BAMClipper: removing primers from alignments to minimize false-negative mutations in amplicon next-generation sequencing

Abstract Background Genomic structural variations (SV) are important determinants of genotypic and phenotypic changes in many organisms. However, the detection of SV from next-generation sequencing data remains challenging. Results In this study, DNA from a Chinese family quartet is sequenced at three different sequencing centers in triplicate. A total of 288 derivative data sets are generated utilizing different analysis pipelines and compared to identify sources of analytical variability. Mapping methods provide the major contribution to variability, followed by sequencing centers and replicates. Interestingly, SV supported by only one center or replicate often represent true positives with 47.02% and 45.44% overlapping the long-read SV call set, respectively. This is consistent with an overall higher false negative rate for SV calling in centers and replicates compared to mappers (15.72%). Finally, we observe that the SV calling variability also persists in a genotyping approach, indicating the impact of the underlying sequencing and preparation approaches. Conclusions This study provides the first detailed insights into the sources of variability in SV identification from next-generation sequencing and highlights remaining challenges in SV calling for large cohorts. We further give recommendations on how to reduce SV calling variability and the choice of alignment methodology.

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667. Next Generation Sequencing of Microbial Cell Free DNA in the Diagnosis and Treatment of Infectious Disease in Children: When Does the Result Justify the Cost?

Open Forum Infectious Diseases ◽

10.1093/ofid/ofab466.864 ◽

2021 ◽

Vol 8 (Supplement_1) ◽

pp. S436-S436

Author(s):

Rachel Downey Quick ◽

Kelli A Martinez ◽

Susan M Russo ◽

Sarah E McGwier ◽

Rachel A Quirt ◽

...

Keyword(s):

Next Generation Sequencing ◽

Antimicrobial Resistance ◽

False Negative ◽

Next Generation ◽

Cell Free Dna ◽

Negative Results ◽

Free Dna ◽

False Negative Results ◽

The Cost ◽

Generation Sequencing

Abstract Background Pathogen testing using next-generation sequencing of microbial cell-free DNA (NGS cfDNA) is a promising diagnostic tool to identify pathogens that might not be detected using conventional lab evaluation. Considering the cost of this test, it is important to determine when it is most useful to the plan of care (POC). Figure 1. Unit of admission among cases Figure 2. Patient characteristics in cases determined to be valuable and not valuable to the plan of care (POC) Methods In this retrospective study, we collected data from the medical charts of 50 consecutive NGS cfDNA tests in a free-standing children’s hospital. We evaluated patients for demographics, underlying conditions, diagnosis at time of testing, conventional laboratory testing and timing, medical treatment, and NGS cfDNA test results for clinical relevance or false negative results compared to conventional testing. The primary goal was to identify patients for whom the NGS cfDNA testing affected the POC. Charts were reviewed, and determinations regarding whether the result influenced the POC were confirmed by a provider. Results We were unable to differentiate patients with clinically valuable NGS cfDNA results (Fig 1 & 2). Among those with NGS cfDNA results valuable to the POC (n=22), both negative and positive testing guided POC (13 valuable negative vs. 9 diagnostic cases). In the total sample, 5 cases (10%) had a clinically relevant pathogen identified through conventional testing, but not through NGS cfDNA and 2 cases had antimicrobial resistance on culture, which is not detected by NGS cfDNA. Conclusion While we did not find a specific clinical profile for NGS cfDNA use, positive results were essential to the diagnosis in 18% of cases with otherwise negative laboratory evaluation for the pathogen identified in NGS cfDNA. Negative tests affected the POC in 26% of cases by avoiding unnecessary antimicrobials in high risk immunocompromised patients and patients that presented with low-risk of infection, but unclear disease process. Caution must be exercised with reliance on this test with respect to antimicrobial resistance and risk of false negative results. Disclosures All Authors: No reported disclosures

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Proficiency Testing of Standardized Samples Shows Very High Interlaboratory Agreement for Clinical Next-Generation Sequencing–Based Oncology Assays

Archives of Pathology & Laboratory Medicine ◽

10.5858/arpa.2018-0336-cp ◽

2018 ◽

Vol 143 (4) ◽

pp. 463-471 ◽

Cited By ~ 9

Author(s):

Jason D. Merker ◽

Kelly Devereaux ◽

A. John Iafrate ◽

Suzanne Kamel-Reid ◽

Annette S. Kim ◽

...

