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Literature Review: NGS

Two novel mutations in the PPIB gene cause a rare pedigree of osteogenesis imperfecta type IX
BACKGROUND: Osteogenesis imperfecta (OI) is a rare genetic skeletal disorder characterized by increased bone fragility and vulnerability to fractures. PPIB is identified as a candidate gene for OI-IX, here we detect two pathogenic mutations in PPIB and analyze the genotype-phenotype correlation in a Chinese family with OI.
METHODS: Next-generation sequencing (NGS) was used to screen the whole exome of the parents of proband. Screening of variation frequency, evolutionary conservation comparisons, pathogenicity evaluation, and protein structure prediction were conducted to assess the pathogenicity of the novel mutations. Sanger sequencing was used to confirm the candidate variants. RTQ-PCR was used to analyze the PPIB gene expression.
RESULTS: All mutant genes screened out by NGS were excluded except PPIB. Two novel heterozygous PPIB mutations (father, c.25A>G; mother, c.509G>A) were identified in relation to osteogenesis imperfecta type IX. Both mutations were predicted to be pathogenic by bioinformatics analysis and RTQ-PCR analysis revealed downregulated PPIB expression in the two carriers.
CONCLUSION: We report a rare pedigree with an autosomal recessive osteogenesis imperfecta type IX (OI-IX) caused by two novel PPIB mutations identified for the first time in China. The current study expands our knowledge of PPIB mutations and their associated phenotypes, and provides new information on the genetic defects associated with this disease for clinical diagnosis.
Application of next generation sequencing in clinical microbiology and infection prevention
Deurenberg RH, Bathoorn E, Chlebowicz MA, Couto N, Ferdous M et al. J Biotechnol 2017; 243: 16–24
Current molecular diagnostics of human pathogens provide limited information that is often not sufficient for outbreak and transmission investigation. Next generation sequencing (NGS) determines the DNA sequence of a complete bacterial genome in a single sequence run, and from these data, information on resistance and virulence, as well as information for typing is obtained, useful for outbreak investigation. The obtained genome data can be further used for the development of an outbreak-specific screening test. In this review, a general introduction to NGS is presented, including the library preparation and the major characteristics of the most common NGS platforms, such as the MiSeq (Illumina) and the Ion PGM™ (ThermoFisher). An overview of the software used for NGS data analyses used at the medical microbiology diagnostic laboratory in the University Medical Center Groningen in The Netherlands is given. Furthermore, applications of NGS in the clinical setting are described, such as outbreak management, molecular case finding, characterization and surveillance of pathogens, rapid identification of bacteria using the 16S-23S rRNA region, taxonomy, metagenomics approaches on clinical samples, and the determination of the transmission of zoonotic micro-organisms from animals to humans. Finally, we share our vision on the use of NGS in personalised microbiology in the near future, pointing out specific requirements.
A targeted high-throughput next-generation sequencing panel for clinical screening of mutations, gene amplifications, and fusions in solid tumours
Luthra R, Patel KP, Routbort MJ, Broaddus RR, Yau J, Simien C, Chen W, Hatfield DZ, Medeiros LJ, Singh RR. J Mol Diagn 2017; 19(2): 255–264
Clinical next-generation sequencing (NGS) assay choice requires careful consideration of panel size, inclusion of appropriate markers, ability to detect multiple genomic aberration types, compatibility with low quality and quantity of nucleic acids, and work flow feasibility. Herein, in a high-volume clinical molecular diagnostic laboratory, we have validated a targeted high-multiplex PCR-based NGS panel (OncoMine Comprehensive Assay) coupled with high-throughput sequencing using Ion Proton sequencer for routine screening of solid tumours. The panel screens 143 genes using low amounts of formalin-fixed, paraffin-embedded DNA (20 ng) and RNA (10 ng). A large cohort of 121 tumour samples representing 13 tumour types and 6 cancer cell lines was used to assess the capability of the panel to detect 148 single-nucleotide variants, 49 insertions or deletions, 40 copy number aberrations, and a subset of gene fusions. High levels of analytic sensitivity and reproducibility and robust detection sensitivity were observed. Furthermore, we demonstrated the critical utility of sequencing paired normal tissues to improve the accuracy of detecting somatic mutations in a background of germline variants. We also validated use of the Ion Chef automated bead templating and chip loading system, which represents a major work flow improvement. In summary, we present data establishing the OncoMine Comprehensive Assay-Ion Proton platform to be well suited for implementation as a routine clinical NGS test for solid tumours.
