ABSTRACT
Aerobic granular sludge (AGS) consists of a microbial consortium that has an important role in wastewater treatment. This study investigates AGS microorganisms cultivated in a laboratory-scale sequencing batch reactor. Metagenomic sequencing was conducted using PacBio and Illumina, resulting in 759 metagenome-assembled genomes, 331 of which remained after dereplication.
KEYWORDS: biofilms, aerobic granular sludge, metagenome-assembled genomes, wastewater, PacBio, accumulibacter, illumina
ANNOUNCEMENT
Aerobic granular sludge (AGS) is a promising wastewater treatment process for efficient biological carbon, nitrogen, and phosphorus removal (1). This study collected activated sludge from a wastewater treatment plant (ARA Thunersee, Switzerland) and cultivated it with different carbon source mixtures in a laboratory-scale sequential batch reactor (2).
Sample collection, DNA extraction, and sequencing protocols have previously been described in detail (3, 4). Briefly, biomass was collected on four different days and for each day, two DNA extractions were carried out, and used for Illumina (extraction A and B) and PacBio sequencing (extraction B) (Table 1). DNA “extraction (A)” was based on a customized CTAB protocol, while “extraction (B)” used the Maxwell 16 Tissue DNA Purification Kit (3, 4). Illumina samples were processed at the University of Lausanne, including library preparation and multiplex sequencing, using the HiSeq 2500 platform with paired-end mode and read lengths of 2 × 100 bp (4). PacBio libraries, generated with the SMRTbell Template Prep Kit 1, underwent size-based selection using the Blue Pippin system (Sage Science, MA, USA). This involved choosing DNA molecules between 7 and 10 Kb size as per the methodology (4). Sequenced samples (Table 1) were processed using two workflows, referred to as “workflow A” and “workflow B” with default parameters of software unless specified otherwise.
TABLE 1.
Twelve metagenomic samples with accessions, sequencing technology, DNA extraction, sampling day, carbon substrates, sequenced reads, and assembly statistics
| Accessiona | Sequencing technologyb | DNA extractionc | Dayd | Carbon susbstratese | Number of readsf | Average read lengthg | Assemblies size (Mbp)h | Assemblies (N50)i | Assemblies (number of contigs)j | |||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| ERR5621421 | Illumina | A | 71 | Volatile fatty acids | 61,579,556 | 101 | 260 | 258 | 28,719 | 29,682 | 19,427 | 19,361 |
| ERR5621422 | Illumina | B | 51,182,028 | 101 | ||||||||
| ERR5621427 | PacBio | A | 1,201,009 | 5,911 | 69 | 65,018 | 1,484 | |||||
| ERR5621419 | Illumina | A | 322 | Complex monomeric | 60,911,008 | 101 | 260 | 257 | 18,714 | 19,478 | 24,455 | 24,064 |
| ERR5621420 | Illumina | B | 49,723,068 | 101 | ||||||||
| ERR5621428 | PacBio | A | 775,085 | 4,238 | 30 | 76,706 | 548 | |||||
| ERR5621423 | Illumina | A | 427 | Complex monomeric | 56,540,510 | 101 | 360 | 358 | 17,470 | 17,265 | 32,953 | 33,060 |
| ERR5621424 | Illumina | B | 49,921,504 | 101 | ||||||||
| ERR5621429 | PacBio | A | 1,329,387 | 5,286 | 76 | 55,509 | 1,997 | |||||
| ERR5621425 | Illumina | A | 740 | Complex polymeric | 62,194,586 | 101 | 497 | 493 | 24,172 | 23,187 | 37,625 | 38,376 |
| ERR5621426 | Illumina | B | 57,070,578 | 101 | ||||||||
| ERR5621430 | PacBio | A | 1,149,988 | 7,878 | 124 | 211,407 | 1,299 | |||||
Accession numbers with download links associated with BioProject accession number PRJEB38840.
Type of metagenomic sequencing technology: Illumina short reads and PacBio long reads.
DNA extraction method.
Day of sampling.
Type of carbon source mixtures.
Number of Illumina and PacBio reads calculated using the SeqKit.