Keyword(s):

Next Generation Sequencing ◽

Proficiency Testing ◽

False Negative ◽

High Specificity ◽

Variant Allele ◽

Next Generation ◽

Single Nucleotide Variants ◽

Sequencing Platform ◽

Negative Results ◽

Generation Sequencing

Context.— Next-generation sequencing–based assays are being increasingly used in the clinical setting for the detection of somatic variants in solid tumors, but limited data are available regarding the interlaboratory performance of these assays. Objective.— To examine proficiency testing data from the initial College of American Pathologists (CAP) Next-Generation Sequencing Solid Tumor survey to report on laboratory performance. Design.— CAP proficiency testing results from 111 laboratories were analyzed for accuracy and associated assay performance characteristics. Results.— The overall accuracy observed for all variants was 98.3%. Rare false-negative results could not be attributed to sequencing platform, selection method, or other assay characteristics. The median and average of the variant allele fractions reported by the laboratories were within 10% of those orthogonally determined by digital polymerase chain reaction for each variant. The median coverage reported at the variant sites ranged from 1922 to 3297. Conclusions.— Laboratories demonstrated an overall accuracy of greater than 98% with high specificity when examining 10 clinically relevant somatic single-nucleotide variants with a variant allele fraction of 15% or greater. These initial data suggest excellent performance, but further ongoing studies are needed to evaluate the performance of lower variant allele fractions and additional variant types.

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False-negative errors in next-generation sequencing contribute substantially to inconsistency of mutation databases

PLoS ONE ◽

10.1371/journal.pone.0222535 ◽

2019 ◽

Vol 14 (9) ◽

pp. e0222535 ◽

Cited By ~ 3

Author(s):

Young-Ho Kim ◽

Yura Song ◽

Jong-Kwang Kim ◽

Tae-Min Kim ◽

Hye Won Sim ◽

...

Keyword(s):

Next Generation Sequencing ◽

False Negative ◽

Next Generation ◽

Generation Sequencing ◽

Mutation Databases

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Proficiency Testing of Standardized Samples Shows High Interlaboratory Agreement for Clinical Next-Generation Sequencing–Based Hematologic Malignancy Assays With Survey Material–Specific Differences in Variant Frequencies

Archives of Pathology & Laboratory Medicine ◽

10.5858/arpa.2019-0352-cp ◽

2020 ◽

Vol 144 (8) ◽

pp. 959-966 ◽

Cited By ~ 1

Author(s):

Alissa Keegan ◽

Julia A. Bridge ◽

Neal I. Lindeman ◽

Thomas A. Long ◽

Jason D. Merker ◽

...

Keyword(s):

Next Generation Sequencing ◽

Proficiency Testing ◽

Hematologic Malignancy ◽

False Negative ◽

False Negative Rate ◽

Hematologic Malignancies ◽

Variant Allele ◽

Next Generation ◽

Single Nucleotide Variants ◽

Generation Sequencing

Context.— As laboratories increasingly turn from single-analyte testing in hematologic malignancies to next-generation sequencing–based panel testing, there is a corresponding need for proficiency testing to ensure adequate performance of these next-generation sequencing assays for optimal patient care. Objective.— To report the performance of laboratories on proficiency testing from the first 4 College of American Pathologists Next-Generation Sequencing Hematologic Malignancy surveys. Design.— College of American Pathologists proficiency testing results for 36 different engineered variants and/or allele fractions as well as a sample with no pathogenic variants were analyzed for accuracy and associated assay performance characteristics. Results.— The overall sensitivity observed for all variants was 93.5% (2190 of 2341) with 99.8% specificity (22 800 of 22 840). The false-negative rate was 6.5% (151 of 2341), and the largest single cause of these errors was difficulty in identifying variants in the sequence of CEBPA that is rich in cytosines and guanines. False-positive results (0.18%; 40 of 22 840) were most likely the result of preanalytic or postanalytic errors. Interestingly, the variant allele fractions were almost uniformly lower than the engineered fraction (as measured by digital polymerase chain reaction). Extensive troubleshooting identified a multifactorial cause for the low variant allele fractions, a result of an interaction between the linearized nature of the plasmid and the Illumina TruSeq chemistry. Conclusions.— Laboratories demonstrated an overall accuracy of 99.2% (24 990 of 25 181) with 99.8% specificity and 93.5% sensitivity when examining 36 clinically relevant somatic single-nucleotide variants with a variant allele fraction of 10% or greater. The data also highlight an issue with artificial linearized plasmids as survey material for next-generation sequencing.

