scholarly journals A cloud-compatible bioinformatics pipeline for ultrarapid pathogen identification from next-generation sequencing of clinical samples

2014 ◽  
Vol 24 (7) ◽  
pp. 1180-1192 ◽  
Author(s):  
S. N. Naccache ◽  
S. Federman ◽  
N. Veeraraghavan ◽  
M. Zaharia ◽  
D. Lee ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Clinical Samples ◽  
Next Generation ◽  
Pathogen Identification ◽  
Bioinformatics Pipeline ◽  
Generation Sequencing

scholarly journals Investigation of neglected protists Blastocystis sp. and Dientamoeba fragilis in immunocompetent and immunodeficient diarrheal patients using both conventional and molecular methods

2021 ◽  
Vol 15 (10) ◽  
pp. e0009779
Author(s):  
Fakhriddin Sarzhanov ◽  
Funda Dogruman-Al ◽  
Monica Santin ◽  
Jenny G. Maloney ◽  
Ayse Semra Gureser ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Gastrointestinal Symptoms ◽  
Molecular Methods ◽  
Clinical Samples ◽  
Rrna Gene ◽  
Next Generation ◽  
Dientamoeba Fragilis ◽  
Significant Difference ◽  
Generation Sequencing ◽  
Immunocompetent Patients

Introduction The clinical significance of Blastocystis sp. and Dientamoeba fragilis in patients with gastrointestinal symptoms is a controversial issue. Since the pathogenicity of these protists has not been fully elucidated, testing for these organisms is not routinely pursued by most laboratories and clinicians. Thus, the prevalence of these organisms and the subtypes of Blastocystis sp. in human patients in Turkey are not well characterized. This study aimed to determine the prevalence of Blastocystis sp. and D. fragilis in the diarrheic stool samples of immunodeficient and immunocompetent patients using conventional and molecular methods and to identify Blastocystis sp. subtypes using next generation sequencing. Material and methods Individual stool specimens were collected from 245 immunodeficient and 193 immunocompetent diarrheic patients between March 2017 and December 2019 at the Gazi University Training and Research Hospital in Ankara, Turkey. Samples were screened for Blastocystis sp. and D. fragilis by conventional and molecular methods. Molecular detection of both protists was achieved by separate qPCRs targeting a partial fragment of the SSU rRNA gene. Next generation sequencing was used to identify Blastocystis sp. subtypes. Results The prevalence of Blastocystis sp. and D. fragilis was 16.7% and 11.9%, respectively as measured by qPCR. The prevalence of Blastocystis sp. and D. fragilis was lower in immunodeficient patients (12.7% and 10.6%, respectively) compared to immunocompetent patients (21.8% and 13.5%, respectively). Five Blastocystis sp. subtypes were identified and the following subtype distribution was observed: ST3 54.4% (n = 37), ST2 16.2% (n = 11), ST1 4.4% (n = 3), ST6 2.9% (n = 2), ST4 1.5% (n = 1), ST2/ST3 11.8% (n = 8) and ST1/ST3 8.8% (n = 6). There was no statistically significant difference in the distribution of Blastocystis sp. subtypes between immunocompetent and immunodeficient patients. Conclusion and recommendation Our findings demonstrated that Blastocystis sp. and D. fragilis are commonly present in immunocompetent and immunodeficient patients with diarrhea. This study is the first to use next generation sequencing to address the presence of Blastocystis sp. mixed subtypes and intra-subtype variability in clinical samples in Turkey.

scholarly journals Next generation sequencing of RB1gene for the molecular diagnosis of ethnic minority with retinoblastoma in Yunnan

2020 ◽  
Author(s):  
Huaiyu Gu ◽  
Zhen Zhang ◽  
Yi-shuang Xiao ◽  
Ru Shen ◽  
Hong-chao Jiang ◽  
...  
Keyword(s):  
Genetic Testing ◽  
Next Generation Sequencing ◽  
Low Income ◽  
Eastern China ◽  
Low Income Countries ◽  
Next Generation ◽  
Single Nucleotide Variants ◽  
Bioinformatics Pipeline ◽  
Generation Sequencing

