Abstract LB-227: Guideline adherent clinical validation of a comprehensive DNA/RNA panel (523 genes-TruSight Oncology 500) for determination of single nucleotide variants (SNV’s), small insertions or deletions (Indels), copy number variations (CNV’s), splice variations (SV’s), gene fusions (GF’s), tumor mutation burden (TMB) and micro-satellite instability (MSI) on anext-generation sequencing (NGS)platform in a CLIA setting

Context.— An increasing number of molecular laboratories are implementing next-generation sequencing platforms to identify clinically actionable and relevant genomic alterations for precision oncology. Objective.— To describe the validation studies as per New York State–Department of Health (NYS-DOH) guidelines for the Oncomine Comprehensive Panel v2, which was originally tailored to the National Cancer Institute Molecular Analysis for Therapy Choice (NCI-MATCH) trial. Design.— Accuracy, precision, and reproducibility were investigated by using 130 DNA and 18 RNA samples from cytology cell blocks; formalin-fixed, paraffin-embedded tissues; and frozen samples. Analytic sensitivity and specificity were tested by using ATCC and HapMap cell lines. Results.— High accuracy and precision/reproducibility were observed for single nucleotide variants and insertion/deletions. We also share our experience in the detection of gene fusions and copy number alterations from an amplicon-based sequencing platform. After sequencing analysis, variant annotation and report generation were performed by using the institutional knowledgebase. Conclusions.— This study serves as an example for validating a comprehensive targeted next-generation sequencing assay with both DNASeq and RNASeq components for NYS-DOH.

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Combination of Genome-Wide Polymorphisms and Copy Number Variations of Pharmacogenes in Koreans

Journal of Personalized Medicine ◽

10.3390/jpm11010033 ◽

2021 ◽

Vol 11 (1) ◽

pp. 33

Author(s):

Nayoung Han ◽

Jung Mi Oh ◽

In-Wha Kim

Keyword(s):

Copy Number ◽

Genome Wide Association Study ◽

Copy Number Gain ◽

Copy Number Variations ◽

Gene Gain ◽

Single Nucleotide Variants ◽

Single Nucleotide ◽

Haplotype Blocks ◽

Genome Wide ◽

Control And Prevention

For predicting phenotypes and executing precision medicine, combination analysis of single nucleotide variants (SNVs) genotyping with copy number variations (CNVs) is required. The aim of this study was to discover SNVs or common copy CNVs and examine the combined frequencies of SNVs and CNVs in pharmacogenes using the Korean genome and epidemiology study (KoGES), a consortium project. The genotypes (N = 72,299) and CNV data (N = 1000) were provided by the Korean National Institute of Health, Korea Centers for Disease Control and Prevention. The allele frequencies of SNVs, CNVs, and combined SNVs with CNVs were calculated and haplotype analysis was performed. CYP2D6 rs1065852 (c.100C>T, p.P34S) was the most common variant allele (48.23%). A total of 8454 haplotype blocks in 18 pharmacogenes were estimated. DMD ranked the highest in frequency for gene gain (64.52%), while TPMT ranked the highest in frequency for gene loss (51.80%). Copy number gain of CYP4F2 was observed in 22 subjects; 13 of those subjects were carriers with CYP4F2*3 gain. In the case of TPMT, approximately one-half of the participants (N = 308) had loss of the TPMT*1*1 diplotype. The frequencies of SNVs and CNVs in pharmacogenes were determined using the Korean cohort-based genome-wide association study.

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Whole Genome Sequencing Identifies Single Nucleotide Variants, Structural and Copy Number Abnormalities with a High Sensitivity in Hematological Neoplasm: An All in One Diagnostic Tool Tested on 3241 Cases

Blood ◽

10.1182/blood-2018-99-113321 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 1525-1525

Author(s):

Claudia Haferlach ◽

Sven O. Twardziok ◽

Stephan Hutter ◽

Wencke Walter ◽

Wolfgang Kern ◽

...

