Technical validation of a high-sensitivity target capture NGS assay using unique molecular identifier approach.

2020 ◽  
Vol 38 (15_suppl) ◽  
pp. e13657-e13657
Author(s):  
Ruifang Mao ◽  
Shanshan Xiao ◽  
Rui Lin ◽  
Yuchen Wang ◽  
Tao Wang
Keyword(s):  
Copy Number ◽  
Somatic Mutations ◽  
Limit Of Detection ◽  
Sequence Data ◽  
Nsclc Patient ◽  
High Sensitivity ◽  
Alternative Methods ◽  
Driver Mutations ◽  
Single Nucleotide Variants ◽  
Illumina Hiseq

e13657 Background: Identification of a broad spectrum of somatic mutations is crucial to guide targeted therapy such as for non-small cell lung cancer (NSCLC) patients. In the clinical environment, it requires well validated NGS workflow both for the wet-lab and dry-lab procedures. Here we describe a high sensitivity target NGS assay to accurately capture single nucleotide variants (SNVs), short insertions and deletions (indels), copy number alterations and gene rearrangements for formalin-fixed paraffin-embedded (FFPE) NSCLC patient samples. Extensive analytical validation was performed following the checklists of College of American Pathologists. Methods: Next generation sequencing (NGS) libraries were prepared using extracted DNA from FFPE tissue NSCLC patient samples. The protocol for library generation was optimized in several steps and incorporated 10bp unique molecular identifiers (UMIs). The libraries were sequenced on Illumina HiSeq X-Ten platform. The sequence data was analyzed by an in-house bioinformatics pipeline to call somatic mutations at an average depth of 4000X. Results: We tested the accuracy of 68 clinical tumor samples that were also validated by conventional or alternative methods in the third party CAP accredited labs. We observed 100% sensitivity and 100% specificity compared with the other lab¡¯s validation results. To define the limit of detection (LOD) for different mutation types, clinical DNA samples containing different variants were diluted with normal DNA. The LODs for SNV (as in EGFR L858R) and indel (as in EGFR 19del) were 0.5% and 1%, respectively. Addressing the LOD of fusion and copy number alteration is usually challenging. Our NGS assay was able to achieve 2% LOD for gene rearrangement (fusion) and 3.5 copies for copy number amplification. The high reproducibility was also achieved by inter- and intra- replicate experiments. Our NGS assay showed better performance than other widely used commercial NGS assay panels. Conclusions: We have validated an NGS based approach with UMI technology that is able to achieve high accuracy and sensitivity as low as 0.5% for detection of somatic mutations, which will improve the clinical testing performance for NSCLC FFPE samples with low allele frequencies of driver mutations.

Whole Exome Sequencing In Relapsed Pediatric T-ALL: Progression Into Relapse Is Characterized By An Increased Number Of Somatic Mutations and a Conservation Of Mutations In Leukemogenic Driver Genes

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 228-228
Author(s):  
Joachim Kunz ◽  
Tobias Rausch ◽  
Obul R Bandapalli ◽  
Martina U. Muckenthaler ◽  
Adrian M Stuetz ◽  
...  
Keyword(s):  
Exome Sequencing ◽  
Whole Exome Sequencing ◽  
Copy Number ◽  
Lymphoblastic Leukemia ◽  
Initial Diagnosis ◽  
Driver Mutations ◽  
Single Nucleotide Variants ◽  
Single Nucleotide ◽  
Whole Exome ◽  
Recurrent Mutations

