Duplicate Reads Research Articles

Integrating RFID Technology with Intelligent Classifiers for Meaningful Prediction Knowledge

Radio Frequency Identification (RFID) is wireless technology that has been designed to automatically identify tagged objects using a reader. Several applications of this technology have been introduced in past literature such as pet identification and luggage tracking which have increased the efficiency and effectiveness of each environment into which it was integrated. However, due to the ambiguous nature of the captured information with the existence of missing, wrong and duplicate readings, the wide-scale adoption of the architecture is limited to commercial sectors where the integrity of the observations can tolerate ambiguity. In this work, we propose an application of RFID to take the reporting of class attendance and to integrate a predictive classifier to extract high level meaningful information that can be used in diverse areas such as scheduling and low student retention. We conclude by providing an analysis of the core strengths and opportunities that exist for this concept and how we might extend it in future research.

221 RNA-Seq ANALYSIS OF INNER CELL MASS AND TROPHECTODERM CELLS OF EQUINE BLASTOCYSTS

The objective of this study was to characterise the transcriptome profiles of inner cell mass (ICM) and trophectoderm (TE) cells from horse blastocysts. Equine blastocysts were collected by uterine flushing 8 days after ovulation in mares superovulated with reFSH and reLH (Meyers-Brown et al. 2011 Anim. Reprod. Sci. 128, 52–59). The ICM were isolated by immunosurgery, whereas polar TE cells were obtained microsurgically. Individual ICM (n = 2) and TE (n = 3) samples were snap frozen in liquid nitrogen and stored at –80°C until processing. Total RNA was extracted from each individual sample using the Arcturus PicoPure RNA isolation kit including DNAse treatment. The RNA was then amplified using the SPIA-based Ovation RNASeq-II kit (NuGEN Inc., San Carlos, CA, USA). Sequencing libraries were prepared using the TruSeq DNA sample preparation kit (Illumina, San Diego, CA, USA). Libraries bearing unique indexes per sample were pooled and sequenced in a single lane of a HiSEqn 2000 apparatus (Illumina) by a single run of 100 bp. Data analysis was performed using CLC Genomics Workbench. Equus caballus genomic sequences and annotation (EquCab2.0) were obtained from the National Center for Biotechnology Information (NCBI). Sequence analysis was performed using CLC Genomics Workbench and differential expression by DeSeq analysis. A total of 196 669 501 reads were produced. After discarding duplicated and bad quality reads, an average of 22 508 594 reads per sample was used for analysis. Using the RNASeq algorithms, 77% of reads mapped to annotated transcripts. Among the 22 380 annotated genes, 11 677 and 11 919 we detected as expressed [reads per kilobase of exon model per million sequences (RPKM) > 0.3] in all ICM and TE samples, respectively. The correlation of RPKM values for all the genes analysed between any pair of ICM or TE samples was >0.97, indicating a high repeatability of the assay. Global analysis of the transcriptome by means of unsupervised clustering indicated that ICM and TE samples clustered to different groups. Genes known to be specific to ICM and TE were expressed primarily in their respective tissue, including KLF4, SOX2, POU5F1, NANOG, DNMT3B, LIN28A, FOXA2, SALL4, and HNF4A for ICM, and CDX2, KRT8, ATP12A, GRHL2, and GRHL1 for TE. In addition, genes related to primitive endoderm development were more highly expressed in ICM than in TE and included GATA4, GATA6, and PDGFRA. The DeSeq analysis between ICM and TE samples indicated that 1934 genes were differentially expressed (adjusted P < 0.01 and fold change >2). Biological functions overrepresented among genes that were overexpressed in ICM cells (n = 1374) included cell-cell adhesion, cell morphogenesis involved in differentiation, negative regulation of DNA binding, and cell proliferation. Among genes overexpressed in TE samples (n = 560), gene ontology analysis indicated that the most overrepresented biological processes were lipid localization, placenta development, and organic acid transport. In summary, this study provides a comprehensive analysis of genes expressed in ICM and TE of equine embryos, which is a fundamental resource to understanding early embryo development in this species.

