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Nucleotide sequence and taxonomy reference databases are critical resources for widespread applications including marker-gene and metagenome sequencing for microbiome analysis, diet metabarcoding, and environmental DNA (eDNA) surveys. Reproducibly generating, managing, using, and evaluating nucleotide sequence and taxonomy reference databases creates a significant bottleneck for researchers aiming to generate custom sequence databases. Furthermore, database composition drastically influences results, and lack of standardization limits cross-study comparisons. To address these challenges, we developed RESCRIPt, a Python 3 software package and QIIME 2 plugin for reproducible generation and management of reference sequence taxonomy databases, including dedicated functions that streamline creating databases from popular sources, and functions for evaluating, comparing, and interactively exploring qualitative and quantitative characteristics across reference databases. To highlight the breadth and capabilities of RESCRIPt, we provide several examples for working with popular databases for microbiome profiling (SILVA, Greengenes, NCBI-RefSeq, GTDB), eDNA and diet metabarcoding surveys (BOLD, GenBank), as well as for genome comparison. We show that bigger is not always better, and reference databases with standardized taxonomies and those that focus on type strains have quantitative advantages, though may not be appropriate for all use cases. Most databases appear to benefit from some curation (quality filtering), though sequence clustering appears detrimental to database quality. Finally, we demonstrate the breadth and extensibility of RESCRIPt for reproducible workflows with a comparison of global hepatitis genomes. RESCRIPt provides tools to democratize the process of reference database acquisition and management, enabling researchers to reproducibly and transparently create reference materials for diverse research applications. RESCRIPt is released under a permissive BSD-3 license at https://github.com/bokulich-lab/RESCRIPt .

Metabarcoding studies provide a powerful approach to estimate the diversity and abundance of organisms in mixed communities in nature. While strategies exist for optimizing sample and sequence library preparation, best practices for bioinformatic processing of amplicon sequence data are lacking in animal diet studies. Here we evaluate how decisions made in core bioinformatic processes, including sequence filtering, database design, and classification, can influence animal metabarcoding results. We show that denoising methods have lower error rates compared to traditional clustering methods, although these differences are largely mitigated by removing low‐abundance sequence variants. We also found that available reference datasets from GenBank and BOLD for the animal marker gene cytochrome oxidase I (COI) can be complementary, and we discuss methods to improve existing databases to include versioned releases. Taxonomic classification methods can dramatically affect results. For example, the commonly used Barcode of Life Database (BOLD) Classification API assigned fewer names to samples from order through species levels using both a mock community and bat guano samples compared to all other classifiers (vsearch‐SINTAX and q2‐feature‐classifier's BLAST + LCA, VSEARCH + LCA, and Naive Bayes classifiers). The lack of consensus on bioinformatics best practices limits comparisons among studies and may introduce biases. Our work suggests that biological mock communities offer a useful standard to evaluate the myriad computational decisions impacting animal metabarcoding accuracy. Further, these comparisons highlight the need for continual evaluations as new tools are adopted to ensure that the inferences drawn reflect meaningful biology instead of digital artifacts. Modern animal diet metabarcoding experiments are complicated assortments of many alternative bioinformatic pipelines. We chose to investigate how parameter choices in three core aspects common to any diet analysis (clustering/denoising, classification, and database development) may influence subsequent inferences. Our findings outline both a set of recommended processes and a template for future evaluations as new bioinformatic tools become available.

