This repository provides analysis code and results produced during evaluation of metagenomic sequencing (MGS) data collected through the Mosaic Standards Challenge. The Mosaic Standards Challenge asked participating laboratories analyze the same set of 7 samples using their own favored MGS laboratory methods. Each lab submitted their raw sequencing results and protocol information. The resulting MGS data was analyzed through a common bioinformatic pipeline and then evaluated to determine the effects of methodological choices.

This repository contains all submissions made to the Mosaic Standards challenge from the period of the challenge opening through December 31st, 2019. The Mosaic Standards Challenge asked the microbiome research community to participate in determining the level of variation due to wet-lab protocols by sequencing a set of samples and providing the resulting files. Each participant ordered one or more kits, where each kit contained five fecal samples and two predetermined DNA mixtures. All samples were identical across all kits; in other words, the samples labled "#1" provided to each lab were identical to each other. Participants in the challenge sequenced any number of the provided samples and provided both the raw sequencing result files, and the details of their protocol. Protocol details were provided by answering a set of pre-specified questions in a metadata spreadsheet upon submission of each sample.

This repository contains all submissions made to the Mosaic Standards challenge from the period of the challenge opening through December 31st, 2019. The Mosaic Standards Challenge asked the microbiome research community to participate in determining the level of variation due to wet-lab protocols by sequencing a set of samples and providing the resulting files. Each participant ordered one or more kits, where each kit contained five fecal samples and two predetermined DNA mixtures. All samples were identical across all kits; in other words, the samples labled "#1" provided to each lab were identical to each other. Participants in the challenge sequenced any number of the provided samples and provided both the raw sequencing result files, and the details of their protocol. Protocol details were provided by answering a set of pre-specified questions in a metadata spreadsheet upon submission of each sample.

This repository provides the raw data and analysis code used to demonstrate the implementation of a mathematical framework that uses internal standards to correct for compositionally in MGS datasets. Some of the data contained herein was previously published and is reanalyzed in the context of this new framework, while other datasets represent newly analyzed samples. Please refer to the associated publication for further discussion and a full exploration of the results.

This repository provides the raw data, analysis code, and results generated during a systematic evaluation of the impact of selected experimental protocol choices on the metagenomic sequencing analysis of microbiome samples. Briefly, a full factorial experimental design was implemented varying biological sample (n=5), operator (n=2), lot (n=2), extraction kit (n=2), 16S variable region (n=2), and reference database (n=3), and the main effects were calculated and compared between parameters (bias effects) and samples (real biological differences). A full description of the effort is provided in the associated publication.

To assess the variability of low-abundance oligonucleotide detection across sample matrices, we spiked DNA reference standards (meta sequins) into replicate wastewater DNA extracts at logarithmically decreasing mass-to-mass percentages (m/m%) and deeply sequenced them on the Illumina platform. This dataset summarizes the experimental conditions and results of the detection frequencies of those oligonucleotides as well as detailed descriptions of the DNA reference standards used. This dataset is associated with the following publication: Davis, B., P. Vikesland, and A. Pruden. Evaluating Quantitative Metagenomics for Environmental Monitoring of Antibiotic Resistance and Establishing Detection Limits. ENVIRONMENTAL SCIENCE & TECHNOLOGY. American Chemical Society, Washington, DC, USA, 59(12): 6192-6202, (2025).

The environmental samples were collected with the polyester wipes from eight different locations in the International Space Station (ISS) during two consecutive sampling sessions (three months apart) within the ISS Microbial Observatory Experiment. DNA extracted from each of the samples was used to create amplicon libraries based on customized panel of 500 antimicrobial resistance genes followed by next-generation sequencing. This is the first study of that shows the reservoir of antimicrobial genes in the ISS. The International Space Station (ISS) as a closed built environment has its own environmental microbiome which is shaped by microgravity radiation and limited human presence. The microbial diversity associated with ISS environmental surfaces was investigated during this study. Polyester wipes and contact slides were used for sampling of eight various surface locations on the ISS at different time periods. The samples were retrieved and analyzed immediately upon the return to the Earth (via Soyuz TMA-14M or Dragon capsule from SpaceX). After surface sample collection contact slides containing nutrient media for the growth of bacteria and fungi were incubated at 25 xcb x9aC. The polyester wipes were processed to measure microbial burden (R2A Blood Agar and Potato Dextrose Agar) and recover cultivable bacteria as well as fungi. Subsequently viable microbial burden was assessed using Adenosine Triphosphate (ATP) assay and quantitative polymerase chain reaction (PCR) methods after propidium monoazide (PMA) treatment. The 16S-tag and metagenome analyses were used to elucidate viable microbial diversity. The cultivable bacterial population yield from the polyester wipes was very high (5 to 7-logs) when compared with the contact slides (102 to 103 CFU/m2). The PMA-qPCR analysis showed considerable variation of viable bacterial population (105 to 109 16S rDNA gene copies/m2) among locations sampled. Unlike contact slides polyester wipes cover much larger sample surface (~1 m2) and produce much more reliable results of the microbial diversity of the ISS covering both cultivable and non-cultivable species. The cultivable total and viable microbial diversity was determined utilizing state-of-the art molecular techniques. The implementation of the PMA assay before DNA extraction allowed distinguishing viable microorganisms which is crucial for determining their role to the crew health the ISS maintenance and the general knowledge of the closed environmentally controlled built systems.