Bromodomain-containing Protein 4 (BRD4) Regulates RNA Polymerase II Serine 2 Phosphorylation in Human CD4+ T Cells

Transcriptional elongation by RNA polymerase II (Pol II) is regulated by positive transcription elongation factor b (P-TEFb) in association with bromodomain-containing protein 4 (BRD4). We used genome-wide chromatin immunoprecipitation sequencing in primary human CD4+ T cells to reveal that BRD4 co-localizes with Ser-2-phosphorylated Pol II (Pol II Ser-2) at both enhancers and promoters of active genes. Disruption of bromodomain-histone acetylation interactions by JQ1, a small-molecule bromodomain inhibitor, resulted in decreased BRD4 binding, reduced Pol II Ser-2, and reduced expression of lineage-specific genes in primary human CD4+ T cells. A large number of JQ1-disrupted BRD4 binding regions exhibited diacetylated H4 (lysine 5 and -8) and H3K27 acetylation (H3K27ac), which correlated with the presence of histone acetyltransferases and deacetylases. Genes associated with BRD4/H3K27ac co-occupancy exhibited significantly higher activity than those associated with H3K27ac or BRD4 binding alone. Comparison of BRD4 binding in T cells and in human embryonic stem cells revealed that enhancer BRD4 binding sites were predominantly lineage-specific. Our findings suggest that BRD4-driven Pol II phosphorylation at serine 2 plays an important role in regulating lineage-specific gene transcription in human CD4+ T cells.

Novel Genomic Structural Variations in Relapsed Childhood ETV6/RUNX1 (TEL/AML1) Acute Lymphoblastic Leukemias Identified by Next Generation Sequencing

AbstractAbstract 518 IntroductionThe chimeric fusion gene ETV6/RUNX1 generated by the interchromosomal translocation t(12;21) presents the most frequent chromosomal aberration in childhood acute lymphoblastic leukemia (ALL), occurring in approximately 25% of all patients. This ALL subtype is associated with an overall favorable prognosis, nevertheless 10–20% of children will suffer from relapse. ETV6/RUNX1-positive preleukemic clones arise already in utero, but require additional cooperating oncogenic lesions for the development of overt leukemia. The nature of the assisting genetic alterations and the mechanisms driving the development of leukemia and recurrent disease are still not well understood. MethodsWe applied state-of-the-art whole genome and whole exome next-generation-sequencing to comprehensively analyze the assisting oncogenic alterations in pediatric patients with initial and/or recurrent ETV6/RUNX1+ ALL (primary disease n=11, recurrent disease n=7). Matched sample sets taken at initial diagnosis, remission and relapse were compared. Mate pair and/or paired end sequencing was carried out for whole genome analysis with inserts spanning 2 kb or 500 kb, respectively. Constructed libraries were sequenced from both ends with 36- or 50-bp reads on a Genome Analyzer IIx or a HiSeq 2000 (Illumina/Solexa), respectively. Reads were aligned against the human reference genome (GRCh37) using BWA. Duplicate reads were removed. Unique reads with high mapping quality (q>35) served as input for GASV which detected translocations and inversions based on the mapping coordinates, insert sizes and read orientation. Variations covered by at least three reads in the tumor sample and not detected in the remission sample or the Database of Genomic Variants were reported. For detection of copy number variations, the program FREEC carried out coverage normalization, computation of copy number ratios between paired leukemia and remission samples (with up to 10 kb resolution) and subsequent segmentation. A subset of six selected patients was further investigated by targeted enrichment of whole exomic regions employing SeqCap EZ libraries (Roche) and 100 bp single read next-generation-sequencing on a HighSeq 2000. Mutations were called using GATK and further processed by an in-house bioinformatic pipeline. Putative somatically acquired mutations were validated by PCR, Sanger sequencing and FISH analysis. ResultsGenomes were sequenced to at least 13× physical coverage (mate pair) and 6.7× sequence coverage (paired-end). Exome sequencing achieved a minimum of 25× sequence coverage. In silico we detected 155 tumor-specific intragenic translocations. On average each tumor harbored 9 acquired translocations. With the exception of one case (13 translocations at diagnosis, 9 at relapse), the number of translocations was higher in relapse than in the matched diagnosis sample (additional 3 translocations on average). Ongoing validation studies confirmed the defining ETV6/RUNX1 translocation t(12;21) and identified 13 novel translocations. The genes affected are involved in essential signaling pathways, such as cytokine signaling (LIFR), calcium signaling (RCAN2), insulin and anti-apoptotic signaling (PHIP). Interestingly, also a factor essential for pre-mRNA splicing (IBP160) and genes encoding regulatory RNAs (miRNAs, lincRNAs and RNAs involved in splicing) were rearranged. A validated intragenic deletion of 836 bp leading to a frameshift and premature stop affected a calcium ion sensor of the ferlin protein family. Recurrent deletions in 9 of 11 cases (82%) ranging from 5 to 200 kb were detected in the immunoglobulin lambda variable gene cluster (IGLV) on chromosome 22q11. Some of the deletions were extending into the pre-B lymphocyte 1 gene (VPREB1) locus. In silico the probabilty of illegitimate RAG-mediated recombination at the breakpoint sites was determined by evaluation of RIC scores. RIC scores indicated that aberrant V(D)J rearrangements involving cryptic recombination sequence signals had caused the deletions on chromosome 22q11. ConclusionWe present somatic mutations that are promising novel candicate genes (e.g. LIFR, RCAN2, PHIP, IBP160) for cooperating secondary mutations in ETV6/RUNX1+ ALL and discuss their impact on the molecular pathology of primary and recurrent disease. Disclosures:No relevant conflicts of interest to declare.

