Development of a dual-index sequencing strategy and curation pipeline for analyzing amplicon sequence data on the MiSeq Illumina sequencing platform.

Rapid advances in sequencing technology have changed the experimental landscape of microbial ecology. In the last 10 years, the field has moved from sequencing hundreds of 16S rRNA gene fragments per study using clone libraries to the sequencing of millions of fragments per study using next-generation sequencing technologies from 454 and Illumina. As these technologies advance, it is critical to assess the strengths, weaknesses, and overall suitability of these platforms for the interrogation of microbial communities. Here, we present an improved method for sequencing variable regions within the 16S rRNA gene using Illumina's MiSeq platform, which is currently capable of producing paired 250-nucleotide reads. We evaluated three overlapping regions of the 16S rRNA gene that vary in length (i.e., V34, V4, and V45) by resequencing a mock community and natural samples from human feces, mouse feces, and soil. By titrating the concentration of 16S rRNA gene amplicons applied to the flow cell and using a quality score-based approach to correct discrepancies between reads used to construct contigs, we were able to reduce error rates by as much as two orders of magnitude. Finally, we reprocessed samples from a previous study to demonstrate that large numbers of samples could be multiplexed and sequenced in parallel with shotgun metagenomes. These analyses demonstrate that our approach can provide data that are at least as good as that generated by the 454 platform while providing considerably higher sequencing coverage for a fraction of the cost.

OptiFit: an Improved Method for Fitting Amplicon Sequences to Existing OTUs.

Assigning amplicon sequences to operational taxonomic units (OTUs) is an important step in characterizing microbial communities across large data sets. A notable difference between de novo clustering and database-dependent reference clustering methods is that OTU assignments from de novo methods may change when new sequences are added. However, one may wish to incorporate new samples to previously clustered data sets without clustering all sequences again, such as when comparing across data sets or deploying machine learning models. Existing reference-based methods produce consistent OTUs but only consider the similarity of each query sequence to a single reference sequence in an OTU, resulting in assignments that are worse than those generated by de novo methods. To provide an efficient method to fit sequences to existing OTUs, we developed the OptiFit algorithm. Inspired by the de novo OptiClust algorithm, OptiFit considers the similarity of all pairs of reference and query sequences to produce OTUs of the best possible quality. We tested OptiFit using four data sets with two strategies: (i) clustering to a reference database and (ii) splitting the data set into a reference and query set, clustering the references using OptiClust, and then clustering the queries to the references. The result is an improved implementation of reference-based clustering. OptiFit produces OTUs of a quality similar to that of OptiClust at faster speeds when using the split data set strategy. OptiFit provides a suitable option for users requiring consistent OTU assignments at the same quality as afforded by de novo clustering methods. IMPORTANCE Advancements in DNA sequencing technology have allowed researchers to affordably generate millions of sequence reads from microorganisms in diverse environments. Efficient and robust software tools are needed to assign microbial sequences into taxonomic groups for characterization and comparison of communities. The OptiClust algorithm produces high-quality groups by comparing sequences to each other, but the assignments can change when new sequences are added to a data set, making it difficult to compare different studies. Other approaches assign sequences to groups by comparing them to sequences in a reference database to produce consistent assignments, but the quality of the groups produced is reduced compared to that with OptiClust. We developed OptiFit, a new reference-based algorithm that produces consistent yet high-quality assignments like OptiClust. OptiFit allows researchers to compare microbial communities across different studies or add new data to existing studies without sacrificing the quality of the group assignments.

Assessing and improving methods used in operational taxonomic unit-based approaches for 16S rRNA gene sequence analysis.

