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MicroFisher: Fungal taxonomic classification for metatranscriptomic and metagenomic data using multiple short hypervariable markers

AbstractProfiling the taxonomic and functional composition of microbes using metagenomic (MG) and metatranscriptomic (MT) sequencing is advancing our understanding of microbial functions. However, the sensitivity and accuracy of microbial classification using genome– or core protein-based approaches, especially the classification of eukaryotic organisms, is limited by the availability of genomes and the resolution of sequence databases. To address this, we propose the MicroFisher, a novel approach that applies multiple hypervariable marker genes to profile fungal communities from MGs and MTs. This approach utilizes the hypervariable regions of ITS and large subunit (LSU) rRNA genes for fungal identification with high sensitivity and resolution. Simultaneously, we propose a computational pipeline (MicroFisher) to optimize and integrate the results from classifications using multiple hypervariable markers. To test the performance of our method, we applied MicroFisher to the synthetic community profiling and found high performance in fungal prediction and abundance estimation. In addition, we also used MGs from forest soil and MTs of root eukaryotic microbes to test our method and the results showed that MicroFisher provided more accurate profiling of environmental microbiomes compared to other classification tools. Overall, MicroFisher serves as a novel pipeline for classification of fungal communities from MGs and MTs.

Wang, Haihua↗

Age-related telomere attrition causes aberrant gene expression in sub-telomeric regions

Telomere attrition has been proposed as a biomarker and causal factor in aging. In addition to causing cellular senescence and apoptosis, telomere shortening has been found to affect gene expression in subtelomeric regions. Here, we analyzed the distribution of age-related differentially expressed genes from the GTEx RNA sequencing database of 54 tissue types from 979 human subjects and found significantly more upregulated than downregulated genes in subtelomeric regions as compared to the genome-wide average. Our data demonstrate spatial relationships between telomeres and gene expression in aging.

59 BASIC BIOLOGICAL SCIENCES↗

Web of Registries Search (WoRS) v1.0.0

The Web of Registries Search (WoRs) is a web based software application that enables users to search for publicly available biological parts using keywords or sequence fragments. For the initial version (1.0.0) of the application, WoRS targets 10 sources of biological part data: the GenBank NIH genetic sequence database (https://www.ncbi.nlm.nih.gov/genbank/), the iGem parts registry (parts.igem.org), the Addgene plasmid repository (https://www.addgene.org/), and 7 Inventory of Composable Elements (ACS Synbio, JGI, JBEI, JBEI Public, ABF, SynBerc, ABF Public) registry instances. WoRs has built-in automated web scrapers which extract data from sources that do not have a public or well-defined application programming interface (API). They extract as much public data as they can find and create a searchable index to speed up searches. Included in the indexed information is the source of the information.

Plahar, Hector↗

What Should Health Departments Do with HIV Sequence Data?

Many countries and US states have mandatory statues that require reporting of HIV clinical data including genetic sequencing results to the public health departments. Because genetic sequencing is a part of routine care for HIV infected persons, health departments have extensive sequence collections spanning years and even decades of the HIV epidemic. How should these data be used (or not) in public health practice? This is a complex, multi-faceted question that weighs personal risks against public health benefit. The answer is neither straightforward nor universal. However, to make that judgement—of how genetic sequence data should be used in describing and combating the HIV epidemic—we need a clear image of what a phylogenetically enhanced HIV surveillance system can do and what benefit it might provide. In this paper, we present a positive case for how up-to-date analysis of HIV sequence databases managed by health departments can provide unique and actionable information of how HIV is spreading in local communities. We discuss this question broadly, with examples from the US, as it is globally relevant for all health authorities that collect HIV genetic data.