Abstract Background: Retinoblastoma is a rare intraocular malignancy and typically initiated by inactivating biallelic mutations of RB1 gene. Each year, ~8,000 children worldwide are diagnosed for retinoblastoma. In high-income countries, patient survival is over 95% while low-income countries is ~30%.If disease is diagnosed early and treated in centers specializing in retinoblastoma, the survival might exceed 95% and many eyes could be safely treated and support a lifetime of good vision. In China, approximate 1,100 newly diagnosed cases are expected annually and 28 hospitals covering 25 provinces established centers classified by expertise and resources for better treatment options and follow-up. Comparing with other province of eastern China, Yunnan province is remote geographically. This might result that healthcare staff have low awareness of the role of genetic testing in management and screening in families.Methods: The patients with retinoblastoma were selected in Yunnan. DNA from blood was used for targeted gene sequencing. Then, an in-house bioinformatics pipeline was done to detect both single nucleotide variants and small insertions/deletions. The pathogenic mutations were identified and further confirmed by conventional methods and cosegregation in families.Results: Using our approach, targeted next generation sequencing was used to detect the mutation of these 12 probands. Bioinformatic predictions showed that nine mutations were found in our study and four were novel pathogenic variants in these nine mutations.Conclusions: It’s the first report to describe RB1 mutations in Yunnan children with retinoblastoma. This study would improve role of genetic testing for management and family screening.

Microsatellite instability detection using a large next-generation sequencing cancer panel across diverse tumour types

2019 ◽  
Vol 73 (2) ◽  
pp. 83-89 ◽  
Author(s):  
Jiuhong Pang ◽  
Tatyana Gindin ◽  
Mahesh Mansukhani ◽  
Helen Fernandes ◽  
Susan Hsiao
Keyword(s):  
Next Generation Sequencing ◽  
Microsatellite Instability ◽  
Microsatellite Loci ◽  
Clinical Samples ◽  
Next Generation ◽  
Diverse Range ◽  
Tree Classifier ◽  
Generation Sequencing ◽  
Cancer Panel ◽  
Clinical Cases

AimMicrosatellite instability (MSI), a hallmark of DNA mismatch repair deficiency, is a key molecular biomarker with multiple clinical implications including the selection of patients for immunotherapy, identifying patients who may have Lynch syndrome and predicting prognosis in patients with colorectal tumours. Next-generation sequencing (NGS) provides the opportunity to interrogate large numbers of microsatellite loci concurrently with genomic variants. We sought to develop a method to detect MSI that would not require paired normal tissue and would leverage the sequence data obtained from a broad range of tumours tested using our 467-gene NGS Columbia Combined Cancer Panel (CCCP).MethodsAltered mononucleotide and dinucleotide microsatellite loci across the CCCP region of interest were evaluated in clinical samples encompassing a diverse range of tumour types. The number of altered loci was used to develop a decision tree classifier model trained on the retrospectively collected cohort of 107 clinical cases sequenced by the CCCP assay.ResultsThe classifier was able to correctly classify all cases and was then used to analyse a test set of clinical cases (n=112) and was able to correctly predict their MSI status with 100% sensitivity and specificity. Analysis of recurrently altered loci identified alterations in genes involved in DNA repair, signalling and transcriptional regulation pathways, many of which have been implicated in MSI tumours.ConclusionThis study highlights the utility of this approach, which should be applicable to laboratories performing similar testing.

Next-generation sequencing for tumor mutation quantification using liquid biopsies

2020 ◽  
Vol 58 (2) ◽  
pp. 306-313 ◽  
Author(s):  
Mariano Provencio ◽  
Clara Pérez-Barrios ◽  
Miguel Barquin ◽  
Virginia Calvo ◽  
Fabio Franco ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Next Generation Sequencing ◽  
Correlation Coefficient ◽  
Resistance Mutation ◽  
Response To Treatment ◽  
Clinical Samples ◽  
Next Generation ◽  
Liquid Biopsies ◽  
Nsclc Patients ◽  
Generation Sequencing

AbstractBackgroundNon-small cell lung cancer (NSCLC) patients benefit from targeted therapies both in first- and second-line treatment. Nevertheless, molecular profiling of lung cancer tumors after first disease progression is seldom performed. The analysis of circulating tumor DNA (ctDNA) enables not only non-invasive biomarker testing but also monitoring tumor response to treatment. Digital PCR (dPCR), although a robust approach, only enables the analysis of a limited number of mutations. Next-generation sequencing (NGS), on the other hand, enables the analysis of significantly greater numbers of mutations.MethodsA total of 54 circulating free DNA (cfDNA) samples from 52 NSCLC patients and two healthy donors were analyzed by NGS using the Oncomine™ Lung cfDNA Assay kit and dPCR.ResultsLin’s concordance correlation coefficient and Pearson’s correlation coefficient between mutant allele frequencies (MAFs) assessed by NGS and dPCR revealed a positive and linear relationship between the two data sets (ρc = 0.986; 95% confidence interval [CI] = 0.975–0.991; r = 0.987; p < 0.0001, respectively), indicating an excellent concordance between both measurements. Similarly, the agreement between NGS and dPCR for the detection of the resistance mutation p.T790M was almost perfect (K = 0.81; 95% CI = 0.62–0.99), with an excellent correlation in terms of MAFs (ρc = 0.991; 95% CI = 0.981–0.992 and Pearson’s r = 0.998; p < 0.0001). Importantly, cfDNA sequencing was successful using as low as 10 ng cfDNA input.ConclusionsMAFs assessed by NGS were highly correlated with MAFs assessed by dPCR, demonstrating that NGS is a robust technique for ctDNA quantification using clinical samples, thereby allowing for dynamic genomic surveillance in the era of precision medicine.