Abstract Acute precursor T-lymphoblastic leukemia (T-ALL) remains a serious challenge in pediatric oncology, because relapses carry a particularly poor prognosis with high rates of induction failure and death despite generally excellent treatment responses of the initial disease. It is critical, therefore, to understand the molecular evolution of pediatric T-ALL and to elucidate the mechanisms leading to T-ALL relapse and to understand the differences in treatment response between the two phases of the disease. We have thus subjected DNA from bone marrow samples obtained at the time of initial diagnosis, remission and relapse of 14 patients to whole exome sequencing (WES). Eleven patients suffered from early relapse (duration of remission 6-19 months) and 3 patients from late relapse (duration of remission 29-46 months).The Agilent SureSelect Target Enrichment Kit was used to capture human exons for deep sequencing. The captured fragments were sequenced as 100 bp paired reads using an Illumina HiSeq2000 sequencing instrument. All sequenced DNA reads were preprocessed using Trimmomatic (Lohse et al., Nucl. Acids Res., 2012) to clip adapter contaminations and to trim reads for low quality bases. The remaining reads greater than 36bp were mapped to build hg19 of the human reference genome with Stampy (Lunter & Goodson, Genome Res. 2011), using default parameters. Following such preprocessing, the number of mapped reads was >95% for all samples. Single-nucleotide variants (SNVs) were called using SAMtools mpileup (Li et al., Bioinformatics, 2009). The number of exonic SNVs varied between 23,741 and 31,418 per sample. To facilitate a fast classification and identification of candidate driver mutations, all identified coding SNVs were comprehensively annotated using the ANNOVAR framework (Wang et al., Nat. Rev. Genet., 2010). To identify possible somatic driver mutations, candidate SNVs were filtered for non-synonymous, stopgain or stoploss SNVs, requiring an SNV quality greater or equal to 50, and requiring absence of segmental duplications. Leukemia-specific mutations were identified by filtering against the corresponding remission sample and validated by Sanger sequencing of the genomic DNA following PCR amplification. We identified on average 9.3 somatic single nucleotide variants (SNV) and 0.6 insertions and deletions (indels) per patient sample at the time of initial diagnosis and 21.7 SNVs and 0.3 indels in relapse. On average, 6.3 SNVs were detected both at the time of initial diagnosis and in relapse. These SNVs were thus defined as leukemia specific. Further to SNVs, we have also estimated the frequency of copy number variations (CNV) at low resolution. Apart from the deletions resulting from T-cell receptor rearrangement, we identified on average for each patient 0.7 copy number gains and 2.2 copy number losses at the time of initial diagnosis and 0.5 copy number gains and 2.4 copy number losses in relapse. We detected 24/27 copy number alterations both in initial diagnosis and in relapse. The most common CNV detected was the CDKN2A/B deletion on chromosome 9p. Nine genes were recurrently mutated in 2 or more patients thus indicating the functional leukemogenic potential of these SNVs in T-ALL. These recurrent mutations included known oncogenes (Notch1), tumor suppressor genes (FBXW7, PHF6, WT1) and genes conferring drug resistance (NT5C2). In several patients one gene (such as Notch 1, PHF6, WT1) carried different mutations either at the time of initial diagnosis and or in relapse, indicating that the major leukemic clone had been eradicated by primary treatment, but that a minor clone had persisted and expanded during relapse. The types of mutations did not differ significantly between mutations that were either already present at diagnosis or those that were newly acquired in relapse, indicating that the treatment did not cause specific genomic damage. We will further characterize the clonal evolution of T-ALL into relapse by targeted re-sequencing at high depth of genes with either relapse specific or initial-disease specific mutations. In conclusion, T-ALL relapse differs from primary disease by a higher number of leukemogenic SNVs without gross genomic instability resulting in large CNVs. Disclosures: No relevant conflicts of interest to declare.

scholarly journals High Sensitivity of Plasma Cell-Free DNA Genotyping in Cases With Evidence of Adequate Tumor Content

2021 ◽  
pp. 921-930
Author(s):  
Catherine B. Meador ◽  
Marina S. D. Milan ◽  
Emmy Y. Hu ◽  
Mark M. Awad ◽  
Michael S. Rabin ◽  
...  
Keyword(s):  
Plasma Cell ◽  
Nsclc Patient ◽  
False Negative ◽  
High Sensitivity ◽  
Driver Mutation ◽  
Driver Mutations ◽  
Resistance Mutations ◽  
Assay Sensitivity ◽  
Cell Free Dna ◽  
Free Dna