Keyword(s):

Diagnostic Tool ◽

Copy Number ◽

Amplicon Sequencing ◽

Whole Genome ◽

Single Nucleotide Variants ◽

Employment Equity ◽

Single Nucleotide ◽

Hematological Neoplasm ◽

Equity Ownership

Abstract Background: Novel targeted treatment approaches for hematological malignancies require a comprehensive genetic characterization of patient samples. So far combinations of various techniques are used in different entities. As gross structural variants (SV) and copy number aberrations (CNA) as well as molecular mutations have to be assessed - in best case genome wide - to date no single technique is able to provide all information in a routine diagnostic setting. Whole genome sequencing (WGS) is a technology able to provide all this information in a single approach. Aim: To evaluate whether WGS qualifies as a diagnostic tool in a routine setting. Patients and Methods: 3241 bone marrow or blood samples from patients (pts) diagnosed with hematological neoplasm (including AML, ALL, MDS, CML, CLL) were evaluated by WGS. Samples had been sent for routine diagnostic work-up to our laboratory between 2005 and 2017. For WGS, 150bp paired-end sequences where generated on Illumina HiseqX and NovaSeq 6000 (Illumina, San Diego, CA). A mixture genomic DNA from multiple anonymous donors was used as normal controls. The median coverage was 104x (range: 47-196). Only cases with an estimated tumor fraction of at least 20% were included. WGS was validated against chromosome banding analysis (CBA), which was available in 2752 pts with an aberrant karyotype detected in 1513. For 334 pts genomic array data (GA) was available. CNA were called using GATK4 and SV using MANTA software accounting for missing matched-normal samples. For the validation of single nucleotide variants (SNV) and Indels we compared WGS data produced by BaseSpace WGS and Tumor/Normal app to variants classified as pathogenic during routine diagnostics using targeted amplicon sequencing (median coverage 1800x) in 70 genes known to be recurrently mutated including ASXL1, DNMT3A, RUNX1, SRSF2, TET2 , and TP53. Results: In total 475 recurrent reciprocal structural rearrangements (38 different rearrangements including BCR-ABL1, PML-RARA, CBFB-MYH11, RUNX1-RUNX1T1, IGH-BCL2, IGH-MYC, IGH-CCND1) were identified by CBA. Of these 455 (96%) rearrangements were identified by WGS. Due to the significantly lower resolution of CBA compared to WGS and the fact that in complex karyotype the precise determination of CNA in CBA is not possible the comparison with respect to CNA between CBA and WGS was restricted to 843 cases with non-complex karyotype (<4 abnormalities). 289 trisomies, 48 monosomies and 464 recurrent deletions (del) (including del(5q), del(7q), del(11q), del(17p)) were identified by CBA. Of these WGS detected 210 (73%) trisomies and 42 (88%) monosomies. For 74 of the 79 trisomies undetected by WGS the percentage of cells harboring the respective trisomy was determined by interphase FISH and was in median 8%. FISH data was available for all 6 missed monosomies, median clone size was 14%. WGS identified 420/446 (81.5%) del detected by CBA. FISH data was available for 31/44 del missed by WGS. The median proportion of cells harboring the respective del was 11%. In order to test the CNA detection of WGS on a higher resolution level GA data from 334 cases was compared to WGS data. These included 135 cases with normal and 194 with aberrant karyotype in CBA (no CBA: 5), respectively. Comparing 18,337,602 positions 18,031,728 (98%) yielded the same result with both technologies with respect to gain, loss or normal, respectively.For SNV/Indel calls we investigated 2074 mutations in 1022 pts (harboring at least 1 pathogenic mutation (range 1 - 12)). 1892/2074 (91%) were concordant between amplicon sequencing and WGS. 132 from the missed 182 mutations had a variant allele frequency of <10%, which is on the verge of the limit of detection for 100x WGS data. Only 50 cases were missed due to low coverage or very complex alterations. Conclusions: WGS can provide in an "all in one test" all relevant information required for classification and treatment decisions in hematological neoplasms with a high potential to substitute current genetic evaluation based on CBA, FISH and targeted mutation analysis. The next steps on the road towards a diagnostic tool are the validation of CNA, SV and SNV/Idel identified in addition to standard diagnostics and the determination of the coverage necessary to detect small clones relevant for patient care. Thus, a first step is taken towards a completely automated genotyping enabling a broad access to state of the art diagnostics. Disclosures Haferlach: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Twardziok:MLL Munich Leukemia Laboratory: Employment. Hutter:MLL Munich Leukemia Laboratory: Employment. Walter:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Meggendorfer:MLL Munich Leukemia Laboratory: Employment. Nadarajah:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