Association between annual visit-to-visit blood pressure variability and stroke in postmenopausal women: data from the Women's Health Initiative.

Accumulating evidence suggests that increased visit-to-visit variability (VVV) of blood pressure is associated with stroke. No study has examined the association between VVV of blood pressure and stroke in postmenopausal women, and scarce data exist as to whether this relation is independent of the temporal trend of blood pressure. We examined the association of VVV of blood pressure with stroke in 58,228 postmenopausal women enrolled in the Women's Health Initiative. Duplicate blood pressure readings, which were averaged, were taken at baseline and at each annual visit. VVV was defined as the SD for the participant's mean systolic blood pressure (SBP) across visits (SD) and about the participant's regression line with SBP regressed across visits (SDreg). Over a median follow-up of 5.4 years, 997 strokes occurred. In an adjusted model including mean SBP over time, the hazard ratios (95% CI) of stroke for higher quartiles of SD of SBP compared with the lowest quartile (referent) were 1.39 (1.03-1.89) for quartile 2, 1.52 (1.13-2.03) for quartile 3, and 1.72 (1.28-2.32) for quartile 4 (P trend <0.001). The relation was similar for SDreg of SBP quartiles in a model that additionally adjusted for the temporal trend in SBP (P trend <0.001). The associations did not differ by stroke type (ischemic versus hemorrhagic). There was a significant interaction between mean SBP and SDreg on stroke with the strongest association seen below 120 mmHg. In postmenopausal women, greater VVV of SBP was associated with increased risk of stroke, particularly in the lowest range of mean SBP.

A Platform-Independent Method for Detecting Errors in Metagenomic Sequencing Data: DRISEE

We provide a novel method, DRISEE (duplicate read inferred sequencing error estimation), to assess sequencing quality (alternatively referred to as “noise” or “error”) within and/or between sequencing samples. DRISEE provides positional error estimates that can be used to inform read trimming within a sample. It also provides global (whole sample) error estimates that can be used to identify samples with high or varying levels of sequencing error that may confound downstream analyses, particularly in the case of studies that utilize data from multiple sequencing samples. For shotgun metagenomic data, we believe that DRISEE provides estimates of sequencing error that are more accurate and less constrained by technical limitations than existing methods that rely on reference genomes or the use of scores (e.g. Phred). Here, DRISEE is applied to (non amplicon) data sets from both the 454 and Illumina platforms. The DRISEE error estimate is obtained by analyzing sets of artifactual duplicate reads (ADRs), a known by-product of both sequencing platforms. We present DRISEE as an open-source, platform-independent method to assess sequencing error in shotgun metagenomic data, and utilize it to discover previously uncharacterized error in de novo sequence data from the 454 and Illumina sequencing platforms.