In spite of technical advances that have provided increases in orders of magnitude in sequencing coverage, microbial ecologists still grapple with how to interpret the genetic diversity represented by the 16S rRNA gene. Two widely used approaches put sequences into bins based on either their similarity to reference sequences (i.e., phylotyping) or their similarity to other sequences in the community (i.e., operational taxonomic units [OTUs]). In the present study, we investigate three issues related to the interpretation and implementation of OTU-based methods. First, we confirm the conventional wisdom that it is impossible to create an accurate distance-based threshold for defining taxonomic levels and instead advocate for a consensus-based method of classifying OTUs. Second, using a taxonomic-independent approach, we show that the average neighbor clustering algorithm produces more robust OTUs than other hierarchical and heuristic clustering algorithms. Third, we demonstrate several steps to reduce the computational burden of forming OTUs without sacrificing the robustness of the OTU assignment. Finally, by blending these solutions, we propose a new heuristic that has a minimal effect on the robustness of OTUs and significantly reduces the necessary time and memory requirements. The ability to quickly and accurately assign sequences to OTUs and then obtain taxonomic information for those OTUs will greatly improve OTU-based analyses and overcome many of the challenges encountered with phylotype-based methods.

Introducing mothur: open-source, platform-independent, community-supported software for describing and comparing microbial communities.

mothur aims to be a comprehensive software package that allows users to use a single piece of software to analyze community sequence data. It builds upon previous tools to provide a flexible and powerful software package for analyzing sequencing data. As a case study, we used mothur to trim, screen, and align sequences; calculate distances; assign sequences to operational taxonomic units; and describe the alpha and beta diversity of eight marine samples previously characterized by pyrosequencing of 16S rRNA gene fragments. This analysis of more than 222,000 sequences was completed in less than 2 h with a laptop computer.

Broadband optical absorbance spectroscopy using a whispering gallery mode microsphere resonator.

We demonstrate the ability to excite and monitor many whispering gallery modes (WGMs) of a microsphere resonator simultaneously in order to make broadband optical absorbance measurements. The 340 microm diameter microsphere is placed in a microfluidic channel. A hemispherical prism is used for coupling the WGMs into and out of the microsphere. The flat surface of the prism seals the microfluidic channel. The slight nonsphericity in the microsphere results in coupling to precessed modes whose emission is spatially separated from the reflected excitation light. The evanescent fields of the light trapped in WGMs interact with the surrounding environment. The change in transmission observed in the precessed modes is used to determine the absorbance of the surrounding environment. In contrast to our broadband optical absorbance measurements, previous WGM sensors have used only a single narrow mode to measure properties such as refractive index. With the microfluidic cell, we have measured the absorbance of solutions of dyes (lissamine green B, sunset yellow, orange G, and methylene blue), aromatic molecules (benzylamine and benzoic acid), and biological molecules (tryptophan, phenylalanine, tyrosine, and o-phospho-L-tyrosine) at visible and ultraviolet wavelengths. The microsphere surface was reacted with organosilane molecules to attach octadecyl groups, amino groups, and fluorogroups to the surface. Both electrostatic and hydrophobic interactions were observed between the analytes and the microsphere surface, as indicated by changes in the measured effective pathlength with different organosilanes. For a given analyte and coated microsphere, the pathlength measurement was repeatable within a few percent. Methylene blue dye had a very strong interaction with the surface and pathlengths of several centimeters were measured. Choosing an appropriate surface coating to interact with a specific analyte should result in the highest sensitivity detection.

Biochemical analysis of a DNA replication origin in the archaeon Aeropyrum pernix.

We have characterised the interaction of the Aeropyrum pernix origin recognition complex proteins (ORC1 and ORC2) with DNA using DNase I footprinting. Each protein binds upstream of its respective gene. However, ORC1 protein alone interacts more tightly with an additional region containing multiple origin recognition box (ORB) sites that we show to be a replication origin. At this origin, there are four ORB elements disposed either side of an A+T-rich region. An ORC1 protein dimer binds at each of these ORB sites. Once all four ORB sites have bound ORC1 protein, there is a transition to a higher-order assembly with a defined alteration in topology and superhelicity. Furthermore, after this transition, the A+T-rich region becomes sensitive to digestion by DNase I and P1 nuclease, revealing that the transition promotes distortion of the DNA in this region, presumably as a prelude to loading of MCM helicase.