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UMI-Gen: a UMI-based reads simulator for variant calling evaluation in paired-end sequencing NGS libraries

10.1101/2020.04.22.027532 ◽

2020 ◽

Author(s):

Vincent Sater ◽

Pierre-Julien Viailly ◽

Thierry Lecroq ◽

Philippe Ruminy ◽

Caroline Bérard ◽

...

Keyword(s):

Variant Calling ◽

Copy Number Variations ◽

Biological Data ◽

Single Nucleotide Variants ◽

Background Error ◽

Single Nucleotide ◽

Low Frequencies ◽

Paired End Sequencing ◽

Very High ◽

Generation Sequencing

AbstractMotivationWith Next Generation Sequencing becoming more affordable every year, NGS technologies asserted themselves as the fastest and most reliable way to detect Single Nucleotide Variants (SNV) and Copy Number Variations (CNV) in cancer patients. These technologies can be used to sequence DNA at very high depths thus allowing to detect abnormalities in tumor cells with very low frequencies. A lot of different variant callers are publicly available and usually do a good job at calling out variants. However, when frequencies begin to drop under 1%, the specificity of these tools suffers greatly as true variants at very low frequencies can be easily confused with sequencing or PCR artifacts. The recent use of Unique Molecular Identifiers (UMI) in NGS experiments offered a way to accurately separate true variants from artifacts. UMI-based variant callers are slowly replacing raw-reads based variant callers as the standard method for an accurate detection of variants at very low frequencies. However, benchmarking done in the tools publication are usually realized on real biological data in which real variants are not known, making it difficult to assess their accuracy.ResultsWe present UMI-Gen, a UMI-based reads simulator for targeted sequencing paired-end data. UMI-Gen generates reference reads covering the targeted regions at a user customizable depth. After that, using a number of control files, it estimates the background error rate at each position and then modifies the generated reads to mimic real biological data. Finally, it will insert real variants in the reads from a list provided by the user.AvailabilityThe entire pipeline is available at https://gitlab.com/vincent-sater/umigen-master under MIT [email protected]

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Analysis of heterozygous PRKN variants and copy number variations in Parkinson’s disease

10.1101/2020.05.07.20072728 ◽

2020 ◽

Author(s):

Eric Yu ◽

Uladzislau Rudakov ◽

Lynne Krohn ◽

Kheireddin Mufti ◽

Jennifer A. Ruskey ◽

...

Keyword(s):