60 APPLIED LIFE SCIENCES↗

RNAcentral 2021: secondary structure integration, improved sequence search and new member databases

RNAcentral is a comprehensive database of non-coding RNA (ncRNA) sequences that provides a single access point to 44 RNA resources and >18 million ncRNA sequences from a wide range of organisms and RNA types. RNAcentral now also includes secondary (2D) structure information for >13 million sequences, making RNAcentral the world’s largest RNA 2D structure database. The 2D diagrams are displayed using R2DT, a new 2D structure visualization method that uses consistent, reproducible and recognizable layouts for related RNAs. The sequence similarity search has been updated with a faster interface featuring facets for filtering search results by RNA type, organism, source database or any keyword. This sequence search tool is available as a reusable web component, and has been integrated into several RNAcentral member databases, including Rfam, miRBase and snoDB. To allow for a more fine-grained assignment of RNA types and subtypes, all RNAcentral sequences have been annotated with Sequence Ontology terms. The RNAcentral database continues to grow and provide a central data resource for the RNA community. RNAcentral is freely available at https://rnacentral.org.

59 BASIC BIOLOGICAL SCIENCES↗

Optimizing metaproteomics database construction: lessons from a study of the vaginal microbiome

Metaproteomics, a method for untargeted, high-throughput identification of proteins in complex samples, provides functional information about microbial communities and can tie functions to specific taxa. Metaproteomics often generates less data than other omics techniques, but analytical workflows can be improved to increase usable data in metaproteomic outputs. Identification of peptides in the metaproteomic analysis is performed by comparing mass spectra of sample peptides to a reference database of protein sequences. Although these protein databases are an integral part of the metaproteomic analysis, few studies have explored how database composition impacts peptide identification. Here, we used cervicovaginal lavage (CVL) samples from a study of bacterial vaginosis (BV) to compare the performance of databases built using six different strategies. We evaluated broad versus sample-matched databases, as well as databases populated with proteins translated from metagenomic sequencing of the same samples versus sequences from public repositories. Smaller sample-matched databases performed significantly better, driven by the statistical constraints on large databases. Additionally, large databases attributed up to 34% of significant bacterial hits to taxa absent from the sample, as determined orthogonally by 16S rRNA gene sequencing. We also tested a set of hybrid databases which included bacterial proteins from NCBI RefSeq and translated bacterial genes from the samples. These hybrid databases had the best overall performance, identifying 1,068 unique human and 1,418 unique bacterial proteins, ~30% more than a database populated with proteins from typical vaginal bacteria and fungi. Our findings can help guide the optimal identification of proteins while maintaining statistical power for reaching biological conclusions.

59 BASIC BIOLOGICAL 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↗

FUSARIUM-ID v.3.0: An Updated, Downloadable Resource for Fusarium Species Identification

Species within Fusarium are of global agricultural, medical, and food/feed safety concern and have been extensively characterized. However, accurate identification of species is challenging and usually requires DNA sequence data. FUSARIUM-ID ( http://isolate.fusariumdb.org/blast.php ) is a publicly available database designed to support the identification of Fusarium species using sequences of multiple phylogenetically informative loci, especially the highly informative ~680-bp 5' portion of the translation elongation factor 1-alpha (TEF1) gene that has been adopted as the primary barcoding locus in the genus. However, FUSARIUM-ID v.1.0 and 2.0 had several limitations, including inconsistent metadata annotation for the archived sequences and poor representation of some species complexes and marker loci. Here, we present FUSARIUM-ID v.3.0, which provides the following improvements: (i) additional and updated annotation of metadata for isolates associated with each sequence, (ii) expanded taxon representation in the TEF1 sequence database, (iii) availability of the sequence database as a downloadable file to enable local BLAST queries, and (iv) a tutorial file for users to perform local BLAST searches using either freely available software, such as SequenceServer, BLAST+ executable in the command line, and Galaxy, or the proprietary Geneious software. FUSARIUM-ID will be updated on a regular basis by archiving sequences of TEF1 and other loci from newly identified species and greater in-depth sampling of currently recognized species.

