Incorporation of unique molecular identifiers in TruSeq adapters improves the accuracy of quantitative sequencing

Sequencing Data ◽

Pcr Duplicates ◽

Positive Rate ◽

Bona Fide ◽

Multiplex Sequencing

Quantitative analysis of next-generation sequencing data requires discriminating duplicate reads generated by PCR from identical molecules that are of unique origin. Typically, PCR duplicates are defined as sequence reads that align to the same genomic coordinates using reference-based alignment. However, identical molecules can be independently generated during library preparation. The false positive rate of coordinate-based deduplication has not been well characterized and may introduce unforeseen biases during analyses. We developed a cost-effective sequencing adapter design by modifying Illumina TruSeq adapters to incorporate a unique molecular identifier (UMI) while maintaining the capacity to undertake multiplexed sequencing. Incorporation of UMIs enables identification of bona fide PCR duplicates as identically mapped reads with identical UMIs. Using TruSeq adapters containing UMIs (TrUMIseq adapters), we find that accurate removal of PCR duplicates results in enhanced data quality for quantitative analysis of allele frequencies in heterogeneous populations and gene expression.Method SummaryTrUMIseq adapters incorporate unique molecular identifiers in TruSeq adapters while maintaining the capacity to multiplex sequencing libraries using existing workflows. The use of UMIs increases the accuracy of quantitative sequencing assays, including RNAseq and allele frequency estimation, by enabling accurate detection of PCR duplicates.

Frozen Section of Thyroid and Parathyroid Specimens

Archives of Pathology & Laboratory Medicine ◽

10.5858/2005-129-1575-fsotap ◽

2005 ◽

Vol 129 (12) ◽

pp. 1575-1584 ◽

Cited By ~ 5

Author(s):

Rose C. Anton ◽

Thomas M. Wheeler

Keyword(s):

Thyroid Nodules ◽

Frozen Section ◽

False Positive Rate ◽

Cost Effective ◽

Needle Aspiration ◽

Thyroid Lobectomy ◽

Parathyroid Lesions ◽

Surgical Evaluation ◽

Positive Rate ◽

Accurate Procedure

Abstract Context.—Preoperative fine-needle aspiration of thyroid lesions has greatly diminished the need for surgical evaluation. However, because thyroid nodules are common lesions, many still require surgical intervention and represent a substantial number of cases that the pathologist encounters in the frozen section laboratory. Objective.—Comprehensive reviews of frozen section indications, as well as gross, cytologic, and histologic features of the most common and diagnostically important thyroid and parathyroid lesions, are presented to provide a guideline for proper triage and management of these cases in the frozen section laboratory. The most common pitfalls are discussed in an attempt to avoid discordant diagnoses. Data Sources.—Thyroid lobectomy, subtotal or total thyroidectomy, and parathyroid biopsy or parathyroidectomy cases are included in this review. Conclusions.—The frozen section evaluation of thyroid and parathyroid lesions remains a highly accurate procedure with a low false-positive rate. Gross inspection, complemented by cytologic and histologic review, provides the surgeon with the rapid, reliable, cost-effective information necessary for optimum patient care.

Clinical and Economic Evaluation after Adopting Contingent Cell-Free DNA Screening for Fetal Trisomies in South Spain

Fetal Diagnosis and Therapy ◽

10.1159/000508306 ◽

2020 ◽

Vol 47 (10) ◽

pp. 749-756

Author(s):

José A. Sainz ◽

María R. Torres ◽

Ignacio Peral ◽

Reyes Granell ◽

Manuel Vargas ◽

...

Keyword(s):

False Positive Rate ◽

Cost Effective ◽

First Trimester ◽

Nuchal Translucency ◽

University Hospitals ◽

Combined Test ◽

Positive Rate ◽

Fetal Malformations ◽

Cfdna Screening ◽

Singleton Pregnancies

Introduction: Contingent cell-free (cf) DNA screening on the basis of the first-trimester combined test (FCT) results has emerged as a cost-effective strategy for screening of trisomy 21 (T21). Objectives: To assess performance, patients’ uptake, and cost of contingent cfDNA screening and to compare them with those of the established FCT. Methods: This is a prospective cohort study including all singleton pregnancies attending to their FCT for screening of T21 at 2 university hospitals in South Spain. When the FCT risk was ≥1:50, there were major fetal malformations, or the nuchal translucency was ≥3.5 mm, women were recommended invasive testing (IT); if the risk was between 1:50 and 1:270, women were recommended cfDNA testing; and for risks bellow 1:270, no further testing was recommended. Detection rate (DR), false-positive rate (FPR), patients’ uptake, and associated costs were evaluated. Results: We analyzed 10,541 women, including 46 T21 cases. DR of our contingent strategy was 89.1% (41/46) at 1.4% (146/10,541) FPR. Uptake of cfDNA testing was 91.2% (340/373), and overall IT rate was 2.0%. The total cost of our strategy was €1,462,895.7, similar to €1,446,525.7 had cfDNA testing not been available. Conclusions: Contingent cfDNA screening shows high DR, low IT rate, and high uptake at a similar cost than traditional screening.

