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At least 217 records · Page 12
All sourcesNASA NTRSDOE OSTI
DOE OSTI2017

Classification of nucleotide sequences by latent semantic analysis

DNA sequences are analyzed using latent semantic analysis. A set of nucleotide sequences is received in which the set has a first number of sequences. A set of basis vectors is determined, in which the set has a second number of basis vectors, the second number being smaller than the first number. Each basis vector represents a specific combination of predetermined nucleotide segments. For each of the nucleotide sequences, an approximate representation of the nucleotide sequence is determined based on a combination of the basis vectors. For each pair of nucleotide sequences, a distance between the pair of nucleotide sequences is determined according the distance between the approximate representation of the pair of nucleotide sequences. The set of nucleotide sequences are classified based on the distances between the pairs of nucleotide sequences.

Sayood, Khalid↗
DOE OSTI2017

Diazotroph Community Characterization via a High-Throughput nifH Amplicon Sequencing and Analysis Pipeline

The dinitrogenase reductase gene ( nifH ) is the most widely established molecular marker for the study of nitrogen-fixing prokaryotes in nature. A large number of PCR primer sets have been developed for nifH amplification, and the effective deployment of these approaches should be guided by a rapid, easy-to-use analysis protocol. Bioinformatic analysis of marker gene sequences also requires considerable expertise. In this study, we advance the state of the art for nifH analysis by evaluating nifH primer set performance, developing an improved amplicon sequencing workflow, and implementing a user-friendly bioinformatics pipeline. Furthermore, the developed amplicon sequencing workflow is a three-stage PCR-based approach that uses established technologies for incorporating sample-specific barcode sequences and sequencing adapters. Based on our primer evaluation, we recommend the Ando primer set be used with a modified annealing temperature of 58°C, as this approach captured the largest diversity of nifH templates, including paralog cluster IV/V sequences. To improve nifH sequence analysis, we developed a computational pipeline which infers taxonomy and optionally filters out paralog sequences. In addition, we employed an empirical model to derive optimal operational taxonomic unit (OTU) cutoffs for the nifH gene at the species, genus, and family levels. A comprehensive workflow script named TaxADivA (TAXonomy Assignment and DIVersity Assessment) is provided to ease processing and analysis of nifH amplicons. Our approach is then validated through characterization of diazotroph communities across environmental gradients in beach sands impacted by the Deepwater Horizon oil spill in the Gulf of Mexico, in a peat moss-dominated wetland, and in various plant compartments of a sugarcane field. IMPORTANCE: Nitrogen availability often limits ecosystem productivity, and nitrogen fixation, exclusive to prokaryotes, comprises a major source of nitrogen input that sustains food webs. The nifH gene, which codes for the iron protein of the nitrogenase enzyme, is the most widely established molecular marker for the study of nitrogen-fixing microorganisms (diazotrophs) in nature. In this study, a flexible sequencing/analysis pipeline, named TaxADivA, was developed for nifH amplicons produced by Illumina paired-end sequencing, and it enables an inference of taxonomy, performs clustering, and produces output in formats that may be used by programs that facilitate data exploration and analysis. Diazotroph diversity and community composition are linked to ecosystem functioning, and our results advance the phylogenetic characterization of diazotroph communities by providing empirically derived nifH similarity cutoffs for species, genus, and family levels. Thus, the utility of our pipeline is validated for diazotroph communities in a variety of ecosystems, including contaminated beach sands, peatland ecosystems, living plant tissues, and rhizosphere soil.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Sequence Impedance Measurement of Utility-Scale Wind Turbines and Inverters – Reference Frame, Frequency Coupling, and MIMO/SISO Forms

Sequence impedance responses with or without considering frequency coupling in both MIMO and SISO forms are increasingly used for the stability analysis of three-phase power electronic systems; however, many aspects of sequence impedance measurement are not fully explored. It is not clear if the sequence impedance has a reference frame similar to the dq impedance. If so, the role of the grid voltage angle estimation in aligning the sequence impedance reference frame has not been discussed. Additionally, existing methods for measuring the sequence impedance with frequency coupling are complicated, are not feasible for large wind turbines and inverters, and provide the sequence impedance responses in only either MIMO or SISO form. This paper presents a sequence impedance measurement method that considers the frequency coupling, performs reference frame alignment, demonstrates the impact of the grid voltage angle estimation, and obtains the sequence impedance response in both MIMO and SISO forms. This paper demonstrates the proposed method and practical problems associated with the sequence impedance measurement of utility-scale wind turbines and inverters on a 1.9-MW Type III wind turbine and a 2.2-MVA inverter using an impedance measurement system built around a 7-MW/13.8-kV grid simulator and a 5-MW dynamometer.