Presence of cancer-associated mutations in exhaled breath condensates of healthy individuals by next generation sequencing
Youssef O, Knuuttila A, Piirilä P, Böhling T, Sarhadi V, Knuutila S. Oncotarget 2017; doi: 0.18632/oncotarget.15233 [Epub ahead of print]
Exhaled breath condensate (EBC) is a non-invasive source that can be used for studying different genetic alterations occurring in lung tissue. However, the low yield of DNA available from EBC has hampered the more detailed mutation analysis by conventional methods. We applied the more sensitive amplicon-based next generation sequencing (NGS) to identify cancer related mutations in DNA isolated from EBC. In order to apply any method for the purpose of mutation screening in cancer patients, it is important to clarify the incidence of these mutations in healthy individuals. Therefore, we studied mutations in hotspot regions of 22 cancer genes of 20 healthy, mainly non-smoker individuals, using AmpliSeq colon and lung cancer panel and sequenced on Ion PGM. In 15 individuals, we detected 35 missense mutations in TP53, KRAS, NRAS, SMAD4, MET, CTNNB1, PTEN, BRAF, DDR2, EGFR, PIK3CA, NOTCH1, FBXW7, FGFR3, and ERBB2: these have been earlier reported in different tumor tissues. Additionally, 106 novel mutations not reported previously were also detected. One healthy non-smoker subject had a KRAS G12D mutation in EBC DNA. Our results demonstrate that DNA from EBC of healthy subjects can reveal mutations that could represent very early neoplastic changes or alternatively a normal process of apoptosis eliminating damaged cells with mutations or altered genetic material. Further assessment is needed to determine if NGS analysis of EBC could be a screening method for high risk individuals such as smokers, where it could be applied in the early diagnosis of lung cancer and monitoring treatment efficacy.
Molecular testing for familial hypercholesterolaemia-associated mutations in a UK-based cohort: development of an NGS-based method and comparison with multiplex polymerase chain reaction and oligonucleotide arrays
Reiman A, Pandey S, Lloyd KL, Dyer N, Khan M, Crockard M, Latten MJ, Watson TL, Cree IA, Grammatopoulos DK. Ann Clin Biochem 2016; 53(6): 654–662
BACKGROUND: Detection of disease-associated mutations in patients with familial hypercholesterolaemia is crucial for early interventions to reduce risk of cardiovascular disease. Screening for these mutations represents a methodological challenge since more than 1200 different causal mutations in the low-density lipoprotein receptor has been identified. A number of methodological approaches have been developed for screening by clinical diagnostic laboratories.
METHODS: Using primers targeting, the low-density lipoprotein receptor, apolipoprotein B, and proprotein convertase subtilisin/kexin type 9, we developed a novel Ion Torrent-based targeted re-sequencing method. We validated this in a West Midlands-UK small cohort of 58 patients screened in parallel with other mutation-targeting methods, such as multiplex polymerase chain reaction (Elucigene FH20), oligonucleotide arrays (Randox familial hypercholesterolaemia array) or the Illumina next-generation sequencing platform. 
RESULTS: In this small cohort, the next-generation sequencing method achieved excellent analytical performance characteristics and showed 100% and 89% concordance with the Randox array and the Elucigene FH20 assay. Investigation of the discrepant results identified two cases of mutation misclassification of the Elucigene FH20 multiplex polymerase chain reaction assay. A number of novel mutations not previously reported were also identified by the next-generation sequencing method.
