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Neurological disorders are clinically heterogeneous group of disorders and are major causes of disability and death. Several of these disorders are caused due to genetic aberration. A precise and confirmatory diagnosis in the patients in a timely manner is essential for appropriate therapeutic and management strategies. Due to the complexity of the clinical presentations across various neurological disorders, arriving at an accurate diagnosis remains a challenge. We sequenced 1012 unrelated patients from India with suspected neurological disorders, using TruSight One panel. Genetic variations were identified using the Strand NGS software and interpreted using the StrandOmics platform. We were able to detect mutations in 197 genes in 405 (40%) cases and 178 mutations were novel. The highest diagnostic rate was observed among patients with muscular dystrophy (64%) followed by leukodystrophy and ataxia (43%, each). In our cohort, 26% of the patients who received definitive diagnosis were primarily referred with complex neurological phenotypes with no suggestive diagnosis. In terms of mutations types, 62.8% were truncating and in addition, 13.4% were structural variants, which are also likely to cause loss of function. In our study, we observed an improved performance of multi-gene panel testing, with an overall diagnostic yield of 40%. Furthermore, we show that NGS (next-generation sequencing)-based testing is comprehensive and can detect all types of variants including structural variants. It can be considered as a single-platform genetic test for neurological disorders that can provide a swift and definitive diagnosis in a cost-effective manner.

Additional file 3: List of heteroplasmy variants identified in subjects. The table lists the mitochondria heteroplasmy variants shortlisted in blood and DBS samples along with the %allele frequencies.

Background Multi–gene panel sequencing using next-generation sequencing (NGS) methods is a key tool for genomic medicine. However, with an estimated 140 000 genomic tests available, current system inefficiencies result in high genetic-testing costs. Reduced testing costs are needed to expand the availability of genomic medicine. One solution to improve efficiency and lower costs is to calculate the most cost-effective set of panels for a typical pattern of test requests. Methods We compiled rare diseases, associated genes, point prevalence, and test-order frequencies from a representative laboratory. We then modeled the costs of the relevant steps in the NGS process in detail. Using a simulated annealing-based optimization procedure, we determined panel sets that were more cost-optimal than whole exome sequencing (WES) or clinical exome sequencing (CES). Finally, we repeated this methodology to cost-optimize pharmacogenomics (PGx) testing. Results For rare disease testing, we show that an optimal choice of 4–6 panels, uniquely covering genes that comprise 95% of the total prevalence of monogenic diseases, saves $257–304 per sample compared with WES, and $66–135 per sample compared with CES. For PGx, we show that the optimal multipanel solution saves $6–7 (27%–40%) over a single panel covering all relevant gene–drug associations. Conclusions Laboratories can reduce costs using the proposed method to obtain and run a cost-optimal set of panels for specific test requests. In addition, payers can use this method to inform reimbursement policy.

BackgroundDried blood spots (DBS) are a relatively inexpensive source of nucleic acids and are easy to collect, transport, and store in large-scale field surveys, especially in resource-limited settings. However, their performance in whole-genome sequencing (WGS) relative to that of venous blood DNA has not been analyzed for various downstream applications.MethodsThis study compares the WGS performance of DBS paired with venous blood samples collected from 12 subjects.ResultsResults of standard quality checks of coverage, base quality, and mapping quality were found to be near identical between DBS and venous blood. Concordance for single-nucleotide variants, insertions and deletions, and copy number variants was high between these two sample types. Additionally, downstream analyses typical of population-based studies were performed, such as mitochondrial heteroplasmy detection, haplotype analysis, mitochondrial copy number changes, and determination of telomere lengths. The absolute mitochondrial copy number values were higher for DBS than for venous blood, though the trend in sample-to-sample variation was similar between DBS and blood. Telomere length estimates in most DBS samples were on par with those from venous blood.ConclusionDBS samples can serve as a robust and feasible alternative to venous blood for studies requiring WGS analysis.