T-lex2: genotyping, frequency estimation and re-annotation of transposable elements using single or pooled next-generation sequencing data

Nucleic Acids Research ◽

10.1093/nar/gku1250 ◽

2014 ◽

Vol 43 (4) ◽

pp. e22-e22 ◽

Cited By ~ 50

Author(s):

Anna-Sophie Fiston-Lavier ◽

Maite G. Barrón ◽

Dmitri A. Petrov ◽

Josefa González

Keyword(s):

Transposable Elements ◽

Frequency Estimation ◽

Next Generation ◽

Sequencing Data ◽

Generation Sequencing

QPLOT: A Quality Assessment Tool for Next Generation Sequencing Data

BioMed Research International ◽

10.1155/2013/865181 ◽

2013 ◽

Vol 2013 ◽

pp. 1-4 ◽

Cited By ~ 7

Author(s):

Bingshan Li ◽

Xiaowei Zhan ◽

Mary-Kate Wing ◽

Paul Anderson ◽

Hyun Min Kang ◽

...

Keyword(s):

Quality Assessment ◽

Assessment Tool ◽

Cost Effective ◽

Next Generation ◽

Sequencing Data ◽

Sequence Alignments ◽

Automated Tool ◽

Generation Sequencing

Background.Next generation sequencing (NGS) is being widely used to identify genetic variants associated with human disease. Although the approach is cost effective, the underlying data is susceptible to many types of error. Importantly, since NGS technologies and protocols are rapidly evolving, with constantly changing steps ranging from sample preparation to data processing software updates, it is important to enable researchers to routinely assess the quality of sequencing and alignment data prior to downstream analyses.Results.Here we describe QPLOT, an automated tool that can facilitate the quality assessment of sequencing run performance. Taking standard sequence alignments as input, QPLOT generates a series of diagnostic metrics summarizing run quality and produces convenient graphical summaries for these metrics. QPLOT is computationally efficient, generates webpages for interactive exploration of detailed results, and can handle the joint output of many sequencing runs.Conclusion.QPLOT is an automated tool that facilitates assessment of sequence run quality. We routinely apply QPLOT to ensure quick detection of diagnostic of sequencing run problems. We hope that QPLOT will be useful to the community as well.

Comparing the clinical and economic effects of clinical examination, pulse oximetry, and echocardiography in newborn screening for congenital heart defects: A probabilistic cost-effectiveness model and value of information analysis

International Journal of Technology Assessment in Health Care ◽

10.1017/s0266462307070304 ◽

2007 ◽

Vol 23 (2) ◽

pp. 192-204 ◽

Cited By ~ 40

Author(s):

Ingolf Griebsch ◽

Rachel L. Knowles ◽

Jacqueline Brown ◽

Catherine Bull ◽

Christopher Wren ◽

...

Keyword(s):

Cost Effectiveness ◽

Newborn Screening ◽

Pulse Oximetry ◽

Clinical Examination ◽

Heart Defects ◽

False Positive Rate ◽

Research Priorities ◽

Cost Effective ◽

Positive Rate ◽

Clinically Significant

Objectives: Congenital heart defects (CHD) are an important cause of death and morbidity in early childhood, but the effectiveness of alternative newborn screening strategies in preventing the collapse or death—before diagnosis—of infants with treatable but life-threatening defects is uncertain. We assessed their effectiveness and efficiency to inform policy and research priorities.Methods: We compared the effectiveness of clinical examination alone and clinical examination with either pulse oximetry or screening echocardiography in making a timely diagnosis of life-threatening CHD or in diagnosing clinically significant CHD. We contrasted their cost-effectiveness, using a decision-analytic model based on 100,000 live births, and assessed future research priorities using value of information analysis.Results: Clinical examination alone, pulse oximetry, and screening echocardiography achieved 34.0, 70.6, and 71.3 timely diagnoses per 100,000 live births, respectively. This finding represents an additional cost per additional timely diagnosis of £4,894 and £4,496,666 for pulse oximetry and for screening echocardiography. The equivalent costs for clinically significant CHD are £1,489 and £36,013, respectively. Key determinants of cost-effectiveness are detection rates and screening test costs. The false-positive rate is very high with screening echocardiography (5.4 percent), but lower with pulse oximetry (1.3 percent) or clinical examination alone (.5 percent).Conclusions: Adding pulse oximetry to clinical examination is likely to be a cost-effective newborn screening strategy for CHD, but further research is required before this policy can be recommended. Screening echocardiography is unlikely to be cost-effective, unless the detection of all clinically significant CHD is considered beneficial and a 5 percent false-positive rate acceptable.

