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KBase Narrative - Complete genome sequence of a novel Microbacterium sp. strain Clip185.

We have isolated a new species of Microbacterium, an Actinobacterium. We have temporarily named this bacterium as Microbacterium sp. strain Clip185 (hereafter called strain Clip185) from a contaminated Tris-Acetate-Phosphate (TAP) medium culture plate of a green micro-alga Chlamydomonas reinhardtii strain LMJ.RY0402.185141 (a Chlamydomonas Library project CLiP strain). We sequenced the whole genome of strain Clip185 using the PacBio Sequel II Continuous Long Read technology and have submitted it to NCBI along with the SRA and PacBio methylation motif data. Additionally, we have submitted the PacBio methylome to REBASE, Ref#35996. We present the whole genome sequence of this new Microbacterium species that offers insights into its coding and non-coding genes and its nearest taxonomic neighbors.

Mitra, Mautusi↗

Multi-choice Viromics Pipeline (MVP) v1

MVP stands for Multi-choice Viromics Pipeline. It is a pipeline that utilizes a suite of state-of-art tools: geNomad to identify viruses, proviruses, and plasmids in sequencing data, CheckV to assess the quality, and completeness of identified viral genomes, including identification of host contamination for integrated proviruses, A custom code for a rapid genome clustering based on pairwise ANI, Bowtie2, Samtools, and CoverM to calculate coverage of individual viral genomes by read mapping, A custom code to create a vOTU table of abundance, MMseqs2 to compare viral proteins to multiple databases. It provides a quick, and intuitive pipeline to get viral sequences and corresponding properties that can be used for downstream analyses.

Roux, Simon↗

DNase Treatment Improves Viral Enrichment in Agricultural Soil Viromes

Viromics is becoming an increasingly popular method for characterizing soil viral communities. DNase treatment of the viral size fraction prior to DNA extraction is meant to reduce contaminating free DNA and is a common step within viromics protocols to ensure that sequences are of viral origin.

59 BASIC BIOLOGICAL SCIENCES↗

Complete genome sequence of Sphingobium yanoikuyae strain CC4533

We have isolated a new strain of Sphingobium yanoikuyae , which belongs to the class Alphaproteobacteria, order Sphingomonadales, and family Sphingomonadaceae. This carotenoid-producing strain is capable of degrading xenobiotics and is tolerant to toxic levels of six heavy metals. We have designated the newly isolated strain of S. yanoikuyae as S. yanoikuyae strain CC4533 (hereafter called strain CC4533) because it was isolated from a contaminated Tris-Acetate-Phosphate (TAP) medium culture plate of a green micro-alga Chlamydomonas reinhardtii wild type strain CC4533. We sequenced the whole genome of strain CC4533 using the PacBio Sequel II Continuous Long Read technology and have submitted it to NCBI along with the SRA and PacBio methylation motif data. Additionally, we have submitted the PacBio methylome to REBASE, Ref#35996. We present the whole genome sequence of S. yanoikuyae strain CC4533 that offers insights into its coding and non-coding genes and its nearest taxonomic neighbors.

59 BASIC BIOLOGICAL SCIENCES↗

Metagenome-assembled genomes from Slate River floodplain sediments near Crested Butte, CO, USA (September 2019)

