MinION Nanopore Sequencing Enables Correlation between Resistome Phenotype and Genotype of Coliform Bacteria in Municipal Sewage

Wastewater treatment plants (WWTPs) functioned as the intersection between the human society and nature environment, are receiving increasingly more attention on risk assessment of the acquisition of environmental antibiotic resistance genes (ARGs) by pathogenetic populations during treatment. However, because of the general lack of robust resistome profiling methods, genotype, and resistance phenotype is still poorly correlated in human pathogens of sewage samples. Here we applied MinION sequencing to quantify the resistance genes of multiple antibiotic resistant (MAR) coliform bacteria, a common indicator for human enteric pathogens in sewage samples. Our pipeline could deliver the results within 30 h from sample collection and the resistome quantification was consistent to that based on the Illumina platform. Additionally, the long nanopore reads not only enabled a simultaneous identification of the carrier populations of ARGs detected, but also facilitated the genome reconstruction of a representative MAR strain, from which we identified an instance of chromosomal integration of environmental resistance gene obtained by plasmid exchange with a porcine pathogen. This study demonstrated the utilization of MinION sequencing in quick monitoring and simultaneous phylogenetic tracking of environmental ARGs to address potential health risk associated with them.

reaction buffer into the sheared DNA solution and then incubating at room temperature for 20 min. The end repaired DNA fragments were then purified by adding 100 μl resuspended AMPure XP beads (Beckman Coulter Inc., cat. no. A63880) at room temperature and mixing by hand rotation for 5 minutes. The DNA binding beads were pelleted on magnet (Invitrogen MagnaRack, cat. no. CS15000) and washed twice with 200μl fresh 70% ethanol without disturbing the pellet.
DNA was eluted in 26 μl of nuclease-free water after the washing. Next, the dA-tailing was then performed using NEBNext dA-Talling Module (New England BioLabs, cat. no. E6054A) in a total volume of 31μl by adding 3μl buffer and 2μl enzyme to the clean DNA. The system was then mixed by inversion and incubated at 37 ℃ for 10 min. The library was purified for another time using the AMPure XP beads and eluted with 31 μl nuclease-free water. A ligation reaction was then performed by adding 50 μl Blunt/TA DNA ligase (New England BioLabs M0367L), 10 μl adaptor mix, 10 μl HP adaptor into the dA-tailed DNA. The reaction was gently pipetted up and down to mix and incubated at room temperature for 10 min. The adapted-ligated DNA was purified using the prepared MyOne C1 beads (Invitrogen Life Technologies) and finally eluted with 25μl elution buffer.

MinION sequencing
During the last half-hour of library preparation, MinION device was connected to the computer via USB3. After inserting flow cell (MAP005 Chemistry) into the MinION, a flow cell quality control protocol was run to assess pore activity: 180 single pores were detected for the flow cell used. Next, flow cell was equilibrated by pipetting two aliquots of 500 μl priming buffer (prepared by: 500 μl running buffer, 475 μl nuclease-free water and 27 μl fuel mix) into the flow cell, incubating for 10 min after each addition. Immediately after the library preparation, 6 μl of the prepared library was mixed with 75 μl running buffer, 65 μl nuclease-free water and 3 μl fuel mix in a fresh tube. The mixed sequencing library was gentle loaded into the flow cell via sample port using a vertical 1000 μl pipette and tip in a continuous flow avoiding introducing air bubbles or disturbing the sensor array. The 24 h sequencing protocol was selected for sequencing.
PCR system and condition used for close the circular structure of NODE5 and NODE7 The 50 μl PCR system included: 25 μl of Takara reaction mix, 1 μl extracted plasmid, 1.5 μl (10 μM each) primer mix and 21μl of nuclease free water. PCR condition were: initial denaturation at 95 ℃ for 2 min, followed by 35 cycles of (denaturation at 95 ℃ for 30 s, annealing at 55 ℃ for 30 s, and extension at 72 ℃ for 4 min), and then final extension at 72 ℃ for 10 min. The circular structure of NODE5 and NODE7 could be confirmed as PCR products of 7kb and 5kb were respectively amplified by the designed primers.

Quality-control of Illumina reads
Short paired-end reads delivered by Illumina sequencing (Q20 ≥ 85% and Q30 ≥ 80%) were trimmed with the following parameters: (1) remove reads with adapter, (2) remove reads containing N in more than 5% of the bases, (3) remove reads with Q ≤ 19 base percentage larger than 15%. 2.6% of raw reads were filtered during the quality control step resulting in 28,963,774 clean reads.  1) Only genera taking more than 1% of the coliform community are shown 2) Percentage within the 852 Nanopore reads that could be phylogenetic assigned at genus level.   Figure S3. Box-and-whiskers plot of the z-score for inserted (left) and deleted (right) k-mers of 3-6 bp in length, grouped by the proportion of GC content. A higher value of zscore indicates higher-than-expected tendency for insertion or deletion.   Figure S8 BRIG mapping of COL1 genome to reference genomes. Reference genomes were selected if they showed closest match to 16S rRNA genes (Shigella flexneri 2a, Shigella boydii sb227) or plasmid sequences of COL1 strain (E. coli PCN033, E coli SF468 and E fergusonii ATCC35469). The inner circle represents the five chromosomal contigs of COL1 genome with the same color scheme to that of Figure 1. Genomic positions of ARGs were highlighted in the out most circle.