Circular metagenome-assembled genome of Candidatus Patescibacteria recovered from anaerobic digestion sludge

A recent study revealed that Candidatus (Ca.) Patescibacteria functions in anaerobic digestion, interacting with methanogens (1). Understanding the role of Ca. Patescibacteria is crucial for maximizing the efficiency of methanogenesis.

Here, we present a circular metagenome-assembled genome (cMAG) of Ca. Patescibacteria, which was recovered from the sludge collected from a full-scale mesophilic food waste treatment plant in Tokyo, Japan, in 2022 (stored at −20°C until use). Genomic DNA was extracted using a FastDNA SPIN Kit for Soil (MP Biomedicals, USA). Sequencing was performed on PacBio Sequel IIe (Pacific Biosciences of California, USA) and DNBSEQ-G400 (MGI Tech, China) platforms, respectively. For PacBio sequencing, the quality of DNA extract was confirmed using the 5200 Fragment Analyzer System and Agilent HS Genomic DNA 50 kb Kit (Agilent Technologies, USA). The library was constructed using SMRTbell Express Template Prep Kit 2.0 (Pacific Biosciences of California). Sequencing polymerase was bound to SMRTbell libraries using Binding kit 2.2 (Pacific Biosciences of California). Adaptor sequences from PacBio raw sequencing data were trimmed using SMRT Link version 10.1.0.119528. The HiFi reads were generated by aligning the trimmed reads with an average quality of 20 or more using pancake with KSW2 (2–4), resulting in 157,984 reads (average: 6,957 bp). For DNBSEQ sequencing, the library was prepared by the MGIEasy FS DNA library prep set (MGI Tech, China), using the same DNA extract. The quality was confirmed using the Fragment Analyzer and dsDNA 915 Reagent Kit (Agilent Technologies). Circular DNA was constructed using a MGIEasy Circularization Kit. Subsequently, DNA nanoballs (DNBs) were constructed using a DNBSEQ-G400RS High-throughput Sequencing Kit (MGI Tech). 2  ×  200 bp paired-end sequencing was performed on DNBSEQ-G400. Trimming of adaptor sequences and quality checks were performed using Trimmomatic v. 0.39. and Fastqc v. 0.11.9 (5), respectively.

Hybrid assembly was performed by metaSPAdes v. 3.10.1 (6), and the HiFi reads were assembled with hifiasm_meta v. 0.3.1 (7). Subsequently, quickmerge v. 0.3 (8) was utilized to merge them. Coverage was calculated by mapping DNBseq reads to the assembly using Bowtie2 v. 2.4.1 (9). Binning was performed using SemiBin2 v. 1.5.1 with built-in model “wastewater” (10). The resulting bin.765 was composed of one contig, with a completeness of 100% (0% contamination), and had one copy each of the 16S rRNA, 23S rRNA, and 5S rRNA gene as reported by MDMcleaner pipeline v. 0.8.7 (11). This MAG was curated by mapping DNBseq and HiFi reads and circularized by identifying the repeat region using Repeat Finder, in Geneious Prime 2023.2.1 (https://www.geneious.com). Default parameters were used for all software unless otherwise noted.

The recovered cMAG has a size of 717,294 bp, GC content of 41%, and 40× coverage. GC skew evaluation using gc_skew.py (https://github.com/christophertbrown/iRep) (12) revealed a well-defined pattern (13) (Fig. 1). Taxonomic analysis by GTDB-Tk v. 2.3.2 (14) indicates that this cMAG belongs to Ca. Patescibacteria (NCBI taxonomy: Ca. Parcubacteria). Annotation was performed by the Prokaryotic Genome Annotation Pipeline (15). The pathway analysis by GhostKOALA (16) suggested that de novo nucleotide synthesis, amino acid synthesis, and phospholipid synthesis pathways were incomplete, similar to other Ca. Patescibacteria species (17).

This work was supported by the Project of Integrated Compost Science (PICS) and JSPS KAKENHI (grant no: 23KJ0151). We would like to thank BIOENERGY (Tokyo, Japan) for providing us the anaerobic digester sludge sample.