Dataset includes the SRA accessions of the sequencing data used to benchmark the MetaCompare 2.0 pipeline as well as tables of bacterial taxa and antibiotic resistance genes used to perform the risk assessments. A link to the GitHub page where the pipeline source code can be found is also provided. This dataset is associated with the following publication: Rumi, M., M. Oh, B. Davis, C. Brown, J. Adeesh, P. Vikesland, A. Pruden, and L. Zhang. MetaCompare 2.0: Differential ranking of ecological and human health resistome risks. FEMS Microbiology Ecology. Oxford University Press, OXFORD, UK, 100(12): fiae155, (2024).

The environmental samples were collected with the polyester wipes from eight different locations in the International Space Station (ISS) during two consecutive sampling sessions (three months apart) within the ISS Microbial Observatory Experiment. DNA extracted from each of the samples was used to create amplicon libraries based on customized panel of 500 antimicrobial resistance genes followed by next-generation sequencing. This is the first study of that shows the reservoir of antimicrobial genes in the ISS. The International Space Station (ISS) as a closed built environment has its own environmental microbiome which is shaped by microgravity radiation and limited human presence. The microbial diversity associated with ISS environmental surfaces was investigated during this study. Polyester wipes and contact slides were used for sampling of eight various surface locations on the ISS at different time periods. The samples were retrieved and analyzed immediately upon the return to the Earth (via Soyuz TMA-14M or Dragon capsule from SpaceX). After surface sample collection contact slides containing nutrient media for the growth of bacteria and fungi were incubated at 25 xcb x9aC. The polyester wipes were processed to measure microbial burden (R2A Blood Agar and Potato Dextrose Agar) and recover cultivable bacteria as well as fungi. Subsequently viable microbial burden was assessed using Adenosine Triphosphate (ATP) assay and quantitative polymerase chain reaction (PCR) methods after propidium monoazide (PMA) treatment. The 16S-tag and metagenome analyses were used to elucidate viable microbial diversity. The cultivable bacterial population yield from the polyester wipes was very high (5 to 7-logs) when compared with the contact slides (102 to 103 CFU/m2). The PMA-qPCR analysis showed considerable variation of viable bacterial population (105 to 109 16S rDNA gene copies/m2) among locations sampled. Unlike contact slides polyester wipes cover much larger sample surface (~1 m2) and produce much more reliable results of the microbial diversity of the ISS covering both cultivable and non-cultivable species. The cultivable total and viable microbial diversity was determined utilizing state-of-the art molecular techniques. The implementation of the PMA assay before DNA extraction allowed distinguishing viable microorganisms which is crucial for determining their role to the crew health the ISS maintenance and the general knowledge of the closed environmentally controlled built systems.

The environmental samples were collected with the polyester wipes from eight different locations in the International Space Station (ISS) during two consecutive sampling sessions (three months apart) within the ISS Microbial Observatory Experiment. DNA extracted from each of the samples was used to create amplicon libraries based on customized panel of 500 antimicrobial resistance genes followed by next-generation sequencing. This is the first study of that shows the reservoir of antimicrobial genes in the ISS. The International Space Station (ISS) as a closed built environment has its own environmental microbiome which is shaped by microgravity radiation and limited human presence. The microbial diversity associated with ISS environmental surfaces was investigated during this study. Polyester wipes and contact slides were used for sampling of eight various surface locations on the ISS at different time periods. The samples were retrieved and analyzed immediately upon the return to the Earth (via Soyuz TMA-14M or Dragon capsule from SpaceX). After surface sample collection contact slides containing nutrient media for the growth of bacteria and fungi were incubated at 25 xcb x9aC. The polyester wipes were processed to measure microbial burden (R2A Blood Agar and Potato Dextrose Agar) and recover cultivable bacteria as well as fungi. Subsequently viable microbial burden was assessed using Adenosine Triphosphate (ATP) assay and quantitative polymerase chain reaction (PCR) methods after propidium monoazide (PMA) treatment. The 16S-tag and metagenome analyses were used to elucidate viable microbial diversity. The cultivable bacterial population yield from the polyester wipes was very high (5 to 7-logs) when compared with the contact slides (102 to 103 CFU/m2). The PMA-qPCR analysis showed considerable variation of viable bacterial population (105 to 109 16S rDNA gene copies/m2) among locations sampled. Unlike contact slides polyester wipes cover much larger sample surface (~1 m2) and produce much more reliable results of the microbial diversity of the ISS covering both cultivable and non-cultivable species. The cultivable total and viable microbial diversity was determined utilizing state-of-the art molecular techniques. The implementation of the PMA assay before DNA extraction allowed distinguishing viable microorganisms which is crucial for determining their role to the crew health the ISS maintenance and the general knowledge of the closed environmentally controlled built systems.