Multiplex oligos kit

DNA of individual isolates, the mock community, and the human metagenome were subjected to both Illumina short-read and nanopore long-read sequencing. Illumina paired-end libraries were prepared by the focused acoustic shearing method with the NEBNext Ultra DNA Library Prep Kit and the Multiplex Oligos Kit for Illumina (NEB) (Li et al., 2018 ). The libraries were quantified by employing quantitative PCR with P5-P7 primers, and were pooled together and sequenced in the HiSeq X10 platform according to the protocol of the manufacturer (Illumina, San Diego, CA, United States). After read trimming and removal of the human reads, a total of 26 Gb 2 × 150 bp pair-end sequencing data was generated by the Illumina HiSeq X10 apparatus. Libraries of nanopore long-read sequencing were prepared with the Rapid Barcoding Sequencing Kit (SQK-RBK004) and flowcell R9.4 according to the protocols of the manufacturer. The sequencing run was stopped after 8 h, and the flow cell was washed by a Wash Kit (EXP-WSH002) (Li et al., 2018 ).

To obtain high-quality short read data, paired-end (2 × 150 bp) libraries were prepared by the focused acoustic shearing method with the NEBNext Ultra DNA Library Prep Kit and the Multiplex Oligos Kit for Illumina (NEB). The libraries were quantified by employing quantitative PCR with P5-P7 primers, and they were pooled together and sequenced on the NextSeq 500 platform according to the manufacturer's protocol (Illumina).