We are pleased to announce the release of an updated ZymoBIOMICS Fecal Reference dataset to highlight the capabilities of Oxford Nanopore Technologies’ for metagenomic analysis. This release includes: new basecalls (including 6mA, 4mC, and 5mC modified basecalls) for the PromethION Flow Cell data previously released in our LC2024 data release; an additional PromethION Flow Cell of sequencing data from the same sample; and new analysis results highlighted in our Metagenomics Application Note.

Dataset

The dataset is available for anonymous download, without login, from a public Amazon Web Services S3 bucket. The bucket is part of the Open Data on AWS project enabling sharing and analysis of a wide range of data. The data can be downloaded with the AWS CLI command:

aws s3 sync --no-sign-request s3://ont-open-data/zymo_fecal_2025.05 zymo_fecal_2025.05

See the tutorials page for information on downloading the dataset. You can also browse and download the files in your web browser courtesy of 42basepairs.

Folder name	Size	Description
RAW	4.6 TB	POD5/Flowcell files
Basecalls	850 GB	BAM files
Analysis	1.2 TB	Analysis outputs

Sample

Attribute	Value
Sample Name	ZymoBIOMIC D6323
Organism	metagenomic community
Molecule Type	gDNA
Sample Type	inactivated cultured stool
Biological replicates	2
Flow Cell replicates	1
Link to sample source	ZymoBIOMICS Fecal Reference with TruMatrix Technology

Preparation

Sample preparation was performed according to protocols published on the main Oxford Nanopore Technologies’ website.

Attribute	Value
Extraction	QIAamp PowerFecal DNA Kit
Library Prep	Ligation sequencing kit (LSK114)
Kit	LSK114

Further preparation information such as sample storage suggestions can be found on the Oxford Nanopore website.

Sequencing

Sequence data was generated using the following configuration:

Attribute	Value
Flow Cell	FLO-PRO114M
Device	PromethION
Chemistry	R10.4.1
Basecall Model	5.0.0
MinKNOW Version	5.9.7
Modified bases	4mC_5mC, 6mA

Analysis

Analysis outputs are available. The analysis results are located in the S3 bucket under the prefix:

s3://ont-open-data/zymo_fecal_2025.05/analysis

The analysis results include metagenomic assembly contigs from MetaMDBG, metagenome binning results from SemiBin2 and MetaBAT2, consensus metagenome bins from DAS Tool, and quality metrics from CheckM2. We also include a script containing all the commands used to generate these analysis results. For further information on the analysis see the linked Applications Note.

Other software used for analysis:

Tool	Version
Dorado	v0.9.1 (HAC)
Dorado	v0.7.3 (SUP)
MetaMDBG	v1.0
SemiBin2	v2.1.0
MetaBAT2	v2.17
DAS Tool	v1.1.7
CheckM2	v1.0.2
Minimap2	v2.28

Oxford Nanopore Technologies’ provides superior metagenome-assembled genome recovery and strain-level resolution from a complex microbiome Applications Note