17 WIND ENERGY↗
DOE OSTI2023

Whole genome sequencing of Mycobacterium bovis directly from clinical tissue samples without culture

Advancement in next generation sequencing offers the possibility of routine use of whole genome sequencing (WGS) for Mycobacterium bovis (M. bovis) genomes in clinical reference laboratories. To date, the M. bovis genome could only be sequenced if the mycobacteria were cultured from tissue. This requirement for culture has been due to the overwhelmingly large amount of host DNA present when DNA is prepared directly from a granuloma. To overcome this formidable hurdle, we evaluated the usefulness of an RNA-based targeted enrichment method to sequence M. bovis DNA directly from tissue samples without culture. Initial spiking experiments for method development were established by spiking DNA extracted from tissue samples with serially diluted M. bovis BCG DNA at the following concentration range: 0.1 ng/μl to 0.1 pg/μl (10 –1 to 10 –4 ). Library preparation, hybridization and enrichment was performed using SureSelect custom capture library RNA baits and the SureSelect XT HS2 target enrichment system for Illumina paired-end sequencing. The method validation was then assessed using direct WGS of M. bovis DNA extracted from tissue samples from naturally (n = 6) and experimentally (n = 6) infected animals with variable Ct values. Direct WGS of spiked DNA samples achieved 99.1% mean genome coverage (mean depth of coverage: 108×) and 98.8% mean genome coverage (mean depth of coverage: 26.4×) for tissue samples spiked with BCG DNA at 10 –1 (mean Ct value: 20.3) and 10 –2 (mean Ct value: 23.4), respectively. The M. bovis genome from the experimentally and naturally infected tissue samples was successfully sequenced with a mean genome coverage of 99.56% and depth of genome coverage ranging from 9.2× to 72.1×. The spoligoyping and M. bovis group assignment derived from sequencing DNA directly from the infected tissue samples matched that of the cultured isolates from the same sample. Our results show that direct sequencing of M. bovis DNA from tissue samples has the potential to provide accurate sequencing of M. bovis genomes significantly faster than WGS from cultures in research and diagnostic settings.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2015

Reducing Dose Uncertainty for Spot-Scanning Proton Beam Therapy of Moving Tumors by Optimizing the Spot Delivery Sequence

Purpose: To develop and validate a novel delivery strategy for reducing the respiratory motion–induced dose uncertainty of spot-scanning proton therapy. Methods and Materials: The spot delivery sequence was optimized to reduce dose uncertainty. The effectiveness of the delivery sequence optimization was evaluated using measurements and patient simulation. One hundred ninety-one 2-dimensional measurements using different delivery sequences of a single-layer uniform pattern were obtained with a detector array on a 1-dimensional moving platform. Intensity modulated proton therapy plans were generated for 10 lung cancer patients, and dose uncertainties for different delivery sequences were evaluated by simulation. Results: Without delivery sequence optimization, the maximum absolute dose error can be up to 97.2% in a single measurement, whereas the optimized delivery sequence results in a maximum absolute dose error of ≤11.8%. In patient simulation, the optimized delivery sequence reduces the mean of fractional maximum absolute dose error compared with the regular delivery sequence by 3.3% to 10.6% (32.5-68.0% relative reduction) for different patients. Conclusions: Optimizing the delivery sequence can reduce dose uncertainty due to respiratory motion in spot-scanning proton therapy, assuming the 4-dimensional CT is a true representation of the patients' breathing patterns.