BACKGROUND: Oral cavity squamous cell carcinoma (OCSCC) is the most common head and neck malignancy. Although the survival rate of patients with advanced-stage disease remains approximately 20% to 60%, when detected at an early stage, the survival rate approaches 80%, posing a pressing need for a well validated profiling method to assess patients who have a high risk of developing OCSCC.Tumor DNA detection in saliva may provide a robust biomarker platform that overcomes the limitations of current diagnostic tests.However, there is no routine saliva-based screening method for patients with OCSCC. METHODS: The authors designed a custom nextgeneration sequencing panel with unique molecular identifiers that covers coding regions of 7 frequently mutated genes in OCSCC and applied it on DNA extracted from 121 treatment-naive OCSCC tumors and matched preoperative saliva specimens. RESULTS: By using stringent variant-calling criteria, mutations were detected in 106 tumors, consistent with a predicted detection rate ≥88%. Moreover,mutations identified in primary malignancies were also detected in 93% of saliva samples. To ensure that variants are not errors resulting in false-positive calls, a multistep analytical validation of this approach was performed: 1) re-sequencing of 46 saliva samples confirmed 88% of somatic variants; 2) no functionally relevant mutations were detected in saliva samples from 11 healthy individuals without a history of tobacco or alcohol; and 3) using a panel of 7 synthetic loci across 8 sequencing runs, it was confirmed that the platform developed is reproducible and provides sensitivity on par with droplet digital polymerase chain reaction. CONCLUSIONS: The current data highlight the feasibility of somatic mutation identification in driver genes in saliva collected at the time of OCSCC diagnosis

Liquid biopsy is increasingly gaining traction as an alternative to invasive solid tumor biopsies for prognosis, treatment decisions, and disease monitoring. Matched tumor‐plasma samples were collected from 180 patients across different cancers with >90% of the samples below Stage IIIB. Tumors were profiled using next‐generation sequencing (NGS) or quantitative PCR (qPCR), and the mutation status was queried in the matched plasma using digital platforms such as droplet digital PCR (ddCPR) or NGS for concordance. Tumor‐plasma concordance of 82% and 32% was observed in advanced (Stage IIB and above) and early (Stage I to Stage IIA) stage samples, respectively. Interestingly, the overall survival outcomes correlated to presurgical/at‐biopsy ctDNA levels. Baseline ctDNA stratified patients into three categories: (a) high ctDNA correlated with poor survival outcome, (b) undetectable ctDNA with good outcome, and (c) low ctDNA whose outcome was ambiguous. ctDNA could be a powerful tool for therapy decisions and patient management in a large number of cancers across a variety of stages.

Breast cancer (BC) is a heterogeneous disease. Numerous chemotherapeutic agents are available for early stage or advanced/metastatic breast cancer to provide maximum benefit with minimum side effects. However, the clinical outcome of patients with the same clinical and pathological characteristics and treated with similar treatments may show major differences and a vast majority of patients still develop treatment resistance and eventually succumb to disease. It remains an unmet need to identify specific molecular defects, new biomarkers to enable clinicians to adopt individualized treatment for every patient in terms of endocrine, chemotherapy or targeted therapy which will improve clinical outcomes in BC. Our study aimed to identify frequent hotspot mutation profile in BC by targeted deep sequencing in cancer-related genes using Illumina Truseq amplicon/Swift Accel-Amplicon panel and MiSeq technology in an IRB-approved prospective study in a CLIA compliant laboratory. All the cases had pathology review for stage, histological type, hormonal status and Ki-67. Data was processed using Strand NGS™. Mutations identified in the tumor were assessed for ‘actionability’ i.e. response to therapy and impact on prognosis., Highlights • Actionable mutations have been identified in breast cancer by targeted deep sequencing using MiSeq technology. • 570 early stage or advanced breast cancer patients were counselled out if which 275 cases were tested. • The most altered genes included TP53, PIK3CA, AKT1, PTEN deletion, ERBB2, ATM, CDH1, APC, KRAS, NRAS. • Mutations identified in the tumor have been correlated with clinicopathological features and assessed for ‘actionability’. • Importance of genomic profiling has been highlighted in the context of utility, economic impact, availability and access to drugs in India.

Breast and/or ovarian cancers are among the most common cancers in women across the world. In the Indian population, the healthcare burden of breast and/or ovarian cancers has been steadily rising, thus stressing the need for early detection, surveillance, and disease management measures. However, the burden attributable to inherited mutations is not well characterized. We sequenced 1010 unrelated patients and families from across India with an indication of breast and/or ovarian cancers, using the TruSight Cancer panel which includes 14 genes, strongly associated with risk of hereditary breast and/or ovarian cancers. Genetic variations were identified using the StrandNGS software and interpreted using the StrandOmics platform. We were able to detect mutations in 304 (30.1%) cases, of which, 56 mutations were novel. A majority (84.9%) of the mutations were detected in the BRCA1/2 genes as compared to non-BRCA genes (15.1%). When the cases were stratified on the basis of age at diagnosis and family history of cancer, the high rate of 75% of detection of hereditary variants was observed in patients whose age at diagnosis was below 40 years and had first-degree family member(s) affected by breast and/or ovarian cancers. Our findings indicate that in the Indian population, there is a high prevalence of mutations in the high-risk breast cancer genes: BRCA1, BRCA2, TP53, and PALB2. In India, socioeconomic inequality limiting access to treatment is a major factor towards increased cancer burden; therefore, incorporation of a cost-effective and comprehensive multi-gene test will be helpful in ensuring widespread implementation of genetic screening in the clinical practice for hereditary breast and/or ovarian cancers.