Parkinson’S Disease ◽

Parkinson's Disease ◽

Movement Disorder ◽

Copy Number ◽

Copy Number Variations ◽

Mendelian Disease ◽

Single Nucleotide Variants ◽

Single Nucleotide ◽

Association Tests ◽

Molecular Inversion Probes

AbstractImportanceBiallelic PRKN mutation carriers with Parkinson’s disease (PD) typically have an earlier disease onset, slow disease progression and, often, different neuropathology compared to sporadic patients. However, the role of heterozygous PRKN variants in the risk of PD remains unclear.ObjectiveTo examine the association between heterozygous PRKN variants, including single nucleotide variants and copy number variations (CNVs), and PD status.DesignThis case-control study was conducted in Canada using data from 4 centers in Canada, France, the United States and Israel, collected between 2013 and 2020.SettingMulticenter, population matched, unrelated participants.ParticipantsPD patients were compared with healthy controls. Participants were excluded if genetic data quality was poor, PRKN variants were biallelic, or relatedness was discovered. In total, 2,807 patients and 3,627 controls were recruited for the study. Data analyses were performed from January 2019 to March 2020.ExposureInheritance of heterozygous PRKN variants.Main Outcome(s) and Measure(s)PD was diagnosed by movement disorder specialists according to the UK Brain Bank Criteria or the Movement Disorder Society criteria. Targeted next-generation sequencing with molecular inversion probes and multiplex ligation-dependent probe amplification were used to detect rare variants and CNVs. These variants were examined with optimized sequence kernel association tests after accounting for potential pathogenicity. P values of optimized sequence kernel association tests between heterozygous PRKN variants and PD were measured.ResultsDNA of all participants were sequenced, including 1,903 late onset (mean [SD], 64.02±7.81 years, 1,196 men [63%]) and 542 early onset patients (mean [SD], 43.30±6.60, 368 men [68%]). Age at onset was not available for 349 patients. Carriers of two PRKN variants were excluded from the analysis. No associations were found between heterozygous variants and risk of PD. Pathogenic and likely pathogenic heterozygous variants and CNVs were less common among PD patients (1.0%) than among controls (1.3%).Conclusion and RelevanceThis study suggests that heterozygous variants and CNVs in PRKN are not associated with PD. Molecular inversion probes allow for rapid and cost-effective detection of all types of PRKN variants, which may be useful for pre-trial screening and for clinical and basic science studies specifically targeting PRKN patients.Key PointsQuestionAre rare heterozygous PRKN single nucleotide variants and copy number variations associated with Parkinson’s disease?FindingsIn this case-controls study including 2,807 patients and 3,627 controls, we found no significant association between rare heterozygous PRKN variants and risk of Parkinson’s disease.MeaningPRKN-associated parkinsonism is an autosomal recessive Mendelian disease, and based on our data, heterozygous carriers are not likely to be at increased risk of Parkinson’s disease.

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Analytical and Clinical Validation of an Amplicon-based Next Generation Sequencing Assay for Ultrasensitive Detection of Circulating Tumor DNA

10.1101/2021.08.03.21261575 ◽

2021 ◽

Author(s):

Jonathan Poh ◽

Kao Chin Ngeow ◽

Michelle Pek ◽

Kian-Hin Tan ◽

Jing Shan Lim ◽

...

Keyword(s):

Next Generation Sequencing ◽

Copy Number ◽

Point Mutations ◽

Circulating Tumor Dna ◽

Clinical Validation ◽

Gene Fusions ◽

Copy Number Alterations ◽

Next Generation ◽

Tumor Dna ◽

Generation Sequencing

Next-generation sequencing of circulating tumor DNA presents a promising approach to cancer diagnostics, complementing conventional tissue-based diagnostic testing by enabling minimally invasive serial testing and broad genomic coverage through a simple blood draw to maximize therapeutic benefit to patients. LiquidHALLMARK® is an amplicon-based next-generation sequencing assay developed for the genomic profiling of plasma-derived cell-free DNA. The comprehensive 80-gene panel profiles point mutations, insertions/deletions, copy number alterations, and gene fusions, and further detects oncogenic viruses (EBV and HBV) and microsatellite instability. Here, the analytical and clinical validation of the assay is reported. Analytical validation using reference genetic materials demonstrated a sensitivity of 99.38% for point mutations and 95.83% for insertions/deletions at 0.1% variant allele frequency (VAF), and a sensitivity of 91.67% for gene fusions at 0.5% VAF, with high specificity even at 0.1% VAF (99.11% per-base). The limit of detection for copy number alterations, EBV, HBV, and microsatellite instability were also empirically determined. Orthogonal comparison of EGFR variant calls made by LiquidHALLMARK and a reference allele-specific PCR method for 355 lung cancer specimens revealed an overall concordance of 93.80%, while external validation with cobas® EGFR Mutation Test v2 for 50 lung cancer specimens demonstrated an overall concordance of 84.00%, with a 100% concordance rate for EGFR variants above 0.4% VAF. Clinical application of LiquidHALLMARK in 1,592 consecutive patients demonstrated a high detection rate (74.8% alteration-positive in cancer samples) and broad actionability (50.0% of cancer samples harboring alterations with biological evidence for actionability). Among ctDNA-positive lung cancers, 72.5% harbored at least one biomarker with a guideline-approved drug indication. These results establish the high sensitivity, specificity, accuracy, and precision of the LiquidHALLMARK assay and supports its clinical application for blood-based genomic testing.