62 RADIOLOGY AND NUCLEAR MEDICINE↗
DOE OSTI2023

Sequence Design of Random Heteropolymers as Protein Mimics

Random heteropolymers (RHPs) have been computationally designed and experimentally shown to recapitulate protein-like phase behavior and function. However, unlike proteins, RHP sequences are only statistically defined and cannot be sequenced. Recent developments in reversible-deactivation radical polymerization allowed simulated polymer sequences based on the well-established Mayo–Lewis equation to more accurately reflect ground-truth sequences that are experimentally synthesized. This led to opportunities to perform bioinformatics-inspired analysis on simulated sequences to guide the design, synthesis, and interpretation of RHPs. We compared batches on the order of 10000 simulated RHP sequences that vary by synthetically controllable and measurable RHP characteristics such as chemical heterogeneity and average degree of polymerization. Our analysis spans across 3 levels: segments along a single chain, sequences within a batch, and batch-averaged statistics. We discuss simulator fidelity and highlight the importance of robust segment definition. Examples are presented that demonstrate the use of simulated sequence analysis for in-silico iterative design to mimic protein hydrophobic/hydrophilic segment distributions in RHPs and compare RHP and protein sequence segments to explain experimental results of RHPs that mimic protein function. To facilitate the community use of this workflow, the simulator and analysis modules have been made available through an open source toolkit, the RHPapp.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2022

Influence of Fault Architecture on Induced Earthquake Sequence Evolution Revealed by High-Resolution Focal Mechanism Solutions

The increasing seismicity and improved seismic observation network in recent years provide an opportunity to explore factors that influence the triggering processes, spatiotemporal evolution, and maximum magnitude of induced sequences. We map the fault architecture and stress state of four induced sequences in Oklahoma to determine their influence on the seismicity. We systematically relocate the earthquakes and compute hundreds of focal mechanisms of small to medium events (1.0 < M < 5.1) using various techniques, including machine learning, for the Guthrie, Woodward, Cushing, and Fairview sequences. The detailed fault geometry and spatiotemporal evolution of seismicity and stress states reveal different dominant driving forces for each sequence. In Cushing and Fairview (largest event ≥M5.0), the main fault structures are near-vertical narrow strike-slip faults, with most of the small earthquake fault planes optimally oriented. The two sequences exhibit discontinuous temporal migration but strong earthquake self-driven rupture growth. In Guthrie and Woodward (largest event <M5.0), the two sequences show more complex diffuse fault structures with varying dipping angles along depth. The inverted focal mechanisms show a mix of strike-slip faulting and normal faulting in both sequences, and the normal faulting events are less optimally oriented than strike-slip events. The two sequences are dominated by continuous diffusive migration in time driven by pore pressure propagation. The above results suggest that fault architecture and stress state influence sequence evolution, major driving forces, and possibly maximum magnitude.

58 GEOSCIENCES↗
DOE OSTI2022

Benchmarking second and third-generation sequencing platforms for microbial metagenomics

Shotgun metagenomic sequencing is a common approach for studying the taxonomic diversity and metabolic potential of complex microbial communities. Current methods primarily use second generation short read sequencing, yet advances in third generation long read technologies provide opportunities to overcome some of the limitations of short read sequencing. Here, we compared seven platforms, encompassing second generation sequencers (Illumina HiSeq 300, MGI DNBSEQ-G400 and DNBSEQ-T7, ThermoFisher Ion GeneStudio S5 and Ion Proton P1) and third generation sequencers (Oxford Nanopore Technologies MinION R9 and Pacific Biosciences Sequel II). We constructed three uneven synthetic microbial communities composed of up to 87 genomic microbial strains DNAs per mock, spanning 29 bacterial and archaeal phyla, and representing the most complex and diverse synthetic communities used for sequencing technology comparisons. Our results demonstrate that third generation sequencing have advantages over second generation platforms in analyzing complex microbial communities, but require careful sequencing library preparation for optimal quantitative metagenomic analysis. Our sequencing data also provides a valuable resource for testing and benchmarking bioinformatics software for metagenomics.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

A high-throughput skim-sequencing approach for genotyping, dosage estimation and identifying translocations

