NGS-based Method Research Articles

Assessing the aerodynamic diameters of taxon-specific fungal bioaerosols by quantitative PCR and next-generation DNA sequencing

Aerodynamic diameter is an important determinant of the physical processes that act upon airborne fungi. Processes include gravitational settling, respiratory deposition, penetration into buildings, resuspension from surfaces into air, and long-range transport. This study combined next-generation DNA sequencing (NGS) with quantitative PCR (qPCR) to evaluate diverse, taxon-specific, fungal aerodynamic diameters from bioaerosol samples. The accuracy of the method was demonstrated by comparing geometric mean aerodynamic diameters of selected taxa produced by the NGS-based method to the diameters produced by taxon-specific qPCR (r=0.996). Geometric means (dg) and geometric standard deviations (σg) of aerodynamic diameters were characterized for more than 50 fungal taxa, spanning 55 genera, 9 classes, and 2 phyla. The results reported in this study demonstrate the robust nature of this method, provide novel insights into aerodynamic properties of diverse airborne fungal species, and potentially enable a better accounting of taxon-specific fungal fate and exposure both in indoor air and in the atmosphere.

Familial hypercholesterolaemia (FH) in Wales is genetically heterogeneous

Familial hypercholesterolaemia (FH) in Wales is genetically heterogeneous

Targeted next-generation sequencing: A novel diagnostic tool for primary immunodeficiencies

Primary immunodeficiency (PID) disorders are a heterogeneous group of inherited disorders caused by a variety of monogenetic immune defects. Thus far, mutations in more than 170 different genes causing PIDs have been described. A clear genotype-phenotype correlation is often not available, which makes a genetic diagnosis in patients with PIDs complex and laborious. We sought to develop a robust, time-effective, and cost-effective diagnostic method to facilitate a genetic diagnosis in any of 170 known PID-related genes by using next-generation sequencing (NGS). We used both targeted array-based and in-solution enrichment combined with a SOLiD sequencing platform and a bioinformatic pipeline developed in house to analyze genetic changes in the DNA of 41 patients with PIDs with known mutations and 26 patients with undiagnosed PIDs. This novel NGS-based method accurately detected point mutations (sensitivity and specificity >99% in covered regions) and exonic deletions (100% sensitivity and specificity). For the 170 genes of interest, the DNA coverage was greater than 20× in 90% to 95%. Nine PID-related genes proved not eligible for evaluation by using this NGS-based method because of inadequate coverage. The NGS method allowed us to make a genetic diagnosis in 4 of 26 patients who lacked a genetic diagnosis despite routine functional and genetic testing. Three of these patients proved to have an atypical presentation of previously described PIDs. This novel NGS tool facilitates accurate simultaneous detection of mutations in 161 of 170 known PID-related genes. In addition, these analyses will generate more insight into genotype-phenotype correlations for the different PID disorders.

Detection of Circulating Tumor Cells of Nonepithelial Origin: A Bench to Bedside Approach

