genome to generate small eccDNAs.

251 Genomic DNA (gDNA) samples for Nanopore long-read sequencing were extracted from shoots of one-month-old seedlings using a QIAGEN^® Genomic DNA extraction kit (Cat #13323, QIAGEN). DNA purity was measured using a NanoDrop™ One UV-Vis spectrophotometer (Thermo Fisher Scientific, USA), which showed that OD_260/280 ranged from 1.8 to 2.0 and OD_260/230 was between 2.0 and 2.2. DNA samples were accurately quantified using a Qubit^® 3.0 Fluorometer (Invitrogen, USA). Size-selected long DNA fragments were then extracted using the BluePippin system (Sage Science, USA). DNA was then repaired and adapters were attached to the ends using an SQK-LSK109 kit. The concentrations of library fragments were quantified with the Qubit^® 3.0 Fluorometer. The DNA library was then loaded into the primed Nanopore PromethION sequencer (Oxford Nanopore Technologies, UK) flow cell. For each accession, the coverage of ONT reads was 98 ± 24× genome coverage. A total of 9.42 Tb of ONT raw reads were obtained (Supplementary information, Table S1a).

Total RNA was extracted from young leaves, flowers, young stems, and fruits of JZ and SZ. Sequencing libraries were generated using the NEBNext^® Ultra™ RNA Library Prep Kit for Illumina^® (#E7530L, NEB, USA) following the manufacturer’s recommendations. Then, the libraries were subjected to 150-bp paired-end sequencing on an Illumina platform. For Oxford Nanopore cDNA sequencing, RNA samples were pooled to construct a cDNA library and sequenced on a PromethION sequencer (Oxford Nanopore Technologies, Oxford, UK).

Leaf tissue samples were used to isolate genomic DNA with a Genomic DNA extraction kit (Qiagen, USA) according to the manufacturer’s instructions. DNA concentration and purity were determined by a Qubit 3.0 fluorometer (Invitrogen, USA) and a Nanodrop One UV-Vis spectrophotometer (Thermo Fisher Scientific, USA), respectively. DNA fragments > 30 kb were selected using BluePippin electrophoresis (Sage Science, USA), and their ends were repaired, A-tailed, and ligated with sequencing adapters provided in the SQK-LSK109 kit (Oxford Nanopore Technologies). The established DNA library was quantitatively detected by a Qubit 3.0 fluorometer (Invitrogen, USA) and sequenced on a PromethION sequencer (Oxford Nanopore Technologies, UK).
The raw subreads obtained from Oxford Nanopore sequencing were filtered by fastp v0.12.6 with default parameters [37 (link)]. The filtered data were firstly error-corrected by Nextdenovo (read_cuoff = 3k, blocksize = 2g, seed_cutoff = 25k) and then assembled by Smartdenovo (wtpre -J 3000, wtzmo -k 21 -z 10 -Z 16 -U -1 -m 0.1 -A 1000, wtclp -d 3 -k 300 -m 0.1 -FT, wtlay -w 300 -s 200 -m 0.1 -r 0.95 -c 1) (https://github.com/Nextomics/NextDenovo). The Illumina short reads generated for genome survey were mapped to the assembly with BWA [38 (link)] to further improve the base accuracy of the assembly, which was then polished three times with Pilon [39 (link)].

A total amount of 2 µg DNA per sample was used as input material for the ONT library preparations. After the qualification, the size selection of long DNA fragments was performed using the BluePippin system (Sage Science, USA). Next, the ends of DNA fragments were repaired, and a ligation reaction was conducted with the NEBNext Ultra II End Repair/dA-tailing Kit (New England Biolabs, UK). The adapter in the LSK109 kit (Oxford Nanpore Technologies, UK) was used for further ligation reaction, and Qubit® 3.0 Fluorometer (Invitrogen, USA) was used to quantify the size of the library fragments. Sequencing was then performed on a PromethION sequencer (Oxford Nanopore Technologies, UK).

One sludge sample was collected on November 15, 2018, from a well-operated one-stage PNA reactor under the same operating parameters as in our previous study [15 (link)]. Genomic DNA was extracted using the DNeasy PowerSoil Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. DNA concentration and purity were determined using a Qubit 2.0 Fluorometer (Life Technologies, Carlsbad, CA, USA) and NanoDrop 1000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA) as previously described [25 (link)]. The total DNA was divided into two equal parts, with one part sequenced by Novogene Company Limited (Beijing, China) using shotgun sequencing on an Illumina HiSeq 4000 PE150 sequencer (Illumina, CA, USA) to generate 150 bp paired-end reads with a 350-bp insert size, and the other sequenced by the Chinese University of Hong Kong (Hong Kong, China) using a Nanopore PromethION sequencer (Oxford Nanopore Technology, UK).

The blood sample used in the study were obtained from a healthy adult male participant belonging to the BKY ethnic group. The SQK-LSK109 kit from Oxford Nanopore Technologies was utilized for library construction to generate DNA libraries following the manufacturer’s standard protocol (Nanopore). The Oxford Nanopore PromethION sequencer was used to sequence the libraries, resulting in a total of 151,728,810,548 bp of raw sequencing data. The generated sequencing data was subjected to filtration based on sequence filtering criteria, with sequences exhibiting mean mass ≤ 7 being excluded, resulting in 142 Gb of data for BKY genome assembly. MGI Easy was utilized for library construction to generate DNA libraries following the manufacturer’s standard protocol (Illumina), and the high-throughput sequencing platform DNBSEQ was used to sequence the libraries, generating 150-bp pair-end reads totaling 203,476,703,400 bp.