Plant 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↗

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↗

Montane Conifer, Aspen, Meadow, and Sagebrush Metagenome Resolved Genomes and Traits in East River Watershed, Colorado, USA

Climate change is driving vegetation shifts in mountain watersheds, with unknown impacts on biogeochemical cycles. We hypothesize that these shifts will reshape soil microbiomes and associated biogeochemical processes. As a part of Lawrence Berkeley National Laboratory (LBNL) Watershed Science Focus Area (SFA), we assessed microbiome and microbial functional trait differences between soils under conifer, aspen, forby meadows, and sagebrush across the East River Watershed, CO, controlling for elevation and aspect.Here we present metagenome assembled genomes (MAGs) for the bacterial and archaeal communities from soils 0-20cm in depth across three locations in the watershed—Headwaters, Upper Reaches, and Lower Reaches from August 3-11th 2016. Each location was further subdivided into two blocks, with one block on a west facing aspect, and two on the east aspect of the valley. Within blocks, two samples per vegetation type were taken (one at each depth). This resulted in 66 samples, which were sequenced at JGI and can be found under the Joint Genome Institute (JGI) Genomes Online Database (GOLD) sequencing project Gs0118068. Metagenomes were assembled through an inhouse pipeline (see methods), binned using four autobinners (concoct, maxbin2, metabat2, and vamb) and consolidated using dastool. The consolidated bins from all metagenomes were pooled, filtered by completeness (>75%) and contamination (<25%), and dereplicated at 95% ANI using drep. The dataset includes a zip file of 687 genomes (Vegtype_MAGS.zip), the accession numbers for the underlying metagenomes, a csv file with MAG quality metrics and taxonomy from Genome Taxonomy Database (GTDB) and National Center for Biotechnology Information (NCBI) taxonomic representative genome proteins (EastRiver_Vegtype_drep_genome_info.csv), and a file containing MAG quality metrics and taxonomy (gtdb_drep_bin_taxonomy.csv). The dataset additionally includes a sample metadata file (EastRiver_Vegtype_sample_metadata.csv), a metadata file used to register associated samples with IGSNs (International Generic Sample Numbers) (samples.csv), a Google KML file for the sampled locations (sample_collection_sites.kml), a location metadata file (locations.csv), a file-level metadata file (flmd.csv), and a data dictionary (dd.csv) file.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.

54 ENVIRONMENTAL SCIENCES↗

efam: an e xpanded, metaproteome-supported HMM profile database of viral protein fam ilies

Viruses infect, reprogram and kill microbes, leading to profound ecosystem consequences, from elemental cycling in oceans and soils to microbiome-modulated diseases in plants and animals. Although metagenomic datasets are increasingly available, identifying viruses in them is challenging due to poor representation and annotation of viral sequences in databases. Here, we establish efam, an expanded collection of Hidden Markov Model (HMM) profiles that represent viral protein families conservatively identified from the Global Ocean Virome 2.0 dataset. This resulted in 240 311 HMM profiles, each with at least 2 protein sequences, making efam >7-fold larger than the next largest, pan-ecosystem viral HMM profile database. Adjusting the criteria for viral contig confidence from ‘conservative’ to ‘eXtremely Conservative’ resulted in 37 841 HMM profiles in our efam-XC database. To assess the value of this resource, we integrated efam-XC into VirSorter viral discovery software to discover viruses from less-studied, ecologically distinct oxygen minimum zone (OMZ) marine habitats. This expanded database led to an increase in viruses recovered from every tested OMZ virome by ~24% on average (up to ~42%) and especially improved the recovery of often-missed shorter contigs (<5 kb). Additionally, to help elucidate lesser-known viral protein functions, we annotated the profiles using multiple databases from the DRAM pipeline and virion-associated metaproteomic data, which doubled the number of annotations obtainable by standard, single-database annotation approaches. Together, these marine resources (efam and efam-XC) are provided as searchable, compressed HMM databases that will be updated bi-annually to help maximize viral sequence discovery and study from any ecosystem.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