Development of pipeline for exome sequencing data analysis

Motivations. Exome sequencing the targeted sequencing of the subset of the protein coding human genome is a powerful and cost-effective new tool for dissecting the genetic basis of diseases and traits that have proved to be intractable to conventional gene-discovery strategies. Until now many algorithms have been produced, each of them addressing a different task in the downstream analysis of next-generation sequencing (NGS) data. The aim of this work is to combine these algorithms into an analysis pipeline for the detection of SNP and deletion/insertion polymorphisms within DNA sequences obtained by whole exome sequencing. The pipeline tested with data obtained from SRA (http://www.ncbi.nlm.nih.gov/sra), will then be applied to studies undergoing in our laboratory. Methods. Starting from raw sequence data, we first performed quality statistics and filtering of sequence reads and then aligned them to a reference genome. To this end, BWA was used to align both single- and paired-end reads for its computational efficiency and multi-platform compatibility. Post-alignment analysis, including removal of duplicate reads and quality score recalibration, was carried out using GATK, which takes into account several covariates such as machine cycle and dinucleotide context. Next, SNP calling was done using GATK UnifiedGenotyper, that uses a Bayesian model to estimate the most likely genotypes and allele frequency in a population of N samples, giving an annotated VCF file as output. Subsequently, variant quality score was recalibrated to estimate the probability of each variant being a true polymorphism, rather than a sequencer, alignment or data processing artifact, and finally filtered to improve the accuracy of genotype and SNP calling. Results. The results obtained support the accuracy of our pipeline to identify SNP and short indels, to provide a global and quantitative catalog of nucleotide variants in the exome. The next step will be to apply this pipeline to samples sequenced in our laboratory.

Fulcrum: condensing redundant reads from high-throughput sequencing studies

Ultra-high-throughput sequencing produces duplicate and near-duplicate reads, which can consume computational resources in downstream applications. A tool that collapses such reads should reduce storage and assembly complications and costs. We developed Fulcrum to collapse identical and near-identical Illumina and 454 reads (such as those from PCR clones) into single error-corrected sequences; it can process paired-end as well as single-end reads. Fulcrum is customizable and can be deployed on a single machine, a local network or a commercially available MapReduce cluster, and it has been optimized to maximize ease-of-use, cross-platform compatibility and future scalability. Sequence datasets have been collapsed by up to 71%, and the reduced number and improved quality of the resulting sequences allow assemblers to produce longer contigs while using less memory.

Limitations and possibilities of low cell number ChIP-seq

BackgroundChromatin immunoprecipitation coupled with high-throughput DNA sequencing (ChIP-seq) offers high resolution, genome-wide analysis of DNA-protein interactions. However, current standard methods require abundant starting material in the range of 1–20 million cells per immunoprecipitation, and remain a bottleneck to the acquisition of biologically relevant epigenetic data. Using a ChIP-seq protocol optimised for low cell numbers (down to 100,000 cells / IP), we examined the performance of the ChIP-seq technique on a series of decreasing cell numbers.ResultsWe present an enhanced native ChIP-seq method tailored to low cell numbers that represents a 200-fold reduction in input requirements over existing protocols. The protocol was tested over a range of starting cell numbers covering three orders of magnitude, enabling determination of the lower limit of the technique. At low input cell numbers, increased levels of unmapped and duplicate reads reduce the number of unique reads generated, and can drive up sequencing costs and affect sensitivity if ChIP is attempted from too few cells.ConclusionsThe optimised method presented here considerably reduces the input requirements for performing native ChIP-seq. It extends the applicability of the technique to isolated primary cells and rare cell populations (e.g. biobank samples, stem cells), and in many cases will alleviate the need for cell culture and any associated alteration of epigenetic marks. However, this study highlights a challenge inherent to ChIP-seq from low cell numbers: as cell input numbers fall, levels of unmapped sequence reads and PCR-generated duplicate reads rise. We discuss a number of solutions to overcome the effects of reducing cell number that may aid further improvements to ChIP performance.

P5-08-03: How Reader's Training, Software, and Image Formats Impact Percent Dense Area Measures.