Nucleotide sequence and taxonomy reference databases are critical resources for widespread applications including marker-gene and metagenome sequencing for microbiome analysis, diet metabarcoding, and environmental DNA (eDNA) surveys. Reproducibly generating, managing, using, and evaluating nucleotide sequence and taxonomy reference databases creates a significant bottleneck for researchers aiming to generate custom sequence databases. Furthermore, database composition drastically influences results, and lack of standardization limits cross-study comparisons. To address these challenges, we developed RESCRIPt, a Python 3 software package and QIIME 2 plugin for reproducible generation and management of reference sequence taxonomy databases, including dedicated functions that streamline creating databases from popular sources, and functions for evaluating, comparing, and interactively exploring qualitative and quantitative characteristics across reference databases. To highlight the breadth and capabilities of RESCRIPt, we provide several examples for working with popular databases for microbiome profiling (SILVA, Greengenes, NCBI-RefSeq, GTDB), eDNA and diet metabarcoding surveys (BOLD, GenBank), as well as for genome comparison. We show that bigger is not always better, and reference databases with standardized taxonomies and those that focus on type strains have quantitative advantages, though may not be appropriate for all use cases. Most databases appear to benefit from some curation (quality filtering), though sequence clustering appears detrimental to database quality. Finally, we demonstrate the breadth and extensibility of RESCRIPt for reproducible workflows with a comparison of global hepatitis genomes. RESCRIPt provides tools to democratize the process of reference database acquisition and management, enabling researchers to reproducibly and transparently create reference materials for diverse research applications. RESCRIPt is released under a permissive BSD-3 license at https://github.com/bokulich-lab/RESCRIPt. Author summary Generating and managing sequence and taxonomy reference data presents a bottleneck to many researchers, whether they are generating custom databases or attempting to format existing, curated reference databases for use with standard sequence analysis tools. Evaluating database quality and choosing the “best” database can be an equally formidable challenge. We developed RESCRIPt to alleviate this bottleneck, supporting reproducible, streamlined generation, curation, and evaluation of reference sequence databases. RESCRIPt uses QIIME 2 artifact file formats, which store all processing steps as data provenance within each file, allowing researchers to retrace the computational steps used to generate any given file. We used RESCRIPt to benchmark several commonly used marker-gene sequence databases for 16S rRNA genes, ITS, and COI sequences, demonstrating both the utility of RESCRIPt to streamline use of these databases, but also to evaluate several qualitative and quantitative characteristics of each database. We show that larger databases are not always best, and curation steps to reduce redundancy and filter out noisy sequences may be beneficial for some applications. We anticipate that RESCRIPt will streamline the use, management, and evaluation/selection of reference database materials for microbiomics, diet metabarcoding, eDNA, and other diverse applications.

Abstract Background Nucleotide sequence and taxonomy reference databases are critical resources for widespread applications including marker-gene and metagenome sequencing for microbiome analysis, diet metabarcoding, and environmental DNA (eDNA) surveys. Reproducibly generating, managing, using, and evaluating nucleotide sequence and taxonomy reference databases creates a significant bottleneck for researchers aiming to generate custom sequence databases. Furthermore, database composition drastically influences results, and lack of standardizations limits cross-study comparisons. To address these challenges, we developed RESCRIPt, a software package for reproducible generation and management of reference sequence taxonomy databases, including dedicated functions that streamline creating databases from popular sources, and functions for evaluating, comparing, and interactively exploring qualitative and quantitative characteristics across reference databases. Results To highlight the breadth and capabilities of RESCRIPt, we provide several examples for working with popular databases for microbiome profiling (SILVA, Greengenes, NCBI-RefSeq, GTDB), eDNA, and diet metabarcoding surveys (BOLD, GenBank), as well as for genome comparison. We show that bigger is not always better, and reference databases with standardized taxonomies and those that focus on type strains have quantitative advantages, though may not be appropriate for all use cases. Most databases appear to benefit from some curation (quality filtering), though sequence clustering appears detrimental to database quality. Finally, we demonstrate the breadth and extensibility of RESCRIPt for reproducible workflows with a comparison of global hepatitis genomes. Conclusions RESCRIPt provides tools to democratize the process of reference database acquisition and management, enabling researchers to reproducibly and transparently create reference materials for diverse research applications. RESCRIPt is released under a permissive BSD-3 license at https://github.com/bokulich-lab/RESCRIPt. Competing Interest Statement The authors have declared no competing interest. Footnotes * https://github.com/bokulich-lab/RESCRIPt * https://doi.org/10.5281/zenodo.3891931 * https://github.com/bokulich-lab/db-benchmarks-2020 * https://github.com/devonorourke/COIdatabases * https://github.com/mikerobeson/q2-sourmash/tree/use-fasta