Microorganisms play a key role in cycling nutrients and contaminants in the terrestrial environment depending on their genetic potential. Here we present metagenome-assembled genomes (MAGs) for the bacterial and archaeal community in floodplain sediment samples taken September 2019 at one locations (OBJ1) near the confluence of the Oh-Be-Joyful Creek and Slate River. The site is one of the field sites in focus for the SLAC National Accelerator Laboratory Groundwater Quality Science Focus Area (SFA) program. Sediment samples from a deep soil pit were collected from 50 to 150 cm depth below surface at discrete depths every 20 cm for microbial analyses. A total of 6 metagenomes were sequenced through the Joint Genome Institute (JGI) and can be found under Genomes Online Database (GOLD) sequencing project: Gs0142591. Metagenomes were assembled, binned, and refined using metawrap to generate MAGs (>50% complete and < 10% contamination based on checkM scores). This dataset includes a zip file of 2562 MAG fasta files and a csv file with quality, taxonomic classification (Genome Taxonomy Database Release RS220), and metagenome accessions for MAGs. This dataset also includes a file-level metadata (flmd.csv) file that lists each file contained in the dataset with associated metadata and a data dictionary (dd.csv) file that contains column/row headers used throughout the files along with a definition, units, and data type.Part of this work was performed at SLAC Accelerator Laboratory funded by the US Department of Energy, Office of Science, Biological and Environmental Research under Contract No. DE-AC02-76SF00515.

54 ENVIRONMENTAL SCIENCES↗

Metagenome-assembled genomes from Slate River floodplain sediments near Crested Butte, CO, USA (June 2018)

Microorganisms play a key role in cycling nutrients and contaminants in the terrestrial environment depending on their genetic potential. Here, we present metagenome-assembled genomes (MAGs) for the bacterial and archaeal community in floodplain sediment samples taken June 2018 at two locations (OBJ1 and OBJ2) near the confluence of the Oh-Be-Joyful Creek and Slate River. The site is one of the field sites in focus for the SLAC National Accelerator Laboratory Groundwater Quality Science Focus Area (SFA) program. Sediment samples from a deep soil pit were collected from 50 to 150 cm depth below surface at discrete depths every 20 cm for microbial analyses. A total of 12 metagenomes were sequenced through the Joint Genome Institute (JGI) and can be found under Genomes Online Database (GOLD) sequencing project: Gs0142591. Metagenomes were assembled, binned, and refined using metawrap to generate MAGs (>50% complete and < 10% contamination based on checkM scores). This dataset includes a zip file of 1233 MAG fasta files and a csv file with quality, taxonomic classification (Genome Taxonomy Database Release RS220), and metagenome accessions for MAGs. This dataset also includes a file-level metadata (flmd.csv) file that lists each file contained in the dataset with associated metadata and a data dictionary (dd.csv) file that contains column/row headers used throughout the files along with a definition, units, and data type.Part of this work was performed at SLAC Accelerator Laboratory funded by the US Department of Energy, Office of Science, Biological and Environmental Research under Contract No. DE-AC02-76SF00515.

54 ENVIRONMENTAL SCIENCES↗

Metagenome-assembled genomes from East River floodplain sediments near Crested Butte, CO, USA (June to September 2019)

Microorganisms play a key role in cycling nutrients and contaminants in the terrestrial environment depending on their genetic potential. Here, we present metagenome-assembled genomes (MAGs) for the bacterial and archaeal community in floodplain sediment samples taken in 2019 in June (flooded conditions) and September (drained conditions) at two locations (MCB1 and MCB3) near the Meander C/Pumphouse floodplain sites of the East River. Sediment cores were collected from 2 depths, a near-surface, generally unsaturated depth (30-40 centimeter (cm) depth below surface) and a deeper depth influenced by flooding with redoximorphic features (70-80 cm depth below surface). Sediments were homogenized from the 10 cm core for microbial analyses. A total of 24 metagenomes were sequenced through the Joint genome institute (JGI) corresponding to 8 samples sequenced in triplicate. These metagenomes can be found under Genomes Online Database (GOLD) sequencing project: Gs0141020. Metagenomes were assembled, binned, and refined using metawrap to generate MAGs (>50% complete and < 10% contamination based on checkM scores). This dataset includes a zip file of 436 MAG fasta files and a csv file with quality, taxonomic classification (Genome Taxonomy Database Release RS220), and metagenome accessions for MAGs. This dataset also includes a file-level metadata (flmd.csv) file that lists each file contained in the dataset with associated metadata and a data dictionary (dd.csv) file that contains column/row headers used throughout the files along with a definition, units, and data type.This work was supported by the Watershed Function Science Focus Area at Lawrence Berkeley National Laboratory funded by the US Department of Energy, Office of Science, Biological and Environmental Research under Contract No. DE-AC02-05CH11231. Part of this work was performed at SLAC Accelerator Laboratory funded by the US Department of Energy, Office of Science, Biological and Environmental Research under Contract No. DE-AC02-76SF00515.