Abstract BACKGROUND: Mammographic percent dense area, the percent ratio of dense to total breast area in a mammogram, is one of the strongest measures of a woman's risk of breast cancer. However, systematic differences have been observed between readers and mammography technologies (film and digital) that could cause clinically inconsistent associations with risk. The purpose of this study was to evaluate inter- and intra-reproducibility of percent dense area between readers and between film and digital technologies. METHODS: One hundred digitized film mammograms were randomly selected with 25 films in each of quartile of percent density and read by two readers at two different sites (Mayo Clinic and UCSF). The readers had extensive experience and were also jointly trained at university of Toronto using Cumulus software. After training, all films were read twice with at least one year between duplicate readings. The Mayo clinic reading used Cumulus while UCSF used custom semiautomatic software to estimate total and dense tissue area. In addition, digitized films and unprocessed full field digital mammograms of the same women were assessed by one reader. The time between the film and digital acquisitions ranged from nine to twenty-four months. Interclass correlation coefficient (ICC) was calculated for each comparison. RESULTS: The intra- and inter-observer ICCs, consistency for film images, were 0.96 (UCSF) and 0.97 (Mayo), and 0.96 (UCSF vs. Mayo). We found ICC between film and digital mammograms for percent dense area was 0.88. The digital mammogram had 9% significantly higher total breast area and 5% significantly lower percent density area compared to film. CONCLUSIONS: Similarly trained readers had a high reproducibility regardless of the software used. Our results suggest centralized reader training should enable pooling of film breast density results from different clinics. However, pooling film and digital results would need careful calibration due to lower measured percent dense areas than on film. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P5-08-03.

BIGpre: A Quality Assessment Package for Next-Generation Sequencing Data

The emergence of next-generation sequencing (NGS) technologies has significantly improved sequencing throughput and reduced costs. However, the short read length, duplicate reads and massive volume of data make the data processing much more difficult and complicated than the first-generation sequencing technology. Although there are some software packages developed to assess the data quality, those packages either are not easily available to users or require bioinformatics skills and computer resources. Moreover, almost all the quality assessment software currently available didn’t taken into account the sequencing errors when dealing with the duplicate assessment in NGS data. Here, we present a new user-friendly quality assessment software package called BIGpre, which works for both Illumina and 454 platforms. BIGpre contains all the functions of other quality assessment software, such as the correlation between forward and reverse reads, read GC-content distribution, and base Ns quality. More importantly, BIGpre incorporates associated programs to detect and remove duplicate reads after taking sequencing errors into account and trimming low quality reads from raw data as well. BIGpre is primarily written in Perl and integrates graphical capability from the statistics package R. This package produces both tabular and graphical summaries of data quality for sequencing datasets from Illumina and 454 platforms. Processing hundreds of millions reads within minutes, this package provides immediate diagnostic information for user to manipulate sequencing data for downstream analyses. BIGpre is freely available at http://bigpre.sourceforge.net/.

Somatic Structural Variations in Pediatric High Hyperdiploid Acute Lymphoblastic Leukemia Revealed by Paired-End Parallel Sequencing

Abstract 401 Introduction:High hyperdiploidy (51–67 chromosomes) is the most frequent numerical cytogenetic alteration found in pediatric B-cell precursor acute lymphoblastic leukemia (ALL), occurring in 25–30% of patients. It is characterized by nonrandom gains of chromosomes X, 4, 6, 10, 14, 17, 18, or 21. Children suffering from high hyperdiploid ALL have a good prognosis, nevertheless in 15–20% of cases the disease will recur. The mechanisms involved in the pathogenesis of primary and relapsing high hyperdiploid ALL are poorly understood. In some cases, IGH rearrangements arise in utero, indicating an early formation of pre-leukemic clones. However, the cellular origin of these pre-leukemic clones, as well as the molecular mechanism underlying the formation of high hyperdiploid cells, remains to be determined. Further genetic changes assisting in the development of ALL and recurrent disease are still unknown. Objective:By using massive parallel genome-wide next generation sequencing (Illumina/Solexa), we intended to identify specific cytogenetic structural variations (SVs) of high hyperdiploid ALL and possible clonal relationships between paired diagnostic and relapse ALL samples. Method:Paired-end sequencing libraries were generated from genomic DNA of diagnostic and relapse leukemic samples as well as germline DNA from the same patient. Libraries of two patients and one high hyperdiploid ALL cell line (MHH-CALL-2) with insert sizes of 350–400 bp were sequenced with paired end reads. Read lengths of 36 bp (Genome analyzer IIx) or 51 bp (HiSeq 2000) were sequenced, respectively. Sequencing raw data were aligned to the human reference genome hg19 (GRCh 37) by Burrows-Wheeler Aligner (BWA) and duplicate reads were removed. Copy number variants (CNVs), deletions, intrachromosomal inversions and interchromosomal translocations were analyzed by FREEC and GASV. After subtraction of germline SVs, putative leukemia-specific SVs were obtained. These were validated by PCR performed on genomic DNA. Specific breakpoints of SVs at single base resolution were identified by capillary sequencing of the PCR products. Results:Sequencing of different libraries yielded 95–279 million unique reads that mapped with both ends to the reference genome. Sequence coverages of 57–87% and fragment coverages of 4.9–12.3x were achieved (Table 1). [Display omitted] CNV profiles with 10 kb resolution were generated. A comparison of the CNVs of diagnosis and relapse ALL samples demonstrated a high degree of conformity with only few additional alterations present mainly, but not exclusively, in the relapse samples. In one of the patients, a large gain of chromosome 1q was only observed in the relapse sample (Figure 1). [Display omitted] SV analysis of all samples resulted in a total of 375 intragenic deletions, 16 intergenic inversions and 83 translocations (Table 1). PCR validation identified 2 previously unknown somatic translocations in the MHH-CALL-2 cell line concerning chromosomes 3 and 7 as well as chromosomes 15 and 18. Furthermore, 6 novel translocations present at diagnosis and relapse could be validated in patient samples. They were concerning chromosomes 3, 11, 12 and 20. One unique new relapse-specific translocation t(4;7) was identified. Conclusion:Paired-end sequencing of leukemia samples and matched non-tumor materials provides a robust tool for the discovery of genome-wide structural rearrangements. The high degree of conformity of CNVs and SVs detected in paired diagnosis/relapse samples indicate a common origin and a close relationship of the leukemic clones at diagnosis and relapse. The observation of few additional alterations in both diagnostic and relapse samples suggests the presence of different subclones at the time of diagnosis and the evolution of the relapse clone from either the diagnostic clone or a minor subclone. Disclosures:No relevant conflicts of interest to declare.