The development of next-generation sequencing (NGS) enabled a shift from array-based genotyping to directly sequencing genomic libraries for high-throughput genotyping. Even though whole-genome sequencing was initially too costly for routine analysis in large populations such as breeding or genetic studies, continued advancements in genome sequencing and bioinformatics have provided the opportunity to capitalize on whole-genome information. As new sequencing platforms can routinely provide high-quality sequencing data for sufficient genome coverage to genotype various breeding populations, a limitation comes in the time and cost of library construction when multiplexing a large number of samples. Here we describe a high-throughput whole-genome skim-sequencing (skim-seq) approach that can be utilized for a broad range of genotyping and genomic characterization. Using optimized low-volume Illumina Nextera chemistry, we developed a skim-seq method and combined up to 960 samples in one multiplex library using dual index barcoding. With the dual-index barcoding, the number of samples for multiplexing can be adjusted depending on the amount of data required, and could be extended to 3,072 samples or more. Panels of doubled haploid wheat lines ( Triticum aestivum , CDC Stanley x CDC Landmark), wheat-barley ( T . aestivum x Hordeum vulgare ) and wheat-wheatgrass ( Triticum durum x Thinopyrum intermedium ) introgression lines as well as known monosomic wheat stocks were genotyped using the skim-seq approach. Bioinformatics pipelines were developed for various applications where sequencing coverage ranged from 1 × down to 0.01 × per sample. Using reference genomes, we detected chromosome dosage, identified aneuploidy, and karyotyped introgression lines from the skim-seq data. Leveraging the recent advancements in genome sequencing, skim-seq provides an effective and low-cost tool for routine genotyping and genetic analysis, which can track and identify introgressions and genomic regions of interest in genetics research and applied breeding programs.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2013

SEMIREGULAR VARIABLES WITH PERIODS LYING BETWEEN THE PERIOD-LUMINOSITY SEQUENCES C', C, AND D

We analyze the distribution of semiregular variables and Mira stars in the period-luminosity plane. Our sample consists of 6169 oxygen-rich long-period variables in the Large Magellanic Cloud included in the OGLE-III Catalog of Variable Stars. There are many stars with periods that lie between the well-known sequences C and C'. Most of these stars are multi-periodic and the period ratios suggest that these stars oscillate in the same mode as the sequence C stars. Models suggest that this mode is the fundamental radial pulsation mode. The stars with primary periods between sequences C and C' preferentially lie on an additional sequence (named F), and a large fraction of these stars also have long secondary periods (LSPs) that lie between sequences C and D. There are also a small number of stars with primary periods lying between sequences C and D. The origin of this long-period variability is unknown, as is the cause of sequence D variability. In addition, the origin of sequence F is unknown but we speculate that sequence F variability may be excited by the same phenomenon that causes the LSPs.

79 ASTRONOMY AND ASTROPHYSICS↗
DOE OSTI2013

THE MID-INFRARED AND NEAR-ULTRAVIOLET EXCESS EMISSIONS OF QUIESCENT GALAXIES ON THE RED SEQUENCE

We study the mid-infrared (IR) and near-ultraviolet (UV) excess emissions of spectroscopically selected quiescent galaxies on the optical red sequence. We use the Wide-field Infrared Survey Explorer mid-IR and Galaxy Evolution Explorer near-UV data for a spectroscopic sample of galaxies in the Sloan Digital Sky Survey Data Release 7 to study the possible connection between quiescent red-sequence galaxies with and without mid-IR/near-UV excess. Among 648 12 {mu}m detected quiescent red-sequence galaxies without H{alpha} emission, 26% and 55% show near-UV and mid-IR excess emissions, respectively. When we consider only bright (M{sub r} < -21.5) galaxies with an early-type morphology, the fraction of galaxies with recent star formation is still 39%. The quiescent red-sequence galaxies with mid-IR and near-UV excess emissions are optically fainter and have slightly smaller D{sub n} 4000 than those without mid-IR and near-UV excess emissions. We also find that mid-IR weighted mean stellar ages of quiescent red-sequence galaxies with mid-IR excess are larger than those with near-UV excess, and smaller than those without mid-IR and near-UV excess. The environmental dependence of the fraction of quiescent red-sequence galaxies with mid-IR and near-UV excess seems strong even though the trends of quiescent red-sequence galaxies with near-UV excess differ from those with mid-IR excess. These results indicate that the recent star formation traced by near-UV ({approx}< 1 Gyr) and mid-IR ({approx}< 2 Gyr) excess is not negligible among nearby, quiescent, red, early-type galaxies. We suggest a possible evolutionary scenario of quiescent red-sequence galaxies from quiescent red-sequence galaxies with near-UV excess to those with mid-IR excess to those without near-UV and mid-IR excess.