Purpose/Objective(s): No validated biomarkers exist for minimal residual disease (MRD) detection in non-small cell lung cancer (NSCLC). Tumor DNA is continually shed into the circulation, where it can be accessed for analysis by next-generation sequencing (NGS). We aimed to design a NGS-based method for quantifying circulating tumor-derived DNA in NSCLC and other cancers. Materials/Methods: We developed CAPP-Seq (CAncer Personalized Profiling by deep Sequencing), a novel method for noninvasive and sensitive measurement of cancer burden from cfDNA. Our strategy leverages hybrid affinity capture to enrich recurrently mutated genomic regions from tumor genomic DNA and plasma cfDNA. We used mixtures of NSCLC cell line DNA in cfDNA to benchmark CAPP-Seq. We then analyzed a set of NSCLC tumor/normal genomic DNA pairs. Finally, we studied serial plasma samples collected from patients before and after treatment with radiation therapy, chemotherapy, and/or surgery. Results: We show that CAPP-Seq detects multiple types of cancer-specific somatic mutations within NSCLC DNA, including single nucleotide variants, insertions/deletions, and rearrangements. Benchmarking analyses demonstrate that CAPP-Seq accurately enumerates somatic mutations within cfDNA at a fractional abundance of 0.025%. Applying CAPP-Seq to tumor/normal genomic DNA pairs from 11 NSCLC patients uncovered a median of 3 somatic mutations per patient (range, 1-16). Fifteen plasma samples from 7 patients were profiled by CAPP-Seq. The abundance of patient-specific cancer markers within plasma cfDNA was readily measured by CAPP-Seq in both earlyand late-stage NSCLC. Importantly, circulating tumor DNA responded appropriately to anti-cancer therapies, becoming undetectable in 2 patients (after surgery and chemoradiation therapy, respectively) and declining but remaining detectable in 5 patients (after chemotherapy in 4 patients and surgery in 1 patient). CAPP-Seq measurements correlated with tumor volume (R Z 0.97) and allowed for the detection of resistance mutations in patients being treated with targeted therapies. Conclusions: In this study, we described CAPP-Seq, a novel NGS-based approach to MRD testing in cancer patients. CAPP-Seq could have clinical utility for monitoring disease burden and response to therapy, early detection of relapse, distinguishing radiation pneumonitis from relapse, and risk stratification in early-stage NSCLC following surgery or stereotactic body radiation therapy. Ultimately, we envision CAPP-Seq becoming a routine clinical assay for measuring tumor burden in diverse cancers from plasma and other body fluids. Author Disclosure: S.V. Bratman: None. A.M. Newman: None. J. To: None. J.F. Wynne: None. J.W. Neal: None. H. Wakelee: None. J. Shrager: None. B.W. Loo: None. A.A. Alizadeh: None. M. Diehn: None.

Noninvasive and Ultrasensitive Quantitation of Circulating Tumor DNA by Hybrid Capture and Deep Sequencing

No validated biomarkers exist for minimal residual disease (MRD) detection in non-small cell lung cancer (NSCLC). Tumor DNA is continually shed into the circulation, where it can be accessed for analysis by next-generation sequencing (NGS). We aimed to design a NGS-based method for quantifying circulating tumor-derived DNA in NSCLC and other cancers.

Next generation sequencing for human papillomavirus genotyping

BackgroundHuman papillomavirus (HPV) genotyping using next generation sequencing (NGS) could be useful to study the HPV variant-specific epidemiology, including monitoring for possible emergence of new HPV variants after introduction of HPV vaccination programs. ObjectivesWe wished to design and validate a method for rapid HPV detection, typing and sequencing in clinical samples. Study designPlasmids of 15 different HPV types were mixed and serially diluted in human DNA in concentrations ranging from 1 to 100 copies per sample, amplified using the HPV general PCR primer pair PGMY and sequenced using 454 technology. Sixty cervical samples were tested both with the NGS-based method and with a comparison method based on genotyping using type-specific probes bound to fluorescent beads (Luminex). Thirty-three clinical samples were repeat tested using NGS to evaluate reproducibility. ResultsThe NGS-based method correctly identified all 15 mixed HPV types when present in 100 copies/sample and 13/15 types when present in 10 copies/sample. For 36/60 cervical samples genotyping results using NGS and Luminex were identical. For 12/60 samples the NGS method was more sensitive than the Luminex test and most of the remaining discrepancies could be explained by the different type coverage of the assays. Reproducibility testing found complete or partial concordance in 30/33 samples. ConclusionsNGS provides a sensitive and accurate method for genotyping of HPV. The fact that also the amplimer sequence is obtained could be important for studying the epidemiology of viral variants and monitoring of HPV vaccination.

A Comprehensive Next Generation Sequencing–Based Genetic Testing Strategy To Improve Diagnosis of Inherited Pheochromocytoma and Paraganglioma