OptiClust, an Improved Method for Assigning Amplicon-Based Sequence Data to Operational Taxonomic Units.

Assignment of 16S rRNA gene sequences to operational taxonomic units (OTUs) is a computational bottleneck in the process of analyzing microbial communities. Although this has been an active area of research, it has been difficult to overcome the time and memory demands while improving the quality of the OTU assignments. Here, we developed a new OTU assignment algorithm that iteratively reassigns sequences to new OTUs to optimize the Matthews correlation coefficient (MCC), a measure of the quality of OTU assignments. To assess the new algorithm, OptiClust, we compared it to 10 other algorithms using 16S rRNA gene sequences from two simulated and four natural communities. Using the OptiClust algorithm, the MCC values averaged 15.2 and 16.5% higher than the OTUs generated when we used the average neighbor and distance-based greedy clustering with VSEARCH, respectively. Furthermore, on average, OptiClust was 94.6 times faster than the average neighbor algorithm and just as fast as distance-based greedy clustering with VSEARCH. An empirical analysis of the efficiency of the algorithms showed that the time and memory required to perform the algorithm scaled quadratically with the number of unique sequences in the data set. The significant improvement in the quality of the OTU assignments over previously existing methods will significantly enhance downstream analysis by limiting the splitting of similar sequences into separate OTUs and merging of dissimilar sequences into the same OTU. The development of the OptiClust algorithm represents a significant advance that is likely to have numerous other applications. IMPORTANCE The analysis of microbial communities from diverse environments using 16S rRNA gene sequencing has expanded our knowledge of the biogeography of microorganisms. An important step in this analysis is the assignment of sequences into taxonomic groups based on their similarity to sequences in a database or based on their similarity to each other, irrespective of a database. In this study, we present a new algorithm for the latter approach. The algorithm, OptiClust, seeks to optimize a metric of assignment quality by shuffling sequences between taxonomic groups. We found that OptiClust produces more robust assignments and does so in a rapid and memory-efficient manner. This advance will allow for a more robust analysis of microbial communities and the factors that shape them.

Sequencing 16S rRNA gene fragments using the PacBio SMRT DNA sequencing system.

Over the past 10 years, microbial ecologists have largely abandoned sequencing 16S rRNA genes by the Sanger sequencing method and have instead adopted highly parallelized sequencing platforms. These new platforms, such as 454 and Illumina's MiSeq, have allowed researchers to obtain millions of high quality but short sequences. The result of the added sequencing depth has been significant improvements in experimental design. The tradeoff has been the decline in the number of full-length reference sequences that are deposited into databases. To overcome this problem, we tested the ability of the PacBio Single Molecule, Real-Time (SMRT) DNA sequencing platform to generate sequence reads from the 16S rRNA gene. We generated sequencing data from the V4, V3-V5, V1-V3, V1-V5, V1-V6, and V1-V9 variable regions from within the 16S rRNA gene using DNA from a synthetic mock community and natural samples collected from human feces, mouse feces, and soil. The mock community allowed us to assess the actual sequencing error rate and how that error rate changed when different curation methods were applied. We developed a simple method based on sequence characteristics and quality scores to reduce the observed error rate for the V1-V9 region from 0.69 to 0.027%. This error rate is comparable to what has been observed for the shorter reads generated by 454 and Illumina's MiSeq sequencing platforms. Although the per base sequencing cost is still significantly more than that of MiSeq, the prospect of supplementing reference databases with full-length sequences from organisms below the limit of detection from the Sanger approach is exciting.

Synthesis and luminescence of ZnMgS:Mn2+ nanoparticles.