54 ENVIRONMENTAL SCIENCES↗

Metagenome-assembled genomes from East River floodplain sediments near Crested Butte, CO, USA (June to September 2017)

Microorganisms play a key role in cycling nutrients and contaminants in the terrestrial environment depending on their genetic potential. Here, we present metagenome-assembled genomes (MAGs) for the bacterial and archaeal community in floodplain sediment samples taken in 2017 in June (flooded conditions) and September (drained conditions) at two locations (MCB1 and MCB3) in an active meander (Meander C) of the East River. Sediment cores were collected from 2 depths, a near-surface, generally unsaturated depth (15-40 centimeter (cm) depth below surface) and a deeper depth influenced by flooding with redoximorphic features (50-88 cm depth below surface). Sediments were homogenized from the ~10 cm cores for microbial analyses. A total of 24 metagenomes were sequenced through the Joint genome institute (JGI) corresponding to 8 samples sequenced in triplicate. These metagenomes can be found under Genomes Online Database (GOLD) sequencing project: Gs0151851. Metagenomes were assembled, binned, and refined using metawrap to generate MAGs (>50% complete and < 10% contamination based on checkM scores). This dataset includes a zip file of 405 MAG fasta files and a csv file with quality, taxonomic classification (Genome Taxonomy Database Release RS220), and metagenome accessions for MAGs. This dataset also includes a file-level metadata (flmd.csv) file that lists each file contained in the dataset with associated metadata and a data dictionary (dd.csv) file that contains column/row headers used throughout the files along with a definition, units, and data type.This work was supported by the Watershed Function Science Focus Area at Lawrence Berkeley National Laboratory funded by the US Department of Energy, Office of Science, Biological and Environmental Research under Contract No. DE-AC02-05CH11231. Part of this work was performed at SLAC Accelerator Laboratory funded by the US Department of Energy, Office of Science, Biological and Environmental Research under Contract No. DE-AC02-76SF00515.

54 ENVIRONMENTAL SCIENCES↗

Metagenome-assembled genomes from East River floodplain sediments near Crested Butte, CO, USA (May to September 2018)

Microorganisms play a key role in cycling nutrients and contaminants in the terrestrial environment depending on their genetic potential. Here, we present metagenome-assembled genomes (MAGs) for the bacterial and archaeal community in floodplain sediment samples taken in 2018 in May (flooded conditions) and September (drained conditions) at two locations (MCB1 and MCB3) near the Meander C/Pumphouse floodplain sites of the East River. Sediment cores were collected from 2 depths, a near-surface, generally unsaturated depth (30-40 centimeter (cm) depth below surface) and a deeper depth influenced by flooding with redoximorphic features (70-80 cm depth below surface). Sediments were homogenized from the 10 cm core for microbial analyses. A total of 24 metagenomes were sequenced through the Joint genome institute (JGI) corresponding to 8 samples sequenced in triplicate. These metagenomes can be found under Genomes Online Database (GOLD) sequencing project: Gs0141020. Metagenomes were assembled, binned, and refined using metawrap to generate MAGs (>50% complete and < 10% contamination based on checkM scores). This dataset includes a zip file of 478 MAG fasta files and a csv file with quality, taxonomic classification (Genome Taxonomy Database Release RS220), and metagenome accessions for MAGs. This dataset also includes a file-level metadata (flmd.csv) file that lists each file contained in the dataset with associated metadata and a data dictionary (dd.csv) file that contains column/row headers used throughout the files along with a definition, units, and data type.This work was supported by the Watershed Function Science Focus Area at Lawrence Berkeley National Laboratory funded by the US Department of Energy, Office of Science, Biological and Environmental Research under Contract No. DE-AC02-05CH11231. Part of this work was performed at SLAC Accelerator Laboratory funded by the US Department of Energy, Office of Science, Biological and Environmental Research under Contract No. DE-AC02-76SF00515.