Pheochromocytomas and paragangliomas are notable for a high frequency of inherited cases, many of which present as apparently sporadic tumors. The objective of this study was to establish a comprehensive next generation sequencing (NGS)-based strategy for the diagnosis of patients with pheochromocytoma and paraganglioma by testing simultaneously for mutations in MAX, RET, SDHA, SDHB, SDHC, SDHD, SDHAF2, TMEM127, and VHL. After the methodology for the assay was designed and established, it was validated on DNA samples with known genotype and then patients were studied prospectively. The study was performed in a diagnostic genetics laboratory. DNA samples from 205 individuals affected with adrenal or extraadrenal pheochromocytoma/head and neck paraganglioma (PPGL/HNPGL) were analyzed. A proof-of-principle study was performed using 85 samples known to contain a variant in 1 or more of the genes to be tested, followed by prospective analysis of an additional 120 samples. We assessed the ability to use an NGS-based method to perform comprehensive analysis of genes implicated in inherited PPGL/HNPGL. The proof-of-principle study showed that the NGS assay and analysis gave a sensitivity of 98.7%. A pathogenic mutation was identified in 16.6% of the prospective analysis cohort of 120 patients. A comprehensive NGS-based strategy for the analysis of genes associated with predisposition to PPGL and HNPGL was established, validated, and introduced into diagnostic service. The new assay provides simultaneous analysis of 9 genes and allows more rapid and cost-effective mutation detection than the previously used conventional Sanger sequencing-based methodology.

Quantitative and Sensitive Detection of GNAS Mutations Causing McCune-Albright Syndrome with Next Generation Sequencing

Somatic activating GNAS mutations cause McCune-Albright syndrome (MAS). Owing to low mutation abundance, mutant-specific enrichment procedures, such as the peptide nucleic acid (PNA) method, are required to detect mutations in peripheral blood. Next generation sequencing (NGS) can analyze millions of PCR amplicons independently, thus it is expected to detect low-abundance GNAS mutations quantitatively. In the present study, we aimed to develop an NGS-based method to detect low-abundance somatic GNAS mutations. PCR amplicons encompassing exons 8 and 9 of GNAS, in which most activating mutations occur, were sequenced on the MiSeq instrument. As expected, our NGS-based method could sequence the GNAS locus with very high read depth (approximately 100,000) and low error rate. A serial dilution study with use of cloned mutant and wildtype DNA samples showed a linear correlation between dilution and measured mutation abundance, indicating the reliability of quantification of the mutation. Using the serially diluted samples, the detection limits of three mutation detection methods (the PNA method, NGS, and combinatory use of PNA and NGS [PNA-NGS]) were determined. The lowest detectable mutation abundance was 1% for the PNA method, 0.03% for NGS and 0.01% for PNA-NGS. Finally, we analyzed 16 MAS patient-derived leukocytic DNA samples with the three methods, and compared the mutation detection rate of them. Mutation detection rate of the PNA method, NGS and PNA-NGS in 16 patient-derived peripheral blood samples were 56%, 63% and 75%, respectively. In conclusion, NGS can detect somatic activating GNAS mutations quantitatively and sensitively from peripheral blood samples. At present, the PNA-NGS method is likely the most sensitive method to detect low-abundance GNAS mutation.

Identification of an EMS-induced causal mutation in a gene required for boron-mediated root development by low-coverage genome re-sequencing in Arabidopsis

Next-generation sequencing (NGS) technologies enable the rapid production of an enormous quantity of sequence data. These powerful new technologies allow the identification of mutations by whole-genome sequencing. However, most reported NGS-based mapping methods, which are based on bulked segregant analysis, are costly and laborious. To address these limitations, we designed a versatile NGS-based mapping method that consists of a combination of low- to medium-coverage multiplex SOLiD (Sequencing by Oligonucleotide Ligation and Detection) and classical genetic rough mapping. Using only low to medium coverage reduces the SOLiD sequencing costs and, since just 10 to 20 mutant F2 plants are required for rough mapping, the operation is simple enough to handle in a laboratory with limited space and funding. As a proof of principle, we successfully applied this method to identify the CTR1, which is involved in boron-mediated root development, from among a population of high boron requiring Arabidopsis thaliana mutants. Our work demonstrates that this NGS-based mapping method is a moderately priced and versatile method that can readily be applied to other model organisms.