Populus VariantDB v3.2 facilitates CRISPR and functional genomics research

The success of CRISPR genome editing studies depends critically on the precision of guide RNA (gRNA) design. Sequence polymorphisms in outcrossing tree species pose design hazards that can render CRISPR genome editing ineffective. Despite recent advances in tree genome sequencing with haplotype resolution, sequence polymorphism information remains largely inaccessible to various functional genomics research efforts. The Populus VariantDB v3.2 addresses these challenges by providing a user-friendly search engine to query sequence polymorphisms of heterozygous genomes. The database accepts short sequences, such as gRNAs and primers, as input for searching against multiple poplar genomes, including hybrids, with customizable parameters. We provide examples to showcase the utilities of VariantDB in improving the precision of gRNA or primer design. The platform-agnostic nature of the probe search design makes Populus VariantDB v3.2 a versatile tool for the rapidly evolving CRISPR field and other sequence-sensitive functional genomics applications. The database schema is expandable and can accommodate additional tree genomes to broaden its user base.

59 BASIC BIOLOGICAL SCIENCES↗

Relationship and distribution of Salmonella enterica serovar I 4,[5],12:i:- strain sequences in the NCBI Pathogen Detection database

Background: Of the > 2600 Salmonella serovars, Salmonella enterica serovar I 4,[5],12:i:- (serovar I 4,[5],12:i:-) has emerged as one of the most common causes of human salmonellosis and the most frequent multidrug-resistant (MDR; resistance to ≥3 antimicrobial classes) nontyphoidal Salmonella serovar in the U.S. Serovar I 4,[5],12:i:- isolates have been described globally with resistance to ampicillin, streptomycin, sulfisoxazole, and tetracycline (R-type ASSuT) and an integrative and conjugative element with multi-metal tolerance named Salmonella Genomic Island 4 (SGI-4). Results: We analyzed 13,612 serovar I 4,[5],12:i:- strain sequences available in the NCBI Pathogen Detection database to determine global distribution, animal sources, presence of SGI-4, occurrence of R-type ASSuT, frequency of antimicrobial resistance (AMR), and potential transmission clusters. Genome sequences for serovar I 4,[5],12:i:- strains represented 30 countries from 5 continents (North America, Europe, Asia, Oceania, and South America), but sequences from the United States (59%) and the United Kingdom (28%) were dominant. The metal tolerance island SGI-4 and the R-type ASSuT were present in 71 and 55% of serovar I 4,[5],12:i:- strain sequences, respectively. Sixty-five percent of strain sequences were MDR which correlates to serovar I 4,[5],12:i:- being the most frequent MDR serovar. The distribution of serovar I 4,[5],12:i:- strain sequences in the NCBI Pathogen Detection database suggests that swine-associated strain sequences were the most frequent food-animal source and were significantly more likely to contain the metal tolerance island SGI-4 and genes for MDR compared to all other animal-associated isolate sequences. Conclusions: Our study illustrates how analysis of genomic sequences from the NCBI Pathogen Detection database can be utilized to identify the prevalence of genetic features such as antimicrobial resistance, metal tolerance, and virulence genes that may be responsible for the successful emergence of bacterial foodborne pathogens.

59 BASIC BIOLOGICAL SCIENCES↗

Parallel measurement of transcriptomes and proteomes from same single cells using nanodroplet splitting

Single-cell multiomics provides comprehensive insights into gene regulatory networks, cellular diversity, and temporal dynamics. Here, we introduce nanoSPLITS (nanodroplet SPlitting for Linked-multimodal Investigations of Trace Samples), an integrated platform that enables global profiling of the transcriptome and proteome from same single cells via RNA sequencing and mass spectrometry-based proteomics, respectively. Benchmarking of nanoSPLITS demonstrates high measurement precision with deep proteomic and transcriptomic profiling of single-cells. We apply nanoSPLITS to cyclin-dependent kinase 1 inhibited cells and found phospho-signaling events could be quantified alongside global protein and mRNA measurements, providing insights into cell cycle regulation. We extend nanoSPLITS to primary cells isolated from human pancreatic islets, introducing an efficient approach for facile identification of unknown cell types and their protein markers by mapping transcriptomic data to existing large-scale single-cell RNA sequencing reference databases. Accordingly, we establish nanoSPLITS as a multiomic technology incorporating global proteomics and anticipate the approach will be critical to furthering our understanding of biological systems.

59 BASIC BIOLOGICAL SCIENCES↗