CONCLUSIONS: Ion Torrent-based next-generation sequencing can deliver a suitable alternative for the molecular investigation of familial hypercholesterolaemia patients, especially when comprehensive mutation screening for rare or unknown mutations is required. 
Analytical validation of the next-generation sequencing assay for a nationwide signal-finding clinical trial: Molecular Analysis for Therapy Choice clinical trial
Lih CJ, Harrington RD, Sims DJ, Harper KN, Bouk CH, et al. J Mol Diagn 2017; 19(2): 313–327
The National Cancer Institute-Molecular Analysis for Therapy Choice (NCI-MATCH) trial is a national signal-finding precision medicine study that relies on genomic assays to screen and enroll patients with relapsed or refractory cancer after standard treatments. We report the analytical validation processes for the next-generation sequencing (NGS) assay that was tailored for regulatory compliant use in the trial. The Oncomine Cancer Panel assay and the Personal Genome Machine were used in four networked laboratories accredited for the Clinical Laboratory Improvement Amendments. Using formalin-fixed paraffin-embedded clinical specimens and cell lines, we found that the assay achieved overall sensitivity of 96.98% for 265 known mutations and 99.99% specificity. High reproducibility in detecting all reportable variants was observed, with a 99.99% mean interoperator pairwise concordance across the four laboratories. The limit of detection for each variant type was 2.8% for single-nucleotide variants, 10.5% for insertion/deletions, 6.8% for large insertion/deletions (gap ?4 bp), and four copies for gene amplification. The assay system from biopsy collection through reporting was tested and found to be fully fit for purpose. Our results indicate that the NCI-MATCH NGS assay met the criteria for the intended clinical use and that high reproducibility of a complex NGS assay is achievable across multiple clinical laboratories. Our validation approaches can serve as a template for development and validation of other NGS assays for precision medicine. 
Targeted next-generation sequencing of FNA-derived DNA in pancreatic cancer
Sibinga Mulder BG, Mieog JS, Handgraaf HJ, Farina Sarasqueta A, Vasen H et al. J Clin Pathol 2017; 70(2): 174–178
To improve the diagnostic value of fine-needle aspiration (FNA)-derived material, we perform targeted next-generation sequencing (NGS) in patients with a suspect lesion of the pancreas. The NGS analysis can lead to a change in the treatment plan or supports inconclusive or uncertain cytology results. We describe the advantages of NGS using one particular patient with a recurrent pancreatic lesion 7 years after resection of a pancreatic ductal adenocarcinoma (PDAC). Our NGS analysis revealed the presence of a presumed second primary cancer in the pancreatic remnant, which led to a change in treatment: resection with curative intend instead of palliation. Additionally, NGS identified an unexpected germline CDKN2A 19-base pair deletion, which predisposed the patient to developing PDAC. Preoperative NGS analysis of FNA-derived DNA can help identify patients at risk for developing PDAC and define future therapeutic options.
Exome sequencing covers >98% of mutations identified on targeted next generation sequencing panels
LaDuca H, Farwell KD, Vuong H, Lu HM, Mu W, Shahmirzadi L, Tang S, Chen J, Bhide S, Chao EC. PLoS One 2017;12(2): e0170843
BACKGROUND: With the expanded availability of next generation sequencing (NGS)-based clinical genetic tests, clinicians seeking to test patients with Mendelian diseases must weigh the superior coverage of targeted gene panels with the greater number of genes included in whole exome sequencing (WES)  when considering their first-tier testing approach. Here, we use an in silico analysis to predict the analytic sensitivity of WES using pathogenic variants identified on targeted NGS panels as a reference. METHODS: Corresponding nucleotide positions for 1533 different alterations classified as pathogenic or likely pathogenic identified on targeted NGS multi-gene panel tests in our laboratory were interrogated in data from 100 randomly-selected clinical WES samples to quantify the sequence coverage at each position. Pathogenic variants represented 91 genes implicated in hereditary cancer, X-linked intellectual disability, primary ciliary dyskinesia, Marfan syndrome/aortic aneurysms, cardiomyopathies and arrhythmias.