T-lex2: genotyping, frequency estimation and re-annotation of transposable elements using single or pooled next-generation sequencing data

10.1101/002964 ◽

2014 ◽

Cited By ~ 3

Author(s):

Anna-Sophie Fiston-Lavier ◽

Maite G. Barrón ◽

Dmitri A. Petrov ◽

Josefa González

Keyword(s):

Transposable Elements ◽

Frequency Estimation ◽

Next Generation ◽

Sequencing Data ◽

Flexible Tool ◽

Depth Analysis ◽

Individual Strain ◽

Generation Sequencing

Transposable elements (TEs) constitute the most active, diverse and ancient component in a broad range of genomes. Complete understanding of genome function and evolution cannot be achieved without a thorough understanding of TE impact and biology. However, in-depth analysis of TEs still represents a challenge due to the repetitive nature of these genomic entities. In this work, we present a broadly applicable and flexible tool: T-lex2. T-lex2 is the only available software that allows routine, automatic, and accurate genotyping of individual TE insertions and estimation of their population frequencies both using individual strain and pooled next-generation sequencing (NGS) data. Furthermore, T-lex2 also assesses the quality of the calls allowing the identification of miss-annotated TEs and providing the necessary information to re-annotate them. The flexible and customizable design of T-lex2 allows running it in any genome and for any type of TE insertion. Here, we tested the fidelity of T-lex2 using the fly and human genomes. Overall, T-lex2 represents a significant improvement in our ability to analyze the contribution of TEs to genome function and evolution as well as learning about the biology of TEs. T-lex2 is freely available online at http://sourceforge.net/projects/tlex/.

The Integration of Metabolomics and Next-Generation Sequencing Data to Elucidate the Pathways of Natural Product Metabolism in Medicinal Plants

Planta Medica ◽

10.1055/a-0630-1899 ◽

2018 ◽

Vol 84 (12/13) ◽

pp. 855-873 ◽

Cited By ~ 18

Author(s):

Federico Scossa ◽

Maria Benina ◽

Saleh Alseekh ◽

Youjun Zhang ◽

Alisdair Fernie

Keyword(s):

Medicinal Plants ◽

Secondary Metabolites ◽

Cost Effective ◽

Native Plant ◽

In Planta ◽

Sequencing Data ◽

Active Components ◽

Native Plant Species ◽

Historical Developments

AbstractPlants have always been used as medicines since ancient times to treat diseases. The knowledge around the active components of herbal preparations has remained nevertheless fragmentary: the biosynthetic pathways of many secondary metabolites of pharmacological importance have been clarified only in a few species, while the chemodiversity present in many medicinal plants has remained largely unexplored. Despite the advancements of synthetic biology for production of medicinal compounds in heterologous hosts, the native plant species are often the most reliable and economic source for their production. It thus becomes fundamental to investigate the metabolic composition of medicinal plants to characterize their natural metabolic diversity and to define the biosynthetic routes in planta of important compounds to develop strategies to further increase their content. We present here a number of case studies for selected classes of secondary metabolites and we review their health benefits and the historical developments in their structural elucidation and characterization of biosynthetic genes. We cover the cases of benzoisoquinoline and monoterpenoid indole alkaloids, cannabinoids, caffeine, ginsenosides, withanolides, artemisinin, and taxol; we show how the “early” biochemical or the more recent integrative approaches–based on omics-analyses–have helped to elucidate their metabolic pathways and cellular compartmentation. We also summarize how the knowledge generated about their biosynthesis has been used to develop metabolic engineering strategies in heterologous and native hosts. We conclude that following the advent of novel, high-throughput and cost-effective analytical technologies, the secondary metabolism of medicinal plants can now be examined under the lens of systems biology.

Investigating the Evolutionary Importance of Denisovan Introgressions in Papua New Guineans and Australians

10.1101/022632 ◽

2015 ◽

Author(s):

Ya Hu ◽

Qiliang Ding ◽

Yi Wang ◽

Shuhua Xu ◽

Yungang He ◽

...