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Comprehensive Hybrid Capture–Based Next-Generation Sequencing Identifies a Double ALK Gene Fusion in a Patient Previously Identified to Be False-Negative by FISH

Journal of Thoracic Oncology ◽

10.1016/j.jtho.2016.11.2229 ◽

2017 ◽

Vol 12 (3) ◽

pp. e22-e24 ◽

Cited By ~ 6

Author(s):

Johannes M. Heuckmann ◽

Patrick Pauwels ◽

Erik Thunnissen

Keyword(s):

Next Generation Sequencing ◽

Gene Fusion ◽

False Negative ◽

Next Generation ◽

Hybrid Capture ◽

Generation Sequencing

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Application of metagenomic next-generation sequencing in the diagnosis of severe pneumonia caused by Chlamydia psittaci

BMC Pulmonary Medicine ◽

10.1186/s12890-021-01673-6 ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Huan-huan Wu ◽

Lan-fang Feng ◽

Shuang-yan Fang

Keyword(s):

Mechanical Ventilation ◽

Next Generation Sequencing ◽

Invasive Mechanical Ventilation ◽

False Negative ◽

Clinical Manifestations ◽

Severe Pneumonia ◽

Chlamydia Psittaci ◽

Diagnostic Tools ◽

Next Generation ◽

Generation Sequencing

Abstract Purpose Psittacosis is a zoonotic infectious disease caused by the transmission of the bacterium Chlamydia psittaci (C. psittaci) from birds to humans. Infections in humans mainly present as community-acquired pneumonia (CAP). However, most cases are treated without diagnostic testing, and the importance of Chlamydia psittaci infection as a cause of CAP is therefore unclear. Diagnostic tools, including culture, serologic test, and PCR-based methods, are available but prone to false negative results. Metagenomic next-generation sequencing (mNGS) has been increasingly used in the diagnosis of infectious diseases, particularly when conventional diagnostic approaches have limitation. Detection of nucleic acid sequence of C. psittaci in respiratory tract samples by metagenomic next-generation sequencing (mNGS) is effective for early diagnosis of severe C. psittaci pneumonia. Timely treatment based on tetracycline can reduce unnecessary use of antibiotics and improve prognosis of patients with severe C. psittaci pneumonia. Methods Clinical data of thirteen patients with severe C. psittaci pneumonia diagnosed by mNGS were collected. Clinical manifestations, treatment and prognosis of patients were summarized. Results The typical symptoms of pneumonia caused by C. psittaci include fever, headache, myalgia, cough, and dyspnea. In the current study, all patients met the criteria for severe C. psittaci pneumonia and received mechanical ventilation, including noninvasive mechanical ventilation (five/thirteen) and invasive mechanical ventilation (eight/thirteen). The findings showed that patients with C. psittaci pneumonia presented with normal or slightly increased leucocytes and procalcitonin, and high C-reactive protein levels. Computed tomography manifestations included consolidation of lung parenchyma, with air bronchogram and pleural effusion in some patients. mNGS analysis results were obtained within 48–72 h. Eleven patients fully recovered after targeted treatment, however, two patients died from secondary multidrug-resistant Pseudomonas aeruginosa infection. Conclusions The findings of the current study show that mNGS is effective in diagnosis of C. psittaci pneumonia, and has significant diagnosis value in patients with severe infection. Patients responds well to the timely use of appropriate antibiotics.

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Next-Generation Sequencing Somatic and Germline Assay Troubleshooting Guide Derived From Proficiency Testing Data

Archives of Pathology & Laboratory Medicine ◽

10.5858/arpa.2020-0842-cp ◽

2021 ◽

Author(s):

Valentina Nardi ◽

Karen D. Tsuchiya ◽

Annette S. Kim ◽

Lora J. H. Bean ◽

Jaimie G. Halley ◽

...

Keyword(s):

Next Generation Sequencing ◽

Proficiency Testing ◽

False Negative ◽

Gc Content ◽

Next Generation ◽

Improve Performance ◽

Common Causes ◽

Testing Data ◽

Survey Results ◽

Generation Sequencing

Context.— Next-generation sequencing–based assays are increasingly used in clinical molecular laboratories to detect somatic variants in solid tumors and hematologic malignancies and to detect constitutional variants. Proficiency testing data are potential sources of information about challenges in performing these assays. Objective.— To examine the most common sources of unacceptable results from the College of American Pathologists Next-Generation Sequencing Bioinformatics, Hematological Malignancies, Solid Tumor, and Germline surveys, and provide recommendations on how to avoid these pitfalls and improve performance. Design.— The College of American Pathologists next-generation sequencing somatic and germline proficiency testing survey results from 2016 to 2019 were analyzed to identify the most common causes of unacceptable results. Results.— On somatic and germline proficiency testing surveys, 95.9% (18 815/19 623) and 97.8% (33 890/34 641) of all variants were correctly identified, respectively. The most common causes of unacceptable results related to sequencing were false-negative errors in genomic regions that were difficult to sequence because of high GC content. False-positive errors occurred in the context of homopolymers and pseudogenes. Recurrent errors in variant annotation were seen for dinucleotide and duplication variants and included unacceptable transcript selection and outdated variant nomenclature. A small percentage of preanalytic or postanalytic errors were attributed to specimen swaps and transcription errors. Conclusions.— Laboratories demonstrate overall excellent performance for detecting variants in both somatic and germline proficiency testing surveys. Proficiency testing survey results highlight infrequent, but recurrent, analytic and nonanalytic challenges in performing next- generation sequencing–based assays and point to remedies to help laboratories improve performance.