Lengths of Illumina and PacBio reads calculated using the SeqKit.
Size of metagenomic assemblies were calculated using BBMap. The minimum contig length was 2,500 bp.
N50 values of the metagenomic assemblies were calculated using BBMap. The minimum contig length was 2,500 bp.
The number of contigs in the metagenomic assemblies was determined using BBMap with a minimum contig length of 2,500 bp.
Assembly and binning methods for “workflow A” have previously been published (3, 4), which reports six high-quality Accumulibacter-related MAGs, indicating high microdiversity within this genus (4). In “workflow A”, Illumina short reads were trimmed and filtered for quality using Trimmomatic (v 0.36) (5), whereas “workflow B” employed Atlas (v 2.22) (6) and LongQC (v 1.2.0) (7) for adapter, quality trimming, and results visualization. In "workflow B", SPAdes (8) (v 3.15.5, -pacbio and -meta) obtained hybrid assemblies by incorporating all three samples for each day—one PacBio and two Illumina data sets. Binning was employed using CONCOCT (v 1.1.0) (9), MetaBAT 2 (v 2.15) (10), and MaxBin 2 (v 2.2.7) (11) with default settings in Anvi’o (v 7.1) (12), using coverage information from all 12 sequencing samples (Table 1). SeqKit (2.4.0) (13) and BBmap (v 39.01) (14) calculated the statistics on reads, assemblies, and MAGs, where N50 and L50 represent the length and count of the shortest contigs covering at least 50% of the sequences (15).
The two workflows together yielded 759 quality-controlled MAGs after using >50% completion and <10% contamination criteria in CheckM (v 1.12.1) (16). After post-dereplication using dRep (v 3.4.0, -pa 0.99, -ignoreGenomeQuality) (17), 331 MAGs remained. Before and after dereplication (Fig. 1A through J), the data sets had a median genome size, N50 value, L50 value, completion, and contamination of 3.72 and 3.35 Mbp (Size), 39.57 and 23.45 Kbp (N50), and 28 and 43 (L50), 88.47 and 74.45% (completion), and 1.17, 1.42% (contamination), respectively. Accumulibacter (n = 19), Nitrosomonas (n = 12), Propionivibrio (n = 11), Daejeonella (n = 9), and Azonexus (n = 8) were frequently occurring genera based on GTDB (Toolkit v 2.1.1, RS207) post-dereplication (18). The entire genome collection is now available for future studies investigating AGS metabolism.
Fig 1.
A summary of the MAGs collection before (n = 759) and after dereplication (n = 331) as described in two panels. It includes BBMap-based statistics and CheckM-based quality assessment. The statistics comprise (A-B) genome size and (C-F) N50 and L50 values. (G-J) Assessment of MAGs completion and contamination by CheckM. The colors indicate the different strategies described as workflows in methods.
ACKNOWLEDGMENTS
We express our sincere gratitude to the Swiss National Science Foundation (SNSF) for their generous funding of this project under grant 200021-152963. Many thanks to the NCCR microbiome consortium for their unwavering support throughout the duration of this project.
Contributor Information
Christof Holliger, Email: christof.holliger@epfl.ch.
John J. Dennehy, Department of Biology, Queens College, Queens, New York, USA
DATA AVAILABILITY
The National Center for Biotechnology Information (NCBI) database provides access to raw DNA sequences under the project PRJEB38840. These sequences are identified by accession numbers ranging from ERR5621419 to ERR5621430 as listed in Table 1. In addition, the metagenomic assemblies and MAGs collection with metadata, along with the R-script (R-studio v 2023.03.1+446) used for generating the figure, were deposited at Zenodo (10.5281/zenodo.10229272) (19).
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
The National Center for Biotechnology Information (NCBI) database provides access to raw DNA sequences under the project PRJEB38840. These sequences are identified by accession numbers ranging from ERR5621419 to ERR5621430 as listed in Table 1. In addition, the metagenomic assemblies and MAGs collection with metadata, along with the R-script (R-studio v 2023.03.1+446) used for generating the figure, were deposited at Zenodo (10.5281/zenodo.10229272) (19).