scholarly journals Comparison of targeted next-generation sequencing for whole-genome sequencing of Hantaan orthohantavirus in Apodemus agrarius lung tissues

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Jin Sun No ◽  
Won-Keun Kim ◽  
Seungchan Cho ◽  
Seung-Ho Lee ◽  
Jeong-Ah Kim ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Clinical Samples ◽  
Whole Genome ◽  
Target Enrichment ◽  
Next Generation ◽  
Apodemus Agrarius ◽  
Target Capture ◽  
Generation Sequencing

Abstract Orthohantaviruses, negative-sense single-strand tripartite RNA viruses, are a global public health threat. In humans, orthohantavirus infection causes hemorrhagic fever with renal syndrome or hantavirus cardiopulmonary syndrome. Whole-genome sequencing of the virus helps in identification and characterization of emerging or re-emerging viruses. Next-generation sequencing (NGS) is a potent method to sequence the viral genome, using molecular enrichment methods, from clinical specimens containing low virus titers. Hence, a comparative study on the target enrichment NGS methods is required for whole-genome sequencing of orthohantavirus in clinical samples. In this study, we used the sequence-independent, single-primer amplification, target capture, and amplicon NGS for whole-genome sequencing of Hantaan orthohantavirus (HTNV) from rodent specimens. We analyzed the coverage of the HTNV genome based on the viral RNA copy number, which is quantified by real-time quantitative PCR. Target capture and amplicon NGS demonstrated a high coverage rate of HTNV in Apodemus agrarius lung tissues containing up to 103–104 copies/μL of HTNV RNA. Furthermore, the amplicon NGS showed a 10-fold (102 copies/μL) higher sensitivity than the target capture NGS. This report provides useful insights into target enrichment NGS for whole-genome sequencing of orthohantaviruses without cultivating the viruses.

scholarly journals Multiplex Picoliter-Droplet Digital PCR for Quantitative Assessment of DNA Integrity in Clinical Samples

2013 ◽  
Vol 59 (5) ◽  
pp. 815-823 ◽  
Author(s):  
Audrey Didelot ◽  
Steve K Kotsopoulos ◽  
Audrey Lupo ◽  
Deniz Pekin ◽  
Xinyu Li ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Simultaneous Detection ◽  
Digital Pcr ◽  
Clinical Samples ◽  
Dna Integrity ◽  
Next Generation ◽  
Sequencing Data ◽  
Integrity Test ◽  
Picoliter Droplet ◽  
Generation Sequencing

BACKGROUND Assessment of DNA integrity and quantity remains a bottleneck for high-throughput molecular genotyping technologies, including next-generation sequencing. In particular, DNA extracted from paraffin-embedded tissues, a major potential source of tumor DNA, varies widely in quality, leading to unpredictable sequencing data. We describe a picoliter droplet–based digital PCR method that enables simultaneous detection of DNA integrity and the quantity of amplifiable DNA. METHODS Using a multiplex assay, we detected 4 different target lengths (78, 159, 197, and 550 bp). Assays were validated with human genomic DNA fragmented to sizes of 170 bp to 3000 bp. The technique was validated with DNA quantities as low as 1 ng. We evaluated 12 DNA samples extracted from paraffin-embedded lung adenocarcinoma tissues. RESULTS One sample contained no amplifiable DNA. The fractions of amplifiable DNA for the 11 other samples were between 0.05% and 10.1% for 78-bp fragments and ≤1% for longer fragments. Four samples were chosen for enrichment and next-generation sequencing. The quality of the sequencing data was in agreement with the results of the DNA-integrity test. Specifically, DNA with low integrity yielded sequencing results with lower levels of coverage and uniformity and had higher levels of false-positive variants. CONCLUSIONS The development of DNA-quality assays will enable researchers to downselect samples or process more DNA to achieve reliable genome sequencing with the highest possible efficiency of cost and effort, as well as minimize the waste of precious samples.

scholarly journals Rapid Metagenomic Next-Generation Sequencing during an Investigation of Hospital-Acquired Human Parainfluenza Virus 3 Infections