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Profiling of the Causative Bacteria in Infected Lymphocysts after Lymphadenectomy for Gynecologic Cancer by Pyrosequencing the 16S Ribosomal RNA Gene Using Next-Generation Sequencing Technology

Infectious Diseases in Obstetrics and Gynecology ◽

10.1155/2019/9326285 ◽

2019 ◽

Vol 2019 ◽

pp. 1-5 ◽

Cited By ~ 1

Author(s):

Yuya Nogami ◽

Kouji Banno ◽

Masataka Adachi ◽

Haruko Kunitomi ◽

Yusuke Kobayashi ◽

...

Keyword(s):

Next Generation Sequencing ◽

Pathogenic Bacteria ◽

Sequence Data ◽

Bacterial Flora ◽

Gynecologic Cancer ◽

False Negative ◽

Enteric Bacteria ◽

Next Generation ◽

Single Strain ◽

Generation Sequencing

Background. Surgery for gynecologic cancer with lymphadenectomy and pelvic radiotherapy can produce lymphoceles that sometimes complicate with infection, resulting in abscesses. The true pathogenic bacteria of abscesses are not always found because of false-negative results due to administered antibiotics and difficulty with detection, including for anaerobic bacteria. Analyzing bacteria flora by next-generation sequencing (NGS) using 16S ribosomal DNA may reveal the true pathogenic bacteria in abscesses. This is the first report on causative pathogens for infectious lymphocele using this technology. Methods. The subjects were patients who developed infectious lymphocele after surgery for gynecologic cancer at our hospital from July 2015 to September 2016. NGS analyses of bacterial flora were performed using specimens preserved at -80°C. Two steps of PCR were performed for purified DNA samples to obtain sequence libraries. Processing of sequence data, including operational taxonomic unit (OTU) definition, taxonomy assignment, and an OTU BLAST search were performed. All patients gave written informed consent and the study was approved by the institutional research ethics committee. Results. Six patients underwent puncture and drainage. The result in most cases indicated a single causative pathogen, including Staphylococcus lugdunensis, Streptococcus dysgalactiae, Streptococcus equinus, Enterococcus saccharolyticus, and Escherichia coli. Conclusions. NGS revealed that the causative bacteria in lymphocele infection are normally a single strain, such as a surface Gram-positive coccus or enteric bacteria. Antibiotics should be chosen as appropriate for elimination of these respective bacteria.

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Clinical Analysis of Metagenomic Next-generation Sequencing Confirmed Chlamydia psittaci Pneumonia: A Case Series and Literature Review

10.21203/rs.3.rs-189997/v1 ◽

2021 ◽

Author(s):

Xin-Qi Teng ◽

Wen-Cheng Gong ◽

Ting-Ting Qi ◽

Guo-Hua Li ◽

Qiang Qu ◽

...

Keyword(s):

Next Generation Sequencing ◽

Literature Review ◽

Clinical Characteristics ◽

Pathogenic Bacteria ◽

False Negative ◽

False Negative Rate ◽

Case Series ◽

Chlamydia Psittaci ◽

Next Generation ◽

Generation Sequencing

Abstract Introduction: Chlamydia psittaci infection is a zoonotic infectious disease, which mainly inhaled through the lungs when exposed to the secretions of poultry that carry pathogenic bacteria. The traditional respiratory specimens or serological antibody testing is slow and the false-negative rate is high. Metagenomic next-generation sequencing gives a promising rapid diagnosis tool. Methods: We retrospective summarized the clinical characteristics of five C. psittaci pneumonia patients diagnosed by mNGS, conducted a literature review summarizing the clinical characteristics of patients with C. psittaci pneumonia reported since 2010.Results: Five C. psittaci. pneumonia patients confirmed by mNGS aged from 36 to 66 years with three males. 60% of patients had type 2 diabetes mellitus. And 60% of patients had a history of contact with avian or poultry. All patients had a high fever over 38.5 °C, cough, hypodynamia, hypoxemia, and dyspnea on admission. Two patients had invasive ventilator support and Extracorporeal Membrane Oxygenation support. The levels of C-reactive protein, procalcitonin, and erythrocyte sedimentation rate on admission and follow-up were all higher than normal values. Doxycycline or moxifloxacin monotherapy was accounted for 1/5 (20%) and 2/5 (40%) patients, and combination therapy was accounted for 2/5(40%) patients. Four patients improved and were discharged, and one patient died due to multiple organ failure and disseminated intravascular coagulation.Conclusions: mNGS can increase the detection rate of C. psittaci, shorten the diagnosis time of C. psittaci pneumonia and improve the prognosis of patients.

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