RESULTS: When assessing coverage among 100 individual WES samples for each pathogenic variant (153,300 individual assessments), 99.7% (n = 152,798) would likely have been detected on WES. All pathogenic variants had at least some coverage on exome sequencing, with a total of 97.3% (n = 1491) detectable across all 100 individuals. For the remaining 42 pathogenic variants, the number of WES samples with adequate coverage ranged from 35 to 99. Factors such as location in GC-rich, repetitive, or homologous regions likely explain why some of these alterations were not detected across all samples. To validate study findings, a similar analysis was performed against coverage data from 60,706 exomes available through the Exome Aggregation Consortium (ExAC). Results from this validation confirmed that 98.6% (91,743,296/93,062,298) of pathogenic variants demonstrated adequate depth for detection.
CONCLUSIONS: Results from this in silico analysis suggest that exome sequencing may achieve a diagnostic yield similar to panel-based testing for Mendelian diseases.
Validation of an NGS mutation detection panel for melanoma
Reiman A, Kikuchi H, Scocchia D, Smith P, Tsang YW, Snead D, Cree IA. BMC Cancer 2017;17(1): 150
BACKGROUND: Knowledge of the genotype of melanoma is important to guide patient management. Identification of mutations in BRAF and c-KIT lead directly to targeted treatment, but it is also helpful to know if there are driver oncogene mutations in NRAS, GNAQ or GNA11 as these patients may benefit from alternative strategies such as immunotherapy.
METHODS: While polymerase chain reaction (PCR) methods are often used to detect BRAF mutations, next generation sequencing (NGS) is able to determine all of the necessary information on several genes at once, with potential advantages in turnaround time. We describe here an Ampliseq hotspot panel for melanoma for use with the IonTorrent Personal Genome Machine (PGM) which covers the mutations currently of most clinical interest.
RESULTS: We have validated this in 151 cases of skin and uveal melanoma from our files, and correlated the data with PCR based assessment of BRAF status. There was excellent agreement, with few discrepancies, though NGS does have greater coverage and picks up some mutations that would be missed by PCR. However, these are often rare and of unknown significance for treatment.
CONCLUSIONS: PCR methods are rapid, less time-consuming and less expensive than NGS, and could be used as triage for patients requiring more extensive diagnostic workup. The NGS panel described here is suitable for clinical use with formalin-fixed paraffin-embedded (FFPE) samples.
Exome sequencing in a family with luminal-type breast cancer underpinned by variation in the 
methylation pathway
van der Merwe N, Peeters AV, Pienaar FM, Bezuidenhout J, van Rensburg SJ, Kotze MJ. Int J Mol Sci 2017;18(2): E467
Panel-based next generation sequencing (NGS) is currently preferred over whole exome sequencing (WES) for diagnosis of familial breast cancer, due to interpretation challenges caused by variants of uncertain clinical significance (VUS). There is also no consensus on the selection criteria for WES. In this study, a pathology-supported genetic testing (PSGT) approach was used to select two BRCA1/2 mutation-negative breast cancer patients from the same family for WES. Homozygosity for the MTHFR 677 C>T mutation detected during this PSGT pre-screen step was considered insufficient to cause bilateral breast cancer in the index case and her daughter diagnosed with early-onset breast cancer (<30 years). Extended genetic testing using WES identified the RAD50 R385C missense mutation in both cases. This rare variant with a minor allele frequency (MAF) of <0.001 was classified as a VUS after exclusion in an affected cousin and extended genotyping in 164 unrelated breast cancer patients and 160 controls. Detection of functional polymorphisms (MAF > 5%) in the folate pathway in all three affected family members is consistent with inheritance of the luminal-type breast cancer in the family. PSGT assisted with the decision to pursue extended genetic testing and facilitated clinical interpretation of WES aimed at reduction of recurrence risk.