79 ASTRONOMY AND ASTROPHYSICS↗
DOE OSTI2014

D-Tailor: automated analysis and design of DNA sequences

Motivation: Current advances in DNA synthesis, cloning and sequencing technologies afford high-throughput implementation of artificial sequences into living cells. However, flexible computational tools for multi-objective sequence design are lacking, limiting the potential of these technologies. Results: We developed DNA-Tailor (D-Tailor), a fully extendable software framework, for property-based design of synthetic DNA sequences. D-Tailor permits the seamless integration of multiple sequence analysis tools into a generic Monte Carlo simulation that evolves sequences toward any combination of rationally defined properties. As proof of principle, we show that D-Tailor is capable of designing sequence libraries comprising all possible combinations among three different sequence properties influencing translation efficiency in Escherichia coli. The capacity to design artificial sequences that systematically sample any given parameter space should support the implementation of more rigorous experimental designs.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2015

SU-E-J-223: A BOLD Contrast Imaging Sequence to Evaluate Oxygenation Changes Due to Breath Holding for Breast Radiotherapy: A Pilot Study

Purpose: To develop a robust MRI sequence to measure BOLD breath hold induced contrast in context of breast radiotherapy. Methods: Two sequences were selected from prior studies as candidates to measure BOLD contrast attributable to breath holding within the breast: (1) T2* based Gradient Echo EPI (TR/TE = 500/41ms, flip angle = 60°), and (2) T2 based Single Shot Fast Spin Echo (SSFSE) (TR/TE = 3000/60ms). We enrolled ten women post-lumpectomy for breast cancer who were undergoing treatment planning for whole breast radiotherapy. Each session utilized a 1.5T GE MRI and 4 channel breast coil with the subject immobilized prone on a custom board. For each sequence, 1–3 planes of the lumpectomy breast were imaged continuously during a background measurement (1min) and intermittent breath holds (20–40s per breath hold, 3–5 holds per sequence). BOLD contrast was quantified as correlation of changes in per-pixel intensity with the breath hold schedule convolved with a hemodynamic response function. Subtle motion was corrected using a deformable registration algorithm. Correlation with breath-holding was considered significant if p<0.001. Results: The percentage of the breast ROI with positive BOLD contrast measured by the two sequences were in agreement with a correlation coefficient of R=0.72 (p=0.02). While both sequences demonstrated areas with strong BOLD response, the response was more systematic throughout the breast for the SSFSE (T2) sequence (% breast with response in the same direction: 51.2%±0.7% for T2* vs. 68.1%±16% for T2). In addition, the T2 sequence was less prone to magnetic susceptibility artifacts, especially in presence of seroma, and provided a more robust image with little distortion or artifacts. Conclusion: A T2 SSFSE sequence shows promise for measuring BOLD contrast in the context of breast radiotherapy utilizing a breath hold technique. Further study in a larger patient cohort is warranted to better refine this novel technique.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2016

SU-F-J-103: Assessment of Liver Tumor Contrast for Radiation Therapy: Inter-Patient and Inter-Sequence Variability

Purpose: To determine the variation in tumor contrast between different MRI sequences and between patients for the purpose of MRI-based treatment planning. Methods: Multiple MRI scans of 11 patients with cancer(s) in the liver were included in this IRB-approved study. Imaging sequences consisted of T1W MRI, Contrast-Enhanced T1W MRI, T2W MRI, and T2*/T1W MRI. MRI images were acquired on a 1.5T GE Signa scanner with a four-channel torso coil. We calculated the tumor-to-tissue contrast to noise ratio (CNR) for each MR sequence by contouring the tumor and a region of interest (ROI) in a homogeneous region of the liver using the Eclipse treatment planning software. CNR was calculated (I-Tum-I-ROI)/SD-ROI, where I-Tum and I-ROI are the mean values of the tumor and the ROI respectively, and SD-ROI is the standard deviation of the ROI. The same tumor and ROI structures were used in all measurements for different MR sequences. Inter-patient Coefficient of variation (CV), and inter-sequence CV was determined. In addition, mean and standard deviation of CNR were calculated and compared between different MR sequences. Results: Our preliminary results showed large inter-patient CV (range: 37.7% to 88%) and inter-sequence CV (range 5.3% to 104.9%) of liver tumor CNR, indicating great variations in tumor CNR between MR sequences and between patients. Tumor CNR was found to be largest in CE-T1W (8.5±7.5), followed by T2W (4.2±2.4), T1W (3.4±2.2), and T2*/T1W (1.7±0.6) MR scans. The inter-patient CV of tumor CNR was also the largest in CE-T1W (88%), followed by T1W (64.3%), T1W (56.2%), and T2*/T1W (37.7) MR scans. Conclusion: Large inter-sequence and inter-patient variations were observed in liver tumor CNR. CE-T1W MR images on average provided the best tumor CNR. Efforts are needed to optimize tumor contrast and its consistency for MRI-based treatment planning of cancer in the liver. This project is supported by NIH grant: 1R21CA165384.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2016