Preparation of high-quality next-generation sequencing libraries from picogram quantities of target DNA

New sequencing technologies can address diverse biomedical questions but are limited by a minimum required DNA input of typically 1 μg. We describe how sequencing libraries can be reproducibly created from 20 pg of input DNA using a modified transpososome-mediated fragmentation technique. Resulting libraries incorporate in-line bar-coding, which facilitates sample multiplexes that can be sequenced using Illumina platforms with the manufacturer's sequencing primer. We demonstrate this technique by providing deep coverage sequence of the Escherichia coli K-12 genome that shows equivalent target coverage to a 1-μg input library prepared using standard Illumina methods. Reducing template quantity does, however, increase the proportion of duplicate reads and enriches coverage in low-GC regions. This finding was confirmed with exhaustive resequencing of a mouse library constructed from 20 pg of gDNA input (about seven haploid genomes) resulting in ∼0.4-fold statistical coverage of uniquely mapped fragments. This implies that a near-complete coverage of the mouse genome is obtainable with this approach using 20 genomes as input. Application of this new method now allows genomic studies from low mass samples and routine preparation of sequencing libraries from enrichment procedures.

An Approach for Removing Redundant Data from RFID Data Streams

Radio frequency identification (RFID) systems are emerging as the primary object identification mechanism, especially in supply chain management. However, RFID naturally generates a large amount of duplicate readings. Removing these duplicates from the RFID data stream is paramount as it does not contribute new information to the system and wastes system resources. Existing approaches to deal with this problem cannot fulfill the real time demands to process the massive RFID data stream. We propose a data filtering approach that efficiently detects and removes duplicate readings from RFID data streams. Experimental results show that the proposed approach offers a significant improvement as compared to the existing approaches.

Alignment efficiency of superelastic coaxial nickel-titanium vs superelastic single-stranded nickel-titanium in relieving mandibular anterior crowding

To clinically evaluate the alignment efficiency of 0.016-inch coaxial superelastic nickel-titanium (NiTi) and 0.016-inch superelastic NiTi in the lower anterior region over a period of 12 weeks. A sample of 24 patients requiring lower anterior alignment were included in this single-center, single-operator, double-blind clinical trial and were randomly allocated into two groups of 12 patients. The type of wire selected for each patient was not disclosed to the provider or to the patient. Comparison of pretreatment characteristics of the archwire groups revealed no discrimination between two samples, thus verifying the random allocation of the intervention. An initial alginate impression of the lower arch was followed by impressions at 4-, 8-, and 12-week intervals. Casts were measured using the coordinate measuring machine to denote the degree of alignment. Duplicate readings of the cast series were taken to assess measurement variation. A statistically significant difference (P < .05) in the mean values of tooth movement demonstrated the superior aligning efficiency of coaxial superelastic NiTi over single-stranded superelastic NiTi in relieving lower anterior crowding. The measurement error recorded was within acceptable limits, with range values within 95% limits of agreement. Coaxial superelastic NiTi wire proved superior to single-stranded NiTi in its efficiency in relieving lower anterior crowding over a 12-week period.