54 ENVIRONMENTAL SCIENCES↗

Metagenome-assembled genomes from Wind River Basin floodplain sediments Riverton, Wyoming site (June to October 2019)

Microorganisms play a key role in cycling nutrients and contaminants in the terrestrial environment depending on their genetic potential. Here we present metagenome-assembled genomes (MAGs) for the bacterial and archaeal community in floodplain sediment samples taken at three time points from June 12, 2019 to October 23,2019 at a location (PTT1) close to DOE Legacy Management well 855 at the Riverton, Wyoming floodplain site in the Wind River Basin (WRB). The groundwater at this site exhibits persistent U, Mo, and sulfate plumes and is one of the field sites in focus for the SLAC Groundwater Quality SFA program. Sediment samples were collected from 60 to 180 cm below surface every 30cm for microbial analyses through metagenomic sequencing. 15 metagenomes were sequenced through JGI and can be found under Gold sequencing project: Gs0131241. Metagenomes were assembled, binned, and refined using metawrap to generate MAGs (>50% complete and < 10% contamination based on checkM scores). This dataset includes a zip file of 780 MAG fasta files and a csv file with quality, taxonomic classification (GTDB RS220), and metagenome accessions for MAGs. This dataset also includes a file-level metadata (flmd.csv) file that lists each file contained in the dataset with associated metadata and a data dictionary (dd.csv) file that contains column/row headers used throughout the files along with a definition, units, and data type. A sample metadata file (samples.csv) that contains site information has also been included.

54 ENVIRONMENTAL SCIENCES↗

Metagenome-assembled genomes from Wind River Basin floodplain sediments Riverton, Wyoming site (August 2015)

Microorganisms play a key role in cycling nutrients and contaminants in the terrestrial environment depending on their genetic potential. Here we present metagenome-assembled genomes (MAGs) for the bacterial and archaeal community in floodplain sediment samples taken August 29, 2015 at a location (KB1) close to DOE Legacy Management well 855 at the Riverton, Wyoming floodplain site in the Wind River Basin (WRB). The groundwater at this site exhibits persistent U, Mo, and sulfate plumes and is one of the field sites in focus for the SLAC Groundwater Quality SFA program. Sediment samples from a deep soil pit were collected from 0 to 234 cm depth below surface at discrete depths every ~10-20 cm for microbial analyses. 13 metagenomes were sequenced through JGI and can be found under Gold sequencing project: Gs0131241. Metagenomes were assembled, binned, and refined using metawrap to generate MAGs (>50% complete and < 10% contamination based on checkM scores).This dataset includes a zip file of 2216 MAG fasta files and a csv file with quality, taxonomic classification (GTDB RS220), and metagenome accessions for MAGs. This dataset also includes a file-level metadata (flmd.csv) file that lists each file contained in the dataset with associated metadata and a data dictionary (dd.csv) file that contains column/row headers used throughout the files along with a definition, units, and data type

54 ENVIRONMENTAL SCIENCES↗

Metagenome-assembled genomes from Slate River floodplain sediments near Crested Butte, CO, USA (June to October 2020)