PURPOSE Plasma cell-free DNA (cfDNA) sequencing is a compelling diagnostic tool in solid tumors and has been shown to have high positive predictive value. However, limited assay sensitivity means that negative plasma genotyping, or the absence of detection of mutation of interest, still requires reflex tumor biopsy. METHODS We analyzed two independent cohorts of patients with advanced non–small-cell lung cancer (NSCLC) with known canonical driver and resistance mutations who underwent plasma cfDNA genotyping. We measured quantitative features, such as maximum allelic frequency (mAF), as clinically available measures of cfDNA tumor content, and studied their relationship with assay sensitivity. RESULTS In patients with EGFR-mutant NSCLC harboring EGFR T790M, detection of driver mutation at > 1% AF conferred a sensitivity of 97% (368/380) for detection of T790M across three cfDNA genotyping platforms. Similarly, in a second cohort of patients with EGFR or KRAS driver mutations, when the mAF of nontarget mutations was > 1%, sensitivity for driver mutation detection was 100% (43/43). Combining the two NSCLC patient cohorts, the presence of nontarget mutations at mAF > 1% predicts for high sensitivity (> 95%) for identifying the presence of the known driver mutation, whereas mAF of ≤ 1% confers sensitivity of only 26%-54% across platforms. Focusing on 21 false-negative cases where the driver mutation was not detected on plasma next-generation sequencing, other mutations (presumably clonal hematopoiesis) were detected at ≤ 1% AF in 14 (67%). CONCLUSION Plasma cfDNA genotyping is highly sensitive when adequate tumor DNA content is present. The likelihood of a false-negative cfDNA genotyping result is low in a sample with evidence of > 1% tumor content. Bioinformatic approaches are needed to further optimize the assessment of cfDNA tumor content in plasma genotyping assays.

scholarly journals RBV: Read balance validator, a tool for prioritising copy number variations in germline conditions

2018 ◽  
Author(s):  
Whitney Whitford ◽  
Klaus Lehnert ◽  
Russell G. Snell ◽  
Jessie C. Jacobsen
Keyword(s):  
Copy Number ◽  
High Throughput Sequencing ◽  
Sequence Data ◽  
Relative Proportion ◽  
Copy Number Variants ◽  
Read Depth ◽  
Copy Number Variations ◽  
Single Nucleotide Variants ◽  
Software Packages ◽  
Bioinformatic Tool

AbstractBackgroundThe popularisation and decreased cost of genome resequencing has resulted in an increased use in molecular diagnostics. While there are a number of established and high quality bioinfomatic tools for identifying small genetic variants including single nucleotide variants and indels, currently there is no established standard for the detection of copy number variants (CNVs) from sequence data. The requirement for CNV detection from high throughput sequencing has resulted in the development of a large number of software packages. These tools typically utilise the sequence data characteristics: read depth, split reads, read pairs, and assembly-based techniques. However the additional source of information from read balance, defined as relative proportion of reads of each allele at each position, has been underutilised in the existing applications.ResultsWe present Read Balance Validator (RBV), a bioinformatic tool which uses read balance for prioritisation and validation of putative CNVs. The software simultaneously interrogates nominated regions for the presence of deletions or multiplications, and can differentiate larger CNVs from diploid regions. Additionally, the utility of RBV to test for inheritance of CNVs is demonstrated in this report.ConclusionsRBV is a CNV validation and prioritisation bioinformatic tool for both genome and exome sequencing available as a python package from https://github.com/whitneywhitford/RBV

scholarly journals μLAS technology for DNA isolation coupled to Cas9-assisted targeting for sequencing and assembly of a 30 kb region in plant genome

2019 ◽  
Vol 47 (15) ◽  
pp. 8050-8060 ◽  
Author(s):  
Nicolas Milon ◽  
Céline Chantry-Darmon ◽  
Carine Satge ◽  
Margaux-Alison Fustier ◽  
Stephane Cauet ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Sequence Data ◽  
High Sensitivity ◽  
Artificial Chromosome ◽  
Plant Genome ◽  
Reference Sequence ◽  
Target Sequence ◽  
High Molecular Weight Dna ◽  
Continuous Sequence ◽  
Analytical Time