A food web is a model of the feeding relationships among organisms in an environment. The fidelity of this model is limited principally by the ability to detect these interactions. Researchers who study cryptic interactions such as nocturnal insectivory in bats typically rely on fecal samples to identify trophic connections. Historically these diet analyses were limited to morphological inspection of arthropod fragments, however modern metabarcoding techniques have improved the richness and specificity of consumed prey: rather than bats foraging for a few arthropod orders, we observe hundreds of species among guano samples. Animal metabarcoding is not without bias; nevertheless, a decade of improvements upon such biases have focused largely on molecular portions while bioinformatic considerations remain unresolved. When researchers use distinct software to perform their analyses—tools that have not yet been compared in animal metabarcoding studies—it is unclear if distinct perspectives between two experiments represent meaningful biological differences, or if they arise because of the alternative programs and parameters deployed. We investigated three fundamental bioinformatic tasks that impact a metabarcoding experiment: sequence processing, database construction, and classification (Chapter I). These comparisons offer guidance regarding which steps are most sensitive to parameterization and are therefore in need of optimizing for individual experiments, as well as highlight areas that are in need of critical improvement. We applied these bioinformatic lessons to a molecular diet analysis of Indiana bats, the first ever for this endangered species (Chapter II). While management decisions currently focus on protecting roosting habitat, our molecular analyses provide evidence that site-specific data is needed to more effectively inform conservation practices. For example, while these bats forage a broad swath of the arthropod community, the molecular data suggests they rely on particular aquatic habitats that are not currently protected. Finally, we investigated the diets of New Hampshire bats by collaborating with citizen scientist volunteers throughout the state to perform an extensive sampling regime in that spanned 20 locations over 2015 and 2016, and sequenced more than 900 guano samples (Chapter III). Molecular analysis of these data suggested these bats are foraging hundreds of arthropod species, including some turf and forest pests, demonstrating that our local bats provide ecosystem services. Individual diets varied across season and site, providing evidence of highly flexible and local foraging behaviors.

Massively parallel sequencing technologies have made it possible to generate large quantities of sequence data. However, as research-associated information is transferred into clinical practice, cost and throughput constraints generally require sequence-specific targeted analyses. Therefore, sample enrichment methods have been developed to meet the needs of clinical sequencing applications. However, current amplification and hybrid capture enrichment methods are limited in the contiguous length of sequences for which they are able to enrich. PCR based amplification also loses methylation data and other native DNA features. We have developed a novel technology (Negative Enrichment) where we demonstrate targeting long (>10 kb) genomic regions of interest. We use the specificity of CRISPR-Cas9 single guide RNA (Cas9/sgRNA) complexes to define 5' and 3' termini of sequence-specific loci in genomic DNA, targeting 10 to 36 kb regions. The complexes were found to provide protection from exonucleases, by protecting the targeted sequences from degradation, resulting in enriched, double-strand, non-amplified target sequences suitable for next-generation sequencing library preparation or other downstream analyses.

AbstractWe previously evaluated 119 consecutive total knee arthroplasties that were performed in eighty-six patients with use of the cemented LCS (low contact stress) rotating-platform system with an all-polyethylene patellar component. The average age of the patients at the time of surgery was seventy years (range, thirty-seven to eighty-eight years). The purpose of this study was to report the updated results at a minimum follow-up of fifteen years.Thirty-seven patients (fifty-three knees) were living, and no patient was lost to follow-up. No knee was revised because of loosening, osteolysis, or wear. Three knees required a reoperation (two for periprosthetic fractures and one for infection). No component was revised as a part of the reoperations. Osteolysis was present in three knees. No knee had radiographic signs of component loosening, and there were no dislocated bearings. The average range of motion was from 1° of extension to 105° of flexion. The average clinical and functional Knee Society scores were 43 and 49, respectively, at the preoperative evaluation and 85 and 58 at the time of the final follow-up. We concluded that the cemented LCS rotating-platform knee performed well, with durable clinical and radiographic results at a minimum follow-up of fifteen years.Level of EvidenceTherapeutic Level IV. See Instructions to Authors for a complete description of levels of evidence.