Artificial duplicate reads in sequencing data of 454 Genome Sequencer FLX System

The 454 Genome Sequencer (GS) FLX System is one of the next-generation sequencing systems featured by long reads, high accuracy, and ultra-high throughput. Based on the mechanism of emulsion PCR, a unique DNA template would only generate a unique sequence read after being amplified and sequenced on GS FLX. However, biased amplification of DNA templates might occur in the process of emulsion PCR, which results in production of artificial duplicate reads. Under the condition that each DNA template is unique to another, 3.49%-18.14% of total reads in GS FLX-sequencing data were found to be artificial duplicate reads. These duplicate reads may lead to misunderstanding of sequencing data and special attention should be paid to the potential biases they introduced to the data.

Comparison of Automated Analysis of Cirrus HD OCT Spectral-Domain Optical Coherence Tomography with Stereo Photographs of the Optic Disc

To evaluate a new automated analysis of optic disc images obtained by spectral-domain optical coherence tomography (SD OCT). Areas of the optic disc, cup, and neural rim in SD OCT images were compared with these areas from stereoscopic photographs to represent the current traditional optic nerve evaluation. The repeatability of measurements by each method was determined and compared. Evaluation of diagnostic technology. One hundred nineteen healthy eyes, 23 eyes with glaucoma, and 7 glaucoma suspect eyes. Optic disc and cup margins were traced from stereoscopic photographs by 3 individuals independently. Optic disc margins and rim widths were determined automatically in SD OCT. A subset of photographs was examined and traced a second time, and duplicate SD OCT images also were analyzed. Agreement among photograph readers, between duplicate readings, and between SD OCT and photographs were quantified by the intraclass correlation coefficient (ICC), by the root mean square, and by the standard deviation of the differences. Optic disc areas tended to be slightly larger when judged in photographs than by SD OCT, whereas cup areas were similar. Cup and optic disc areas showed good correlation (0.8) between the average photographic reading and SD OCT, but only fair correlation of rim areas (0.4). The SD OCT was highly reproducible (ICC, 0.96-0.99). Each reader also was consistent with himself on duplicate readings of 21 photographs (ICC, 0.80-0.88 for rim area and 0.95-0.98 for all other measurements), but reproducibility was not as good as SD OCT. Measurements derived from SD OCT did not differ from photographic readings more than the readings of photographs by different readers differed from each other. Designation of the cup and optic disc boundaries by an automated analysis of SD OCT was within the range of variable designations by different readers from color stereoscopic photographs, but use of different landmarks typically made the designation of the optic disc size somewhat smaller in the automated analysis. There was better repeatability among measurements from SD OCT than from among readers of photographs. The repeatability of automated measurement of SD OCT images is promising for use both in diagnosis and in monitoring of progression.

Self-rated quality of life and school performance in relation to helminth infections: case study from Yunnan, People's Republic of China

BackgroundExpert opinion-derived disability weights are widely employed for estimating the global burden of diseases and injuries. For chronic diseases such as soil-transmitted helminthiasis and schistosomiasis, it has been suggested that a patient-based quality of life (QoL) approach should be considered for a more accurate appraisal of disability weights.Methods and ResultsWe carried out a cross-sectional survey and assessed the prevalence and intensity of soil-transmitted helminth infections as well as self-rated QoL indicators among 252 students attending grades 5-8 in two schools (Bulangshan and Pu'er) in Yunnan province, People's Republic of China. Each student provided a single stool sample, which was subjected to duplicate Kato-Katz thick smear readings and a single FLOTAC examination for parasitological diagnosis. Prevalence rates for hookworm, Trichuris trichiura and Ascaris lumbricoides were high in Bulangshan (75.9%, 70.0% and 68.2%), while the respective prevalence rates in Pu'er were 66.9%, 56.5% and 9.2%. Students were interviewed with two standardised questionnaires, the EuroQoL-5 Dimensions (EQ-5D) and ShortForm-12 (SF-12) Health Survey. Impairment in any of the five dimensions of the EQ-5D was reported by 87% of the students. However, no clear differences could be observed between individuals with and those without helminth infections, and there were discrepancies between the two schools. A multivariate logistic regression model revealed no differences between students with varying infection status in the domains of the SF-12 (odds ratio close to 1.0). Somewhat more pronounced, yet not statistically significant differences were observed when end-of-school-term marks were compared with students' helminth infection status: infected individuals had lower marks in Chinese, English and mathematics, but not in sports, compared to their helminth-free counterparts.ConclusionsOur results point to unresolved issues and challenges regarding the cultural appropriateness of the widely used standard QoL questionnaires. Hence, new research is needed to further develop these instruments and to validate them in connection with chronic parasitic diseases.