Efficient green emission from ZnMgS:Mn2+ nanoparticles prepared by co-doping Mg2+ and Mn2+ ions into ZnS lattices has been observed. The synthesis is carried out in aqueous solution, followed by a post-annealing process, thus showing the features of less complexity, low cost, and easy incorporation of dopants. In comparison with the emission of ZnS:Mn2+ nanoparticles, which is located generally around 590 nm, the photoluminescence of ZnMgS:Mn2+ nanoparticles is blue-shifted by 14 nm in wavelength, leading to the enhanced green emission. The X-ray diffraction, electron spin resonance, and pressure dependent photoluminescence measurements suggest that the change of the crystal field caused by Mg2+ ionic doping and the lower symmetry in the nanoparticles may account for the blue-shift of the photoluminescence. The ZnMgS:Mn2+ nanoparticles with 1% Mn2+ doping exhibit the strongest luminescence, which could potentially meet the requirements for the construction of green light emitting diodes.

De novo clustering methods outperform reference-based methods for assigning 16S rRNA gene sequences to operational taxonomic units.

Background. 16S rRNA gene sequences are routinely assigned to operational taxonomic units (OTUs) that are then used to analyze complex microbial communities. A number of methods have been employed to carry out the assignment of 16S rRNA gene sequences to OTUs leading to confusion over which method is optimal. A recent study suggested that a clustering method should be selected based on its ability to generate stable OTU assignments that do not change as additional sequences are added to the dataset. In contrast, we contend that the quality of the OTU assignments, the ability of the method to properly represent the distances between the sequences, is more important. Methods. Our analysis implemented six de novo clustering algorithms including the single linkage, complete linkage, average linkage, abundance-based greedy clustering, distance-based greedy clustering, and Swarm and the open and closed-reference methods. Using two previously published datasets we used the Matthew's Correlation Coefficient (MCC) to assess the stability and quality of OTU assignments. Results. The stability of OTU assignments did not reflect the quality of the assignments. Depending on the dataset being analyzed, the average linkage and the distance and abundance-based greedy clustering methods generated OTUs that were more likely to represent the actual distances between sequences than the open and closed-reference methods. We also demonstrated that for the greedy algorithms VSEARCH produced assignments that were comparable to those produced by USEARCH making VSEARCH a viable free and open source alternative to USEARCH. Further interrogation of the reference-based methods indicated that when USEARCH or VSEARCH were used to identify the closest reference, the OTU assignments were sensitive to the order of the reference sequences because the reference sequences can be identical over the region being considered. More troubling was the observation that while both USEARCH and VSEARCH have a high level of sensitivity to detect reference sequences, the specificity of those matches was poor relative to the true best match. Discussion. Our analysis calls into question the quality and stability of OTU assignments generated by the open and closed-reference methods as implemented in current version of QIIME. This study demonstrates that de novo methods are the optimal method of assigning sequences into OTUs and that the quality of these assignments needs to be assessed for multiple methods to identify the optimal clustering method for a particular dataset.

Gold and Silver Nanoparticles Functionalized by the Adsorption of Dialkyl Disulfides.

The formation of three-dimensional self-assembled monolayers (3-D SAMs) generated by the adsorption of n-octadecyl disulfide onto colloidal gold and silver nanoparticles is described. The functionalized nanoparticles were characterized by solubility, transmission electron microscopy, ultraviolet-visible spectroscopy, 1H nuclear magnetic resonance spectroscopy, surface-enhanced Raman spectroscopy, Fourier transform infrared spectroscopy, and X-ray photoelectron spectroscopy. On gold nanoparticles, this new functionalization method affords crystalline 3-D SAMs that are indistinct from those prepared by the analogous adsorption of n-octadecanethiol. On silver nanoparticles, however, the films derived from n-octadecyl disulfide appear to be somewhat less crystalline than those prepared similarly from n-octadecanethiol. The origin of this difference is briefly explored and discussed.

Reducing the effects of PCR amplification and sequencing artifacts on 16S rRNA-based studies.