Abstract Cas9-assisted targeting of DNA fragments in complex genomes is viewed as an essential strategy to obtain high-quality and continuous sequence data. However, the purity of target loci selected by pulsed-field gel electrophoresis (PFGE) has so far been insufficient to assemble the sequence in one contig. Here, we describe the μLAS technology to capture and purify high molecular weight DNA. First, the technology is optimized to perform high sensitivity DNA profiling with a limit of detection of 20 fg/μl for 50 kb fragments and an analytical time of 50 min. Then, μLAS is operated to isolate a 31.5 kb locus cleaved by Cas9 in the genome of the plant Medicago truncatula. Target purification is validated on a Bacterial Artificial Chromosome plasmid, and subsequently carried out in whole genome with μLAS, PFGE or by combining these techniques. PacBio sequencing shows an enrichment factor of the target sequence of 84 with PFGE alone versus 892 by association of PFGE with μLAS. These performances allow us to sequence and assemble one contig of 29 441 bp with 99% sequence identity to the reference sequence.

Genomic predictors of neoadjuvant chemotherapy (NACT) response in breast cancer (BC).

2017 ◽  
Vol 35 (15_suppl) ◽  
pp. e12122-e12122 ◽  
Author(s):  
Andrea Li Ann Wong ◽  
Kar Tong Tan ◽  
Raghav Sundar ◽  
Samuel Ow ◽  
Angela Pang ◽  
...  
Keyword(s):  
Low Dose ◽  
Somatic Mutations ◽  
Target Lesion ◽  
Driver Mutations ◽  
Single Nucleotide Variants ◽  
Single Nucleotide ◽  
Whole Exome ◽  
Mutant Variant ◽  
Poor Outcomes ◽  
Clinical Trial Information

e12122 Background: We assessed effects of NACT on BC mutational landscape. Methods: Baseline (BL) and post-NACT tumor / matched normal DNA from 12 newly diagnosed BC patients on NACT (4 x doxorubicin/cyclophosphamide + low dose sunitinib; NCT01176799) were subject to whole exome sequencing. Nonsynonymous somatic single nucleotide variants from 34 genes in known BC signaling pathways were evaluated for changes in mutant variant allele frequency (VAF) according to clinical outcome. Poor outcome was defined as <50% target lesion reduction after NACT or BC relapse / progression (PD) within 2 years; significant change was defined as > 0.2 difference in BL vs post-NACT mutant VAF. Results: Mean tumor size was 6.4 + 2.9cm; 50% were N+; 8% were M1; 7/12 patients had poor outcomes. Tumors harbored mutations in PI3K (58%), NOTCH (42%), Wnt (42%), TP53 (33%) and FOXA (17%) pathways. Change in no. of somatic mutations post-NACT correlated with outcome (mean percent change +14% vs -30% in patients with poor vs good outcome, p=0.04). 11 patients had >1 of 23 putative driver mutations identified ( Table 1). Mutant VAF declined significantly in those with good outcomes, except for a new NOTCH2 mutation in A2 and rise in mutant VAF in A4. In patients with poor outcomes, mutant VAF persisted or rose, and emergent mutations (AKT1, PIK3CA) occurred in 2 patients. Conclusions: Chemoresistance and emergent mutations were revealed by tracking mutant VAF in BC patients on NACT. Clinical trial information: NCT01176799. [Table: see text]

scholarly journals Detecting and Quantitating Low Fraction DNA Variants with Low-Depth Sequencing

2020 ◽  
Author(s):  
Ping Song ◽  
Sherry X. Chen ◽  
Yan Helen Yan ◽  
Alessandro Pinto ◽  
Lauren Y. Cheng ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
High Sensitivity ◽  
Human Cell Line ◽  
Clinical Samples ◽  
Single Nucleotide Variants ◽  
Tumor Biopsy ◽  
Single Nucleotide ◽  
Tissue Samples ◽  
Acquired Drug Resistance ◽  
Dna Variants