Microorganisms play a key role in cycling nutrients and contaminants in the terrestrial environment depending on their genetic potential. Here we present metagenome-assembled genomes (MAGs) for the bacterial and archaeal community in floodplain sediment samples taken June to October 2020 at two locations (OBJ1 and OBJ2) near the confluence of the Oh-Be-Joyful Creek and Slate River. The site is one of the field sites in focus for the SLAC National Accelerator Laboratory Groundwater Quality Science Focus Area (SFA) program. Sediment samples from a deep soil pit were collected from 30 cm depth below surface to just above the cobble layer (~190-250 cm depth) at discrete depths every 40 cm for microbial analyses. A total of 35 metagenomes were sequenced through the Joint Genome Institute (JGI) and can be found under Genomes Online Database (GOLD) sequencing project: Gs0142591. Metagenomes were assembled, binned, and refined using metawrap to generate MAGs (>50% complete and < 10% contamination based on checkM scores). This dataset includes a zip file of 2848 MAG fasta files and a csv file with quality, taxonomic classification (Genome Taxonomy Database Release RS220), and metagenome accessions for MAGs. This dataset also includes a file-level metadata (flmd.csv) file that lists each file contained in the dataset with associated metadata and a data dictionary (dd.csv) file that contains column/row headers used throughout the files along with a definition, units, and data type.Part of this work was performed at SLAC Accelerator Laboratory funded by the US Department of Energy, Office of Science, Biological and Environmental Research under Contract No. DE-AC02-76SF00515.

54 ENVIRONMENTAL SCIENCES↗

Genome-resolved analysis of Serratia marcescens strain SMTT infers niche specialization as a hydrocarbon-degrader

Abstract Bacteria that are chronically exposed to high levels of pollutants demonstrate genomic and corresponding metabolic diversity that complement their strategies for adaptation to hydrocarbon-rich environments. Whole genome sequencing was carried out to infer functional traits of Serratia marcescens strain SMTT recovered from soil contaminated with crude oil. The genome size (Mb) was 5,013,981 with a total gene count of 4,842. Comparative analyses with carefully selected S. marcescens strains, 2 of which are associated with contaminated soil, show conservation of central metabolic pathways in addition to intra-specific genetic diversity and metabolic flexibility. Genome comparisons also indicated an enrichment of genes associated with multidrug resistance and efflux pumps for SMTT. The SMTT genome contained genes that enable the catabolism of aromatic compounds via the protocatechuate para-degradation pathway, in addition to meta-cleavage of catechol (meta-cleavage pathway II); gene enrichment for aromatic compound degradation was markedly higher for SMTT compared to the other S. marcescens strains analysed. Our data presents a valuable genetic inventory for future studies on strains of S. marcescens and provides insights into those genomic features of SMTT with industrial potential.

Genetics & Heredity↗

Nutrient Exposure Alters Microbial Composition, Structure, and Mercury Methylating Activity in Periphyton in a Contaminated Watershed

The conversion of mercury (Hg) to monomethylmercury (MMHg) is a critical area of concern in global Hg cycling. Periphyton biofilms may harbor significant amounts of MMHg but little is known about the Hg-methylating potential of the periphyton microbiome. Therefore, we used high-throughput amplicon sequencing of the 16S rRNA gene, ITS2 region, and Hg methylation gene pair (hgcAB) to characterize the archaea/bacteria, fungi, and Hg-methylating microorganisms in periphyton communities grown in a contaminated watershed in East Tennes(see United States). Furthermore, we examined how nutrient amendments (nitrate and/or phosphate) altered periphyton community structure and function. We found that bacterial/archaeal richness in experimental conditions decreased in summer and increased in autumn relative to control treatments, while fungal diversity generally increased in summer and decreased in autumn relative to control treatments. Interestingly, the Hg-methylating communities were dominated by Proteobacteria followed by Candidatus Atribacteria across both seasons. Surprisingly, Hg methylation potential correlated with numerous bacterial families that do not contain hgcAB, suggesting that the overall microbiome structure of periphyton communities influences rates of Hg transformation within these microbial mats. To further explore these complex community interactions, we performed a microbial network analysis and found that the nitrate-amended treatment resulted in the highest number of hub taxa that also corresponded with enhanced Hg methylation potential. This work provides insight into community interactions within the periphyton microbiome that may contribute to Hg cycling and will inform future research that will focus on establishing mixed microbial consortia to uncover mechanisms driving shifts in Hg cycling within periphyton habitats.