The advent of next generation sequencing has coincided with a growth in interest in using these approaches to better understand the role of the structure and function of the microbial communities in human, animal, and environmental health. Yet, use of next generation sequencing to perform 16S rRNA gene sequence surveys has resulted in considerable controversy surrounding the effects of sequencing errors on downstream analyses. We analyzed 2.7×10(6) reads distributed among 90 identical mock community samples, which were collections of genomic DNA from 21 different species with known 16S rRNA gene sequences; we observed an average error rate of 0.0060. To improve this error rate, we evaluated numerous methods of identifying bad sequence reads, identifying regions within reads of poor quality, and correcting base calls and were able to reduce the overall error rate to 0.0002. Implementation of the PyroNoise algorithm provided the best combination of error rate, sequence length, and number of sequences. Perhaps more problematic than sequencing errors was the presence of chimeras generated during PCR. Because we knew the true sequences within the mock community and the chimeras they could form, we identified 8% of the raw sequence reads as chimeric. After quality filtering the raw sequences and using the Uchime chimera detection program, the overall chimera rate decreased to 1%. The chimeras that could not be detected were largely responsible for the identification of spurious operational taxonomic units (OTUs) and genus-level phylotypes. The number of spurious OTUs and phylotypes increased with sequencing effort indicating that comparison of communities should be made using an equal number of sequences. Finally, we applied our improved quality-filtering pipeline to several benchmarking studies and observed that even with our stringent data curation pipeline, biases in the data generation pipeline and batch effects were observed that could potentially confound the interpretation of microbial community data.

Structural basis of DNA replication origin recognition by an ORC protein.

DNA replication in archaea and in eukaryotes share many similarities. We report the structure of an archaeal origin recognition complex protein, ORC1, bound to an origin recognition box, a DNA sequence that is found in multiple copies at replication origins. DNA binding is mediated principally by a C-terminal winged helix domain that inserts deeply into the major and minor grooves, widening them both. However, additional DNA contacts are made with the N-terminal AAA+ domain, which inserts into the minor groove at a characteristic G-rich sequence, inducing a 35 degrees bend in the duplex and providing directionality to the binding site. Both contact regions also induce substantial unwinding of the DNA. The structure provides insight into the initial step in assembly of a replication origin and recruitment of minichromosome maintenance (MCM) helicase to that origin.

Physical therapy clinical management recommendations for children with cerebral palsy - spastic diplegia: achieving functional mobility outcomes.

The purpose of this special report is to present recommendations for the clinical management of children with cerebral palsy, spastic diplegia when increased functional mobility is the identified outcome. These recommendations provide a framework that allows physical therapists to increase their accountability and promote effective interventions for improved patient outcomes. The key components of this special report on clinical management are: a) the Major Recommendations that provide the background and evidence for clinical management; b) a flow chart to assist in clinical decision-making; and c) a Table of Tests and Measures for information on useful tools in the management of children with spastic diplegia. These recommendations are suggestions for clinical management, not an all-inclusive document on physical therapy for children with cerebral palsy. These recommendations may help therapists develop systematic approaches to service delivery and documentation.

Responsiveness of the Test of Infant Motor Performance (TIMP) in infants born preterm.

PURPOSE: The responsiveness of the Test of Infant Motor Performance (TIMP) was examined in infants born preterm. Infant variables predictive of changes in TIMP scores were also identified. METHODS: Twenty-five infants born

Exploring physical therapy clinical decision making for children with spastic diplegia: survey of pediatric practice.