Keyword(s):

False Positive Rate ◽

Whole Genome Sequencing Data ◽

Phase Method ◽

Sequencing Data ◽

Male Gonad ◽

Two Phase ◽

Genome Wide ◽

A Genome ◽

Positive Rate ◽

Olfactory Pit

Previous research reported that Papua New Guineans (PNG) and Australians contain introgressions from Denisovans. Here we present a genome-wide analysis of Denisovan introgressions in PNG and Australians. We firstly developed a two-phase method to detect Denisovan introgressions from whole-genome sequencing data. This method has relatively high detection power (79.74%) and low false positive rate (2.44%) based on simulations. Using this method, we identified 1.34 Gb of Denisovan introgressions from sixteen PNG and four Australian genomes, in which we identified 38,877 Denisovan introgressive alleles (DIAs). We found that 78 Denisovan introgressions were under positive selection. Genes located in the 78 introgressions are related to evolutionarily important functions, such as spermatogenesis, fertilization, cold acclimation, circadian rhythm, development of brain, neural tube, face, and olfactory pit, immunity, etc. We also found that 121 DIAs are missense. Genes harboring the 121 missense DIAs are also related to evolutionarily important functions, such as female pregnancy, development of face, lung, heart, skin, nervous system, and male gonad, visual and smell perception, response to heat, pain, hypoxia, and UV, lipid transport, metabolism, blood coagulation, wound healing, aging, etc. Taken together, this study suggests that Denisovan introgressions in PNG and Australians are evolutionarily important, and may help PNG and Australians in local adaptation. In this study, we also proposed a method that could efficiently identify archaic hominin introgressions in modern non-African genomes.

Validation of an ultra-fast CNV calling tool for Next Generation Sequencing data using MLPA-verified copy number alterations

10.1101/340505 ◽

2018 ◽

Author(s):

Bas Tolhuis ◽

Hans Karten

Keyword(s):

Copy Number ◽

False Positive Rate ◽

Copy Number Variations ◽

Next Generation ◽

Sequencing Data ◽

Single Node ◽

Generation Sequencing ◽

Cnv Detection

AbstractDNA Copy Number Variations (CNVs) are an important source for genetic diversity and pathogenic variants. Next Generation Sequencing (NGS) methods have become increasingly more popular for CNV detection, but its data analysis is a growing bottleneck. Genalice CNV is a novel tool for detection of CNVs. It takes care of turnaround time, scalability and cost issues associated with NGS computational analysis. Here, we validate Genalice CNV with MLPA-verified exon CNVs and genes with normal copy numbers. Genalice CNV detects 61 out of 62 exon CNVs and its false positive rate is less than 1%. It analyzes 96 samples from a targeted NGS assay in less than 45 minutes, including read alignment and CNV detection, using a single node. Furthermore, we describe data quality measures to minimize false discoveries. In conclusion, Genalice CNV is highly sensitive and specific, as well as extremely fast, which will be beneficial for clinical detection of CNVs.

Statistical modeling, estimation, and remediation of sample index hopping in multiplexed droplet-based single-cell RNA-seq data

10.1101/617225 ◽

2019 ◽

Cited By ~ 2

Author(s):

Rick Farouni ◽

Haig Djambazian ◽

Jiannis Ragoussis ◽

Hamed S. Najafabadi

Keyword(s):

Single Cell ◽

Statistical Modeling ◽

Experimental Validation ◽

Large Fraction ◽

False Positive Rate ◽

Rna Seq ◽

Sequencing Data ◽

Positive Rate ◽

Computational Workflow ◽

Reproducible Analysis

AbstractWe introduce a probabilistic model for estimation of sample index-hopping rate in multiplexed droplet-based single-cell RNA sequencing data and for inference of the true sample of origin of the hopped reads. Across the datasets we analyzed, we estimate the sample index hopping probability to range between 0.003–0.009, a small number that counter-intuitively gives rise to a large fraction of ‘phantom molecules’ – as high as 85% in a given sample. We demonstrate that our model-based approach can correct for this artifact by accurately purging the majority of phantom molecules from the data. Code and reproducible analysis notebooks are available at https://github.com/csglab/phantom_purge.StructureSection 1 provides a concise summary of the paper. Section 2 provides a brief historical and technical overview of the phenomenon of sample index hopping and an explanation of related concepts. The three sections that follow describe the statistical modeling approach and correspond to the following three goals. (1) Building a generative model that probabilistically describes the phenomenon of sample index hopping of multiplexed sample reads (Section 3). (2) Estimating the index hopping rate from empirical experimental data (Section 4). (3) Correcting for the effects of sample index hopping through a principled probabilistic procedure that reassigns reads to their true sample of origin and discards predicted phantom molecules by optimally minimizing the false positive rate (Section 5). Next, Section 6 details the results of the analyses performed on empirical and experimental validation datasets. The Supplementary Notes consists of three sections: (1) Mathematical Derivations, (2) Overview of Computational Workflow, (3) Method’s Limitations.