59 BASIC BIOLOGICAL SCIENCES↗

In-field bioreactors demonstrate dynamic shifts in microbial communities in response to geochemical perturbations

Subsurface microbial communities mediate the transformation and fate of redox sensitive materials including organic matter, metals and radionuclides. Few studies have explored how changing geochemical conditions influence the composition of groundwater microbial communities over time. We temporally monitored alterations in abiotic forces on microbial community structure using 1L in-field bioreactors receiving background and contaminated groundwater at the Oak Ridge Reservation, TN. Planktonic and biofilm microbial communities were initialized with background water for 4 days to establish communities in triplicate control reactors and triplicate test reactors and then fed filtered water for 14 days. On day 18, three reactors were switched to receive filtered groundwater from a contaminated well, enriched in total dissolved solids relative to the background site, particularly chloride, nitrate, uranium, and sulfate. Biological and geochemical data were collected throughout the experiment, including planktonic and biofilm DNA for 16S rRNA amplicon sequencing, cell counts, total protein, anions, cations, trace metals, organic acids, bicarbonate, pH, Eh, DO, and conductivity. We observed significant shifts in both planktonic and biofilm microbial communities receiving contaminated water. This included a loss of rare taxa, especially amongst members of the Bacteroidetes, Acidobacteria, Chloroflexi, and Betaproteobacteria, but enrichment in the Fe- and nitrate- reducing Ferribacterium and parasitic Bdellovibrio. These shifted communities were more similar to the contaminated well community, suggesting that geochemical forces substantially influence microbial community diversity and structure. These influences can only be captured through such comprehensive temporal studies, which also enable more robust and accurate predictive models to be developed.

59 BASIC BIOLOGICAL SCIENCES↗

Metagenome-assembled genomes from Wind River Basin floodplain sediments Riverton, Wyoming site (May to September 2017)

Microorganisms play a key role in cycling nutrients and contaminants in the terrestrial environment depending on their genetic potential. Here we present metagenome-assembled genomes (MAGs) for the bacterial and archaeal community in floodplain sediment samples taken roughly every month in the period May 18 to September 13 in 2017 at a location (Pit2) close to DOE Legacy Management well 855 at the Riverton, Wyoming floodplain site in the Wind River Basin (WRB). The groundwater at this site exhibits persistent U, Mo, and sulfate plumes and is one of the field sites in focus for the SLAC Groundwater Quality SFA program. Cores were taken with a hand-auger and separated into 5-20 cm segments based on soil horizonation down to 150 cm depth below surface. Each segment was subsampled for microbial analyses. Corresponding 16S rRNA gene amplicon data is available at the NCBI Single Read Archive (SRA) Database BioProject ID PRJNA626616, and soil geochemistry data at doi:10.15485/1631972. 40 metagenomes were sequenced through JGI and can be found under Gold sequencing project: Gs0142591. Metagenomes were assembled, binned, and refined using metawrap to generate MAGs (>50% complete and < 10% contamination based on checkM scores). This dataset includes a zip file of 6993 MAG fasta files and a csv file with quality, taxonomic classification (GTDB RS220), and metagenome accessions for MAGs generated from the Wind River Basin (WRB). This dataset also includes a file-level metadata (flmd.csv) file that lists each file contained in the dataset with associated metadata and a data dictionary (dd.csv) file that contains column/row headers used throughout the files along with a definition, units, and data type.