2016 ◽  
Vol 55 (1) ◽  
pp. 177-182 ◽  
Author(s):  
Alexander L. Greninger ◽  
Danielle M. Zerr ◽  
Xuan Qin ◽  
Amanda L. Adler ◽  
Reigran Sampoleo ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Infection Prevention ◽  
Genomic Sequence ◽  
Clinical Samples ◽  
Whole Genome ◽  
Next Generation ◽  
Sequence Coverage ◽  
Medical Unit ◽  
Hospital Acquired ◽  
Generation Sequencing

ABSTRACT Metagenomic next-generation sequencing (mNGS) is increasingly used for the unbiased detection of viruses, bacteria, fungi, and eukaryotic parasites in clinical samples. Whole-genome sequencing (WGS) of clinical bacterial isolates has been shown to inform hospital infection prevention practices, but this technology has not been utilized during potential respiratory virus outbreaks. Here, we report on the use of mNGS to inform the real-time infection prevention response to a cluster of hospital-acquired human parainfluenza 3 virus (HPIV3) infections at a children's hospital. Samples from 3 patients with hospital-acquired HPIV3 identified over a 12-day period on a general medical unit and 10 temporally associated samples from patients with community-acquired HPIV3 were analyzed. Our sample-to-sequencer time was <24 h, while our sample-to-answer turnaround time was <60 h with a hands-on time of approximately 6 h. Eight (2 cases and 6 controls) of 13 samples had sufficient sequencing coverage to yield the whole genome for HPIV3, while 10 (2 cases and 8 controls) of 13 samples gave partial genomes and all 13 samples had >1 read for HPIV3. Phylogenetic clustering revealed the presence of identical HPIV3 genomic sequence in the two of the cases with hospital-acquired infection, consistent with the concern for recent transmission within the medical unit. Adequate sequence coverage was not recovered for the third case. This work demonstrates the promise of mNGS for providing rapid information for infection prevention in addition to microbial detection.

No-cost next generation sequencing of advanced cancer patients within the Strata Precision Oncology Network supports clinical trial enrollment.

2019 ◽  
Vol 37 (15_suppl) ◽  
pp. 3073-3073
Author(s):  
Marc Ryan Matrana ◽  
Scott A. Tomlins ◽  
Kat Kwiatkowski ◽  
Khalis Mitchell ◽  
Jennifer Marie Suga ◽  
...  
Keyword(s):  
Clinical Trial ◽  
Clinical Trials ◽  
Next Generation Sequencing ◽  
Clinical Samples ◽  
Precision Oncology ◽  
Cancer Type ◽  
Next Generation ◽  
Match Trial ◽  
The Impact ◽  
Generation Sequencing

3073 Background: Widespread integration of systematized next generation sequencing (NGS)-based precision oncology is hindered by numerous barriers. Hence, we developed the Strata trial (NCT03061305), a screening protocol to determine the impact of scaled precision oncology. Methods: We implemented no-cost NGS on formalin fixed paraffin embedded (FFPE) clinical samples for all patients with advanced tumors, a common portfolio of partnered therapeutic clinical trials, and robust infrastructure development across the Strata Precision Oncology Network. Results: Across the network of 17 centers, specimens from 8673/9222 (94%) patients were successfully tested in the Strata CLIA/CAP/NCI-MATCH accredited laboratory using comprehensive amplicon-based DNA and RNA NGS. Patients were tested with one of three StrataNGS test versions; the most recent panel assesses all classes of actionable alterations (mutations, copy number alterations, gene fusions, microsatellite instability, tumor mutation burden and PD-L1 expression). Median surface area of received FFPE tumor samples was 25mm2 (interquartile range 9-95mm2), and the median turnaround time from sample receipt to report was 6 business days. 2577 (27.9%) patients had highly actionable alterations, defined as alterations associated with within-cancer type FDA approved or NCCN guideline recommended therapies (1072 patients), NCI-MATCH trial arms (1467 patients), Strata-partnered therapeutic trials (327 patients), or specific alteration-matched FDA approved therapies in patients with cancers of unknown primary (71 patients). Of the 1467 patients matched to an NCI-MATCH trial arm, 15 enrolled. Of the 327 patients matched to one of nine Strata-partnered clinical trials, 77 (24%) were screen failures, while 250 (76%) have either enrolled or are being actively followed for enrollment upon progression. Conclusions: Through streamlined consent methods, electronic medical record queries, and high throughput laboratory testing at no cost to patients, we demonstrate that scaled precision oncology is feasible across a diverse network of healthcare systems when paired with access to relevant clinical trials. Clinical trial information: NCT03061305.

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