SU-F-J-163: In Vivo Quantification of Sequence Parameter Effect On Geometric Distortion Caused by Implanted Titanium Brachytherapy Applicator

Purpose: The use of MR to plan and evaluate brachytherapy treatment for cervical cancer is increasing given the availability of MR conditional or safe applicators and MRI’s proven superiority to CT for characterizing soft tissue lesions. The titanium applicators, however, cause geometric distortions or imaging artifacts, which reduce the utility of MRI for dosimetry. We sought to quantify the observed volume of the same applicator on a previously optimized T2 sequence in comparison to the conventional T2 sequence and CT obtained for brachytherapy planning. Methods: Prior work with testing in phantoms showed that increases in readout bandwidth yielded reductions in artifact area and distortion measurements even with voxel increases. Following IRB approval, nine patients with titanium tandem & ovoid applicator (Varian Medical Systems) in place were scanned with a standard periprocedural protocol which included sagittal T2 fast spin echo (FSE) acquisition (res 0.98×0.78×4.0 mm{sup 3}; BW 200Hz). An additional T2-weighted FSE sequence (res 0.98×0.98×3–4 mm{sup 3}; BW500Hz) with increased readout bandwidth, readout voxel size, and echo train length was added to the protocol. Volume measurements of the applicator (from tip to cervical stop) were hand-segmented in Velocity AI 3.1 (Velocity Medical Solutions) for the two T2 FSE sequences and a planning CT obtained shortly after MRI. Differences were analyzed using a paired t-test. Results: Average apparent volumes of the applicator on standard T2 sequence, decreased bandwidth T2 sequence and CT were 5.922±1.283 cm{sup 3}, 4.544±1.524 cm3, and 2.304±0.509 cm{sup 3} respectively. Conclusion: Apparent volumes of a brachytherapy applicator can be compared in vivo. The modified sequence results in decreased apparent size of the cervical applicator. Both MR sequence volumes were larger than the planning CT, which was expected. Future work will focus on the diagnostic quality of the new sequence and quantifying any geometric shifts after CT to MRI registration based on anatomical landmarks.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2015

Rapid quantification of mutant fitness in diverse bacteria by sequencing randomly bar-coded transposons

Transposon mutagenesis with next-generation sequencing (TnSeq) is a powerful approach to annotate gene function in bacteria, but existing protocols for TnSeq require laborious preparation of every sample before sequencing. Thus, the existing protocols are not amenable to the throughput necessary to identify phenotypes and functions for the majority of genes in diverse bacteria. Here, we present a method, random bar code transposon-site sequencing (RB-TnSeq), which increases the throughput of mutant fitness profiling by incorporating random DNA bar codes into Tn5 and mariner transposons and by using bar code sequencing (BarSeq) to assay mutant fitness. RB-TnSeq can be used with any transposon, and TnSeq is performed once per organism instead of once per sample. Each BarSeq assay requires only a simple PCR, and 48 to 96 samples can be sequenced on one lane of an Illumina HiSeq system. We demonstrate the reproducibility and biological significance of RB-TnSeq with Escherichia coli , Phaeobacter inhibens , Pseudomonas stutzeri , Shewanella amazonensis , and Shewanella oneidensis . To demonstrate the increased throughput of RB-TnSeq, we performed 387 successful genome-wide mutant fitness assays representing 130 different bacterium-carbon source combinations and identified 5,196 genes with significant phenotypes across the five bacteria. In P. inhibens , we used our mutant fitness data to identify genes important for the utilization of diverse carbon substrates, including a putative D-mannose isomerase that is required for mannitol catabolism. RB-TnSeq will enable the cost-effective functional annotation of diverse bacteria using mutant fitness profiling. A large challenge in microbiology is the functional assessment of the millions of uncharacterized genes identified by genome sequencing. Transposon mutagenesis coupled to next-generation sequencing (TnSeq) is a powerful approach to assign phenotypes and functions to genes. However, the current strategies for TnSeq are too laborious to be applied to hundreds of experimental conditions across multiple bacteria. Here, we describe an approach, random bar code transposon-site sequencing (RB-TnSeq), which greatly simplifies the measurement of gene fitness by using bar code sequencing (BarSeq) to monitor the abundance of mutants. We performed 387 genome-wide fitness assays across five bacteria and identified phenotypes for over 5,000 genes. RB-TnSeq can be applied to diverse bacteria and is a powerful tool to annotate uncharacterized genes using phenotype data.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2013