Comprehensive genetic profiling of tumors using next‐generation sequencing (NGS) is gaining acceptance for guiding treatment decisions in cancer care. We designed a cancer profiling test combining both deep sequencing and immunohistochemistry (IHC) of relevant cancer targets to aid therapy choices in both standard‐of‐care (SOC) and advanced‐stage treatments for solid tumors. The SOC report is provided in a short turnaround time for four tumors, namely lung, breast, colon, and melanoma, followed by an investigational report. For other tumor types, an investigational report is provided. The NGS assay reports single‐nucleotide variants (SNVs), copy number variations (CNVs), and translocations in 152 cancer‐related genes. The tissue‐specific IHC tests include routine and less common markers associated with drugs used in SOC settings. We describe the standardization, validation, and clinical utility of the StrandAdvantage test (SA test) using more than 250 solid tumor formalin‐fixed paraffin‐embedded (FFPE) samples and control cell line samples. The NGS test showed high reproducibility and accuracy of >99%. The test provided relevant clinical information for SOC treatment as well as more information related to investigational options and clinical trials for >95% of advanced‐stage patients. In conclusion, the SA test comprising a robust and accurate NGS assay combined with clinically relevant IHC tests can detect somatic changes of clinical significance for strategic cancer management in all the stages.

Sample processing protocols that enable compatible recovery of differentially expressed (DE) transcripts and proteins are necessary for integration of the multiomics data applied in the analysis of tumors. In this pilot study, we compared two different isolation methods for extracting RNA and protein from laryngo-pharyngeal tumor tissues and the corresponding adjacent normal sections. In Method 1, RNA and protein were isolated from a single tissue section sequentially and in Method 2, the extraction was carried out using two different sections and two independent and parallel protocols for RNA and protein. RNA and protein from both methods were subjected to RNA-seq and iTRAQ-based LC-MS/MS analysis, respectively. Analysis of data revealed that a higher number of differentially expressed transcripts and proteins were concordant in their regulation trends in Method 1 as compared to Method 2. Cross method comparison of concordant entities revealed that RNA and protein extraction from the same tissue section (Method 1) recovered more concordant entities that are missed in the other extraction method (Method 2) indicating heterogeneity in distribution of these entities in different tissue sections. Method 1 could thus be the method of choice for integrated analysis of transcriptome and proteome data. This article is protected by copyright. All rights reserved

High throughput molecular profiling and integrated data analysis with tumor tissues require overcoming challenges like tumor heterogeneity and tissue paucity. This study is an attempt to understand and optimize various steps during tissue processing and in establishing pipelines essential for integrated analysis. Towards this effort, we subjected laryngo-pharyngeal primary tumors and the corresponding adjacent normal tissues (n=2) to two RNA and protein isolation methods, one wherein RNA and protein were isolated from the same tissue sequentially (Method 1) and second, wherein the extraction was carried out using two independent methods (Method 2). RNA and protein from both methods were subjected to RNA-seq and iTRAQ based LC-MS/MS analysis. Transcript and peptide identification and quantification was followed by both individual -ome and integrated data analysis. As a result of this analysis, we identified a higher number of total, as well as differentially expressed (DE) transcripts (1329 vs 1134) and proteins (799 vs 408) with fold change ≥ 2.0, in Method 1. Among these, 173 and 86 entities were identified by both transcriptome and proteome analysis in Method 1 and 2, respectively, with higher concordance in the regulation trends observed in the former. The significant cancer related pathways enriched with the individual DE transcript or protein data were similar in both the methods. However, the entities mapping to them were different, allowing enhanced view of the pathways identified after integration of the data and subsequent mapping. The concordant DE transcripts and proteins also revealed key molecules of the pathways with important roles in cancer development. This study thus demonstrates that sequential extraction of the RNA and proteins from the same tissue allows for better profiling of differentially expressed entities and a more accurate integrated data analysis.

e12539 Background: Personalized cancer treatment can benefit from targeted deep-sequencing of multiple genes using NGS. The StrandAdvantage test panel assays 212 amplicons (48 genes) spanning hotsp...

e12505 Background: Breast and/or ovarian cancer are among the most prevalent forms of hereditary cancers in India causing an increasing societal burden. Early detection and identification of those ...