PURPOSE: The purpose of this special interest report is to describe the outcomes of a research round table discussion regarding the physical therapy management of mobility for children with spastic diplegia. DESCRIPTION: Sixty-two pediatric physical therapists and physical therapists assistants participated in focus groups during the Research Round Table at the American Physical Therapy Association (APTA) 1999 Combined Sections Meeting. A case description of a child with spastic diplegia and guiding questions were used to facilitate discussion. SUMMARY OF EXPERIENCE: Common practices in patient management across the child's life-span emerged from the discussion. Practices in examination, evaluation and prognosis, and intervention differed depending on the age and function of the child and the family's needs. In general, therapists reported that younger children receive examinations that include standardized tests of development and ongoing intervention with a frequency of one to five times per week. In contrast, older children receive therapy services on an episodic basis that address their specific needs. The elements of patient management served as a useful framework for exploring decision making. IMPORTANCE TO PEDIATRIC PHYSICAL THERAPY: The information compiled from this project needs to be validated through systematic inquiry. Therapists may, however, use the practices reported here to reflect on their clinical decision making and to identify questions for further exploration. This descriptive document is the first step in the development of a guideline for evidence-based practice. The development of such a clinical guideline could serve as an education tool for novice therapists, a program evaluation tool to ensure quality care, and a foundation for future research to promote evidence-based practice.

Postural control in children: implications for pediatric practice.

Based on a systems theory of motor control, reactive postural control (RPA) and anticipatory postural control (APA) in children are reviewed from several perspectives in order to develop an evidence-based intervention strategy for improving postural control in children with limitations in motor function. Research on development of postural control, postural control in children with specific motor disabilities, and interventions to improve postural control is analyzed. A strategy for intervention to improve postural control systems at the impairment and functional activity levels based on a systems theoretical perspective is presented. Suggestions for research to improve evidence for best practice are provided.

Timed up and down stairs test: preliminary reliability and validity of a new measure of functional mobility.

PURPOSE: The reliability and validity of a new test, the Timed Up and Down Stairs (TUDS), were examined in children with and without cerebral palsy (CP). METHODS: A convenience sample of 47 children age eight to 14 years participated, 20 with CP and 27 with typical development (TD). Using intraclass correlation coefficients (ICC), interrater, intrarater, and test-retest reliability of the TUDS were examined by two raters simultaneously testing nine children, by one rater who then rescored at a later time a video of the testing of 24 children, and by one rater testing 25 children twice with a two-hour separation, respectively. Concurrent validity was examined using Spearman rank correlations between TUDS scores and performance on the Timed Up and Go (TUG), the Functional Reach Test (FRT), and a Timed One Legged Stance (TOLS), which were completed in a random order on all children. Construct validity was examined by correlation and Kruskal-Wallis analysis of variance of scores across ages and three functional level groups. RESULTS: The TUDS demonstrated excellent intrarater, interrater, and test-retest reliability [ICC (2,1) > or =0.94] and moderate to high concurrent validity (Spearman r(s) = 0.78, -0.57, and -0.77, with the TUG, FRT, and TOLS, respectively). Age accounted for 37% and 56% of the variance in the TUDS for the TD group and for the Gross Motor Function Classification Scale level I CP group, respectively. Significant differences in TUDS scores were found between all three functional level groups. CONCLUSION: The TUDS has adequate reliability and validity in children with and without CP and appears to complement current clinical measures of functional mobility and balance. Further investigations on across larger age ranges and samples are warranted.

Muscle force and range of motion as predictors of standing balance in children with cerebral palsy.

Children with cerebral palsy frequently receive therapeutic intervention to remediate standing balance deficits. Evaluation of the impairments associated with poor balance could facilitate more effective treatment programs. This study evaluated the relationship between lower extremity force production, range of motion and standing balance in thirty-five children between the ages of 6 and 14 years of age with spastic cerebral palsy. Standing balance was evaluated using the Pediatric Clinical Test of Sensory Interaction (P-CTSIB). Hand-held dynamometry was used to assess force production and goniometry was used to assess range of motion. The results indicated that force production and range of motion are highly related to standing balance. Blocked, hierarchical multiple regression analysis revealed that force production explained 41% of the variance in P-CTSIB scores in this sample, while range of motion explained an additional 13%. Therefore, the total variance explained by these variables was 54%. Results of this study suggest that impairment level testing may allow the development of more effective individualized intervention programs to remediate balance deficits. Clinical suggestions are provided.