When Is a Microbial Culture “Pure”? Persistent Cryptic Contaminant Escapes Detection Even with Deep Genome Sequencing

Geobacter sulfurreducens strain KN400 was recovered in previous studies in which a culture of the DL1 strain of G. sulfurreducens served as the inoculum in investigations of microbial current production at low anode potentials (-400 mV versus Ag/AgCl). Differences in the genome sequences of KN400 and DL1 were too great to have arisen from adaptive evolution during growth on the anode. Previous deep sequencing (80-fold coverage) of the DL1 culture failed to detect sequences specific to KN400, suggesting that KN400 was an external contaminant inadvertently introduced into the anode culturing system. In order to evaluate this further, a portion of the gene for OmcS, a c -type cytochrome that both KN400 and DL1 possess, was amplified from the DL1 culture. HiSeq-2000 Illumina sequencing of the PCR product detected the KN400 sequence, which differs from the DL1 sequence at 14 bp, at a frequency of ca. 1 in 10 5 copies of the DL1 sequence. A similar low frequency of KN400 was detected with quantitative PCR of a KN400-specific gene. KN400 persisted at this frequency after intensive restreaking of isolated colonies from the DL1 culture. However, a culture in which KN400 could no longer be detected was obtained by serial dilution to extinction in liquid medium. The KN400-free culture could not grow on an anode poised at -400 mV. Thus, KN400 cryptically persisted in the culture dominated by DL1 for more than a decade, undetected by even deep whole-genome sequencing, and was only fortuitously uncovered by the unnatural selection pressure of growth on a low-potential electrode. IMPORTANCE: Repeated streaking of isolated colonies on solidified medium remains a common strategy for obtaining pure cultures, especially of difficult-to-cultivate microorganisms such as strict anaerobes. The results presented here demonstrate that verifying the purity of cultures obtained in this manner may be difficult because extremely rare variants can persist, undetectable with even deep genomic DNA sequencing. The only way to ensure that a culture is pure is to cultivate it from an initial single cell, which may be technically difficult for many environmentally significant microbes.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

SourceFinder: a Machine-Learning-Based Tool for Identification of Chromosomal, Plasmid, and Bacteriophage Sequences from Assemblies

High-throughput genome sequencing technologies enable the investigation of complex genetic interactions, including the horizontal gene transfer of plasmids and bacteriophages. However, identifying these elements from assembled reads remains challenging due to genome sequence plasticity and the difficulty in assembling complete sequences. In this study, we developed a classifier, using random forest, to identify whether sequences originated from bacterial chromosomes, plasmids, or bacteriophages. The classifier was trained on a diverse collection of 23,211 chromosomal, plasmid, and bacteriophage sequences from hundreds of bacterial species. In order to adapt the classifier to incomplete sequences, each complete sequence was subsampled into 5,000 nucleotide fragments and further subdivided into k-mers. This three-class classifier succeeded in identifying chromosomes, plasmids, and bacteriophages using k-mer distributions of complete and partial genome sequences, including simulated metagenomic scaffolds with minimum performance of 0.939 area under the receiver operating characteristic curve (AUC). This classifier, implemented as SourceFinder, has been made available as an online web service to help the community with predicting the chromosomal, plasmid, and bacteriophage sources of assembled bacterial sequence data (https://cge.food.dtu.dk/services/SourceFinder/).

59 BASIC BIOLOGICAL SCIENCES↗