Use of Four Next-Generation Sequencing Platforms to Determine HIV-1 Coreceptor Tropism

HIV-1 coreceptor tropism assays are required to rule out the presence of CXCR4-tropic (non-R5) viruses prior treatment with CCR5 antagonists. Phenotypic (e.g., Trofile™, Monogram Biosciences) and genotypic (e.g., population sequencing linked to bioinformatic algorithms) assays are the most widely used. Although several next-generation sequencing (NGS) platforms are available, to date all published deep sequencing HIV-1 tropism studies have used the 454™ Life Sciences/Roche platform. In this study, HIV-1 co-receptor usage was predicted for twelve patients scheduled to start a maraviroc-based antiretroviral regimen. The V3 region of the HIV-1 env gene was sequenced using four NGS platforms: 454™, PacBio® RS (Pacific Biosciences), Illumina®, and Ion Torrent™ (Life Technologies). Cross-platform variation was evaluated, including number of reads, read length and error rates. HIV-1 tropism was inferred using Geno2Pheno, Web PSSM, and the 11/24/25 rule and compared with Trofile™ and virologic response to antiretroviral therapy. Error rates related to insertions/deletions (indels) and nucleotide substitutions introduced by the four NGS platforms were low compared to the actual HIV-1 sequence variation. Each platform detected all major virus variants within the HIV-1 population with similar frequencies. Identification of non-R5 viruses was comparable among the four platforms, with minor differences attributable to the algorithms used to infer HIV-1 tropism. All NGS platforms showed similar concordance with virologic response to the maraviroc-based regimen (75% to 80% range depending on the algorithm used), compared to Trofile (80%) and population sequencing (70%). In conclusion, all four NGS platforms were able to detect minority non-R5 variants at comparable levels suggesting that any NGS-based method can be used to predict HIV-1 coreceptor usage.

Application of next-generation sequencing for rapid marker development in molecular plant breeding: a case study on anthracnose disease resistance in Lupinus angustifolius L.

BackgroundIn the last 30 years, a number of DNA fingerprinting methods such as RFLP, RAPD, AFLP, SSR, DArT, have been extensively used in marker development for molecular plant breeding. However, it remains a daunting task to identify highly polymorphic and closely linked molecular markers for a target trait for molecular marker-assisted selection. The next-generation sequencing (NGS) technology is far more powerful than any existing generic DNA fingerprinting methods in generating DNA markers. In this study, we employed a grain legume crop Lupinus angustifolius (lupin) as a test case, and examined the utility of an NGS-based method of RAD (restriction-site associated DNA) sequencing as DNA fingerprinting for rapid, cost-effective marker development tagging a disease resistance gene for molecular breeding.ResultsTwenty informative plants from a cross of RxS (disease resistant x susceptible) in lupin were subjected to RAD single-end sequencing by multiplex identifiers. The entire RAD sequencing products were resolved in two lanes of the 16-lanes per run sequencing platform Solexa HiSeq2000. A total of 185 million raw reads, approximately 17 Gb of sequencing data, were collected. Sequence comparison among the 20 test plants discovered 8207 SNP markers. Filtration of DNA sequencing data with marker identification parameters resulted in the discovery of 38 molecular markers linked to the disease resistance gene Lanr1. Five randomly selected markers were converted into cost-effective, simple PCR-based markers. Linkage analysis using marker genotyping data and disease resistance phenotyping data on a F8 population consisting of 186 individual plants confirmed that all these five markers were linked to the R gene. Two of these newly developed sequence-specific PCR markers, AnSeq3 and AnSeq4, flanked the target R gene at a genetic distance of 0.9 centiMorgan (cM), and are now replacing the markers previously developed by a traditional DNA fingerprinting method for marker-assisted selection in the Australian national lupin breeding program.ConclusionsWe demonstrated that more than 30 molecular markers linked to a target gene of agronomic trait of interest can be identified from a small portion (1/8) of one sequencing run on HiSeq2000 by applying NGS based RAD sequencing in marker development. The markers developed by the strategy described in this study are all co-dominant SNP markers, which can readily be converted into high throughput multiplex format or low-cost, simple PCR-based markers desirable for large scale marker implementation in plant breeding programs. The high density and closely linked molecular markers associated with a target trait help to overcome a major bottleneck for implementation of molecular markers on a wide range of germplasm in breeding programs. We conclude that application of NGS based RAD sequencing as DNA fingerprinting is a very rapid and cost-effective strategy for marker development in molecular plant breeding. The strategy does not require any prior genome knowledge or molecular information for the species under investigation, and it is applicable to other plant species.