54 ENVIRONMENTAL SCIENCES↗

Apatite geochemistry as a tool for understanding the petrogenesis of layered mafic-ultramafic rocks in the Bushveld Complex, South Africa

The sources of the magmas that formed the Rustenburg Layered Suite of the Bushveld Complex in South Africa remain debated, despite decades of research. Vertical and lateral variation in bulk rock and mineral separate Sr-Nd isotopic compositions, which generally indicate enriched sources, demonstrate that the layered sequence was formed by the emplacement of multiple batches of magma, crucially resulting in episodes of PGE-Cr-V mineralisation. The Lu-Hf isotope compositions of zircon are, however, at odds with the bulk rock Sr-Nd isotopic heterogeneity as they show near homogeneous compositions throughout the layered sequence (εHf (2.06 Ga) =−8). This lack of variation in Hf isotope composition has been attributed to deep, continental lithospheric mantle-related and/or crustal contamination of plume-derived Bushveld magmas. In this study, we analysed the major, trace element and Sr-Nd isotope geochemistry of apatite in the Rustenburg Layered Suite. Apatite occurs as an intercumulus mineral in the lowermost regions and a cumulus mineral in the uppermost regions of the layered sequence and can therefore be used to test existing models for the isotopic disequilibrium between bulk rock Sr-Nd and zircon Hf isotopic compositions. Apatite is largely chlorapatite in the lowermost regions and fluorapatite in the uppermost regions of the layered sequence. The Merensky Reef is unusual in that it comprises both chlorapatite and fluorapatite. Apatite throughout the layered sequence is generally unzoned and shows no evidence of late-stage alteration. Trace element data show that apatite is enriched in L/HREE, with common negative Eu-Sr anomalies. These trace element signatures are consistent with a magmatic origin for the apatite grains, with prior, or concurrent, plagioclase crystallization from the same melt. Variability in in situ Sr and Nd isotope compositions of apatite is recorded throughout the layered sequence with εNd (2.06 Ga) compositions varying between −2.5 and − 10.2 and initial 87 Sr/ 86 Sr compositions varying between 0.7079 and 0.7103 (for the Marikana dikes only). The variability in Sr-Nd isotope compositions of apatite is consistent with the bulk rock (and mineral separate) variation in Sr-Nd isotope compositions, suggesting apatite preserves primary magmatic compositions in the Rustenburg Layered Suite.

Apatite↗

Proteomics Analysis of Human Contaminant Proteins

Complete characterization of unknowns via proteomics remains challenging. There exist regions of mass spectrometry-based proteomics data where empirical measurements are not attributed to peptides, and/or sequenced peptides from mass spectra are not attributed to any source. These uncharacterized regions are known as the “dark” proteome. Many proteomics tools rely on some a priori knowledge of sample composition; few tools allow for investigation of unknowns without relying on composition assumptions. Further, the potential low abundance of minor traces in these uncharacterized regions can make elucidation of the “dark” proteome challenging. Herein, we describe the development and evaluation of approaches to study the “dark” proteome and move towards an untargeted approach for more complete characterization, namely by studying minor human protein traces in non-human samples and combining that approach with non-human source organism identification without relying on assumptions. Human protein markers, in the form of genetically variant peptides, have been extensively examined in a variety of human matrices, including blood, plasma, and hair, but have yet to be investigated in non-human samples, such as cell cultures, as human contaminant traces. Genetically variant peptides are those that are found in proteins carrying single nucleotide polymorphisms. In this work, we aimed to (1) investigate the feasibility of detecting human contaminant genetically variant peptides (GVPs) in a diverse set of non-human organisms using public proteomics data and a computational pipeline, as well as to (2) develop a combined capability for untargeted source organism characterization and GVP detection. To our knowledge, this is the first report of applying these approaches towards a more complete proteomic characterization of unknowns. We successfully demonstrate the feasibility of broad human contaminant GVP detection in proteomics data, develop a better understanding of GVP detectability, characterize the sample-to-sample variability in GVP detection, and identify a core set of GVPs that can potentially be used as markers indicative of the human contaminant traces portion of the “dark” proteome. Further, we developed and evaluated a combined pipeline, MARLOWE-GVP, that enables both untargeted source organism characterization and GVP detection. We show high accuracy of correct source organism characterization and high degree of similarity of human contaminant GVP detection compared to the conventional approach. Success on both these efforts have allowed us to advance our understanding and characterization of the “dark” proteome.

59 BASIC BIOLOGICAL SCIENCES↗