DNA sequence variants with low allele frequencies below 1% are difficult to detect and quantitate by sequencing, due to the intrinsic error of sequencing-by-synthesis (NGS). Unique molecular identifier barcodes can in principle help NGS detect mutations down to 0.1% variant allele frequency (VAF), but require extremely high sequencing depths of over 25,000x, rendering high sensitivity mutation detection out of reach for most research and clinical samples. Here, we present the multiplex blocker displacement amplification (mBDA) method to selectively enrich DNA variants by an average of 300-fold in highly multiplexed NGS settings. On a 80-plex human single nucleotide polymorphism panel, mBDA achieves a 0.019% VAF limit of detection for single nucleotide variants, using only 250x sequencing depth, and detects human cell line contamination down to 0.07%. Using this technology, we constructed a 16-plex melanoma NGS panel covering 145 actionable mutations across 9 genes, and applied it to 19 fresh/frozen tumor biopsy tissue samples with high tumor fractions. We found low VAF mutations (0.2% to 5%) in 37% of the samples (7/19, 95% confidence interval 19%-58%). These results suggest that tumor heterogeneity could be significantly more pervasive than previously recognized, and can contribute significantly to acquired drug resistance to targeted therapies. We also validate mBDA panels on clinical cell-free DNA samples from lung cancer patients.

Development of a breast and lung cancer research panel to target therapeutically relevant copy number and gene fusion variants from blood.

2017 ◽  
Vol 35 (15_suppl) ◽  
pp. e12518-e12518
Author(s):  
Varun Bagai ◽  
Jeoffrey Schageman ◽  
Dumitru Brinza ◽  
Yanchun Li ◽  
Jian Gu ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Somatic Mutation ◽  
Copy Number ◽  
Gene Fusion ◽  
Somatic Mutations ◽  
Limit Of Detection ◽  
Negative Impact ◽  
Detection Sensitivity ◽  
Gene Fusions ◽  
Blood Samples

e12518 Background: With recent advances in next-generation sequencing (NGS) technologies, it is now possible to detect somatic mutations with allele frequencies in blood samples as low as 0.1% from circulating tumor DNA. A natural extension to this achievement is adding the ability to simultaneously detect copy number variants and gene fusions. A panel such as this addresses a full repertoire of variant classes found to be linked with certain tumors and would enable researchers additional tools to profile cancer samples more dynamically thus enriching current diagnostic tool sets. Here, we present progress on such an approach and apply current NGS technology to achieve our goals. Methods: Samples were sequecned using the Ion S5™ system. Results: Using control samples, we can reproducibly demonstrate detection of ERBB2 (HER2/neu) gene amplifications with high statistical significance and as low as a 2 fold difference versus non-amplified loci in titration experiments. In addition, this ERBB2 gene amplification was detected in the context of a validated breast cancer somatic mutation panel in which no negative impact was exhibited and mutation detection specificity and sensitivity were both greater than 90%. Lastly, we developed an additional panel to detect gene fusions relevant to lung cancer. Using the titration approach above, the EML4-ALK fusion variant was shown to have a limit of detection near 1% with no negative impact on detection sensitivity and specificity when combined with the validated lung cfDNA somatic mutation panel. Conclusions: From the outcomes of these experiments, we have shown the ability to reproducibly and simultaneously detect copy number and gene fusion variants as well as somatic mutations at very low limits of detection in a cell free DNA background derived from blood samples.

scholarly journals Adaptive archaic introgression of copy number variants and the discovery of previously unknown human genes

Science ◽  
2019 ◽  
Vol 366 (6463) ◽  
pp. eaax2083 ◽  
Author(s):  
PingHsun Hsieh ◽  
Mitchell R. Vollger ◽  
Vy Dang ◽  
David Porubsky ◽  
Carl Baker ◽  
...  
Keyword(s):  
Copy Number ◽  
Large Scale ◽  
Sequence Data ◽  
Local Population ◽  
Copy Number Variants ◽  
Human Populations ◽  
Single Nucleotide Variants ◽  
Human Genes ◽  
Long Read ◽  
Archaic Introgression

Copy number variants (CNVs) are subject to stronger selective pressure than single-nucleotide variants, but their roles in archaic introgression and adaptation have not been systematically investigated. We show that stratified CNVs are significantly associated with signatures of positive selection in Melanesians and provide evidence for adaptive introgression of large CNVs at chromosomes 16p11.2 and 8p21.3 from Denisovans and Neanderthals, respectively. Using long-read sequence data, we reconstruct the structure and complex evolutionary history of these polymorphisms and show that both encode positively selected genes absent from most human populations. Our results collectively suggest that large CNVs originating in archaic hominins and introgressed into modern humans have played an important role in local population adaptation and represent an insufficiently studied source of large-scale genetic variation.

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