Reliability of vertebral fracture assessment using multidetector CT lateral scout views: the Framingham Osteoporosis Study

Two radiologists evaluated images of the spine from computed tomography (CT) scans on two occasions to diagnose vertebral fracture in 100 individuals. Agreement was fair to good for mild fractures, and agreement was good to excellent for more severe fractures. CT scout views are useful to assess vertebral fracture. We investigated inter-reader agreement between two radiologists and intra-reader agreement between duplicate readings for each radiologist, in assessment of vertebral fracture using a semi-quantitative method from lateral scout views obtained by CT. Participants included 50 women and 50 men (age 50-87 years, mean 70 years) in the Framingham Study. T4-L4 vertebrae were assessed independently by two radiologists on two occasions using a semi-quantitative scale as normal, mild, moderate, or severe fracture. Vertebra-specific prevalence of grade ≥ 1 (mild) fracture ranged from 3% to 5%. We found fair (κ = 56-59%) inter-reader agreement for grade ≥ 1 vertebral fractures and good (κ = 68-72%) inter-reader agreement for grade ≥ 2 fractures. Intra-reader agreement for grade ≥ 1 vertebral fracture was fair (κ = 55%) for one reader and excellent for another reader (κ = 77%), whereas intra-reader agreement for grade ≥ 2 vertebral fracture was excellent for both readers (κ = 76% and 98%). Thoracic vertebrae were more difficult to evaluate than the lumbar region, and agreement was lowest (inter-reader κ = 43%) for fracture at the upper (T4-T9) thoracic levels and highest (inter-reader κ = 76-78%) for the lumbar spine (L1-L4). Based on a semi-quantitative method to classify vertebral fractures using CT scout views, agreement within and between readers was fair to good, with the greatest source of variation occurring for fractures of mild severity and for the upper thoracic region. Agreement was good to excellent for fractures of at least moderate severity. Lateral CT scout views can be useful in clinical research settings to assess vertebral fracture.

Artificial and natural duplicates in pyrosequencing reads of metagenomic data

BackgroundArtificial duplicates from pyrosequencing reads may lead to incorrect interpretation of the abundance of species and genes in metagenomic studies. Duplicated reads were filtered out in many metagenomic projects. However, since the duplicated reads observed in a pyrosequencing run also include natural (non-artificial) duplicates, simply removing all duplicates may also cause underestimation of abundance associated with natural duplicates.ResultsWe implemented a method for identification of exact and nearly identical duplicates from pyrosequencing reads. This method performs an all-against-all sequence comparison and clusters the duplicates into groups using an algorithm modified from our previous sequence clustering method cd-hit. This method can process a typical dataset in ~10 minutes; it also provides a consensus sequence for each group of duplicates. We applied this method to the underlying raw reads of 39 genomic projects and 10 metagenomic projects that utilized pyrosequencing technique. We compared the occurrences of the duplicates identified by our method and the natural duplicates made by independent simulations. We observed that the duplicates, including both artificial and natural duplicates, make up 4-44% of reads. The number of natural duplicates highly correlates with the samples' read density (number of reads divided by genome size). For high-complexity metagenomic samples lacking dominant species, natural duplicates only make up <1% of all duplicates. But for some other samples like transcriptomic samples, majority of the observed duplicates might be natural duplicates.ConclusionsOur method is available from http://cd-hit.org as a downloadable program and a web server. It is important not only to identify the duplicates from metagenomic datasets but also to distinguish whether they are artificial or natural duplicates. We provide a tool to estimate the number of natural duplicates according to user-defined sample types, so users can decide whether to retain or remove duplicates in their projects.