Most intermediate and long-term studies of cemented posterior-cruciate-substituting total knee prostheses were performed with nonmodular tibial components. The purpose of this study was to evaluate the intermediate-term results of posterior-cruciate-substituting total knee arthroplasties in which a cemented modular tibial component had been used, with a particular focus on evaluating the prevalence of radiographic osteolysis. Between 1992 and 1995, 176 consecutive primary total knee arthroplasties with use of the Insall-Burstein II system were performed in 134 patients at our institution. A modular metal-backed tibial component was inserted in 145 knees, and an all-polyethylene tibial component of the same design was inserted in thirty-one. Standard-terminology questionnaires were completed or Knee Society and The Hospital for Special Surgery scores were determined preoperatively and at the time of final follow-up, at an average of 6.4 years (range, 5.0 to 7.9 years). Initial postoperative radiographs were compared with those made at the time of final follow-up to assess component position, wear, radiolucent lines, and osteolysis. Ninety-two patients (128 knees) treated with the modular tibial component were alive at the time of final follow-up. No patient was lost to follow-up. Radiographs were available for 105 knees (82%). Three knees had been revised because of instability or infection; none had been revised because of loosening or osteolysis. The mean Knee Society clinical and functional scores were 85 points (range, 41 to 100 points) and 79 points (range, 30 to 100 points), respectively, at the time of final follow-up. According to The Hospital for Special Surgery score, 94% of the knees had a good or excellent result. Knee flexion averaged 113 degrees (range, 90 degrees to 130 degrees ) at the time of final follow-up. Osteolysis was present in seventeen (16%) of the knees with radiographic follow-up. Osteolysis did not develop in any knee in which an all-polyethylene tibial component had been used. Two knees (in one patient) were revised because of osteolytic lesions found at the time of follow-up for the study. Both of these knees had anterior wear of the tibial post due to impingement and backside tibial polyethylene wear. Modular Insall-Burstein II total knee prostheses were found to function well after five to eight years of follow-up. However, the high prevalence of osteolysis in patients who had good or excellent clinical scores is worrisome. Particular attention should be paid to preventing flexion of the femoral component, posterior slope of the tibial component, or hyperextension of the knee when posterior-cruciate-substituting total knee arthroplasty is performed. We also recommend routine follow-up radiographs after all total joint arthroplasties to detect asymptomatic osteolytic changes.

While considerable scholarship has explored responsibilities owed to research participants at the conclusion of explanatory clinical trials, no guidance exists regarding responsibilities owed at the conclusion of a pragmatic clinical trial (PCT). Yet post‐trial responsibilities in PCTs present distinct considerations from those emphasized in existing guidance and prior scholarship. Among these considerations include the responsibilities of the healthcare delivery systems in which PCTs are embedded, and decisions about implementation for interventions that demonstrate meaningful benefit following their integration into usual care settings—or deimplementation for those that fail to do so. In this article, we present an overview of prior scholarship and guidance on post‐trial responsibilities, and then identify challenges for post‐trial responsibilities for PCTs. We argue that, given one of the key rationales for PCTs is that they can facilitate uptake of their results by relevant decision‐makers, there should be a presumptive default that PCT study results be incorporated into future care delivery processes. Fulfilling this responsibility will require prospective planning by researchers, healthcare delivery system leaders, institutional review boards, and sponsors, so as to ensure that the knowledge gained from PCTs does, in fact, influence real‐world practice.

We previously evaluated 119 consecutive total knee arthroplasties that were performed in eighty-six patients with use of the cemented LCS (low contact stress) rotating-platform system with an all-polyethylene patellar component. The average age of the patients at the time of surgery was seventy years (range, thirty-seven to eighty-eight years). The purpose of this study was to report the updated results at a minimum follow-up of fifteen years. Thirty-seven patients (fifty-three knees) were living, and no patient was lost to follow-up. No knee was revised because of loosening, osteolysis, or wear. Three knees required a reoperation (two for periprosthetic fractures and one for infection). No component was revised as a part of the reoperations. Osteolysis was present in three knees. No knee had radiographic signs of component loosening, and there were no dislocated bearings. The average range of motion was from 1 degrees of extension to 105 degrees of flexion. The average clinical and functional Knee Society scores were 43 and 49, respectively, at the preoperative evaluation and 85 and 58 at the time of the final follow-up. We concluded that the cemented LCS rotating-platform knee performed well, with durable clinical and radiographic results at a minimum follow-up of fifteen years.