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A map of copy number variations in the Tunisian population: a valuable tool for medical genomics in North Africa

npj Genomic Medicine ◽

10.1038/s41525-020-00166-5 ◽

2021 ◽

Vol 6 (1) ◽

Author(s):

Lilia Romdhane ◽

Nessrine Mezzi ◽

Hamza Dallali ◽

Olfa Messaoud ◽

Jingxuan Shan ◽

...

Keyword(s):

North Africa ◽

Copy Number ◽

Single Nucleotide Polymorphism Array ◽

Copy Number Variations ◽

Tunisian Population ◽

North African ◽

Single Nucleotide Variants ◽

Single Nucleotide ◽

Human Phenotype ◽

Mendelian Diseases

AbstractCopy number variation (CNV) is considered as the most frequent type of structural variation in the human genome. Some CNVs can act on human phenotype diversity, encompassing rare Mendelian diseases and genomic disorders. The North African populations remain underrepresented in public genetic databases in terms of single-nucleotide variants as well as for larger genomic mutations. In this study, we present the first CNV map for a North African population using the Affymetrix Genome-Wide SNP (single-nucleotide polymorphism) array 6.0 array genotyping intensity data to call CNVs in 102 Tunisian healthy individuals. Two softwares, PennCNV and Birdsuite, were used to call CNVs in order to provide reliable data. Subsequent bioinformatic analyses were performed to explore their features and patterns. The CNV map of the Tunisian population includes 1083 CNVs spanning 61.443 Mb of the genome. The CNV length ranged from 1.017 kb to 2.074 Mb with an average of 56.734 kb. Deletions represent 57.43% of the identified CNVs, while duplications and the mixed loci are less represented. One hundred and three genes disrupted by CNVs are reported to cause 155 Mendelian diseases/phenotypes. Drug response genes were also reported to be affected by CNVs. Data on genes overlapped by deletions and duplications segments and the sequence properties in and around them also provided insights into the functional and health impacts of CNVs. These findings represent valuable clues to genetic diversity and personalized medicine in the Tunisian population as well as in the ethnically similar populations from North Africa.

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Tumor mutation burden testing: a survey of the International Quality Network for Pathology (IQN Path)

Virchows Archiv ◽

10.1007/s00428-021-03093-7 ◽

2021 ◽

Author(s):

Francesca Fenizia ◽

Nicola Wolstenholme ◽

Jennifer A. Fairley ◽

Etienne Rouleau ◽

Melanie H. Cheetham ◽

...

Keyword(s):

Current Practice ◽

Checkpoint Inhibitors ◽

Testing Methods ◽

Single Nucleotide Variants ◽

Single Nucleotide ◽

Tumor Mutation Burden ◽

Formalin Fixed Paraffin ◽

Mutation Burden ◽

Formalin Fixed Paraffin Embedded ◽

Formalin Fixed

AbstractWhile tumour mutation burden (TMB) is emerging as a possible biomarker for immune-checkpoint inhibitors (ICI), methods for testing have not been standardised as yet. In April 2019, the International Quality Network for Pathology (IQN Path) launched a survey to assess the current practice of TMB testing. Of the 127 laboratories that replied, 69 (54.3%) had already introduced TMB analysis for research purposes and/or clinical applications. Fifty laboratories (72.5%) used targeted sequencing, although a number of different panels were employed. Most laboratories tested formalin-fixed paraffin-embedded material (94.2%), while 18/69 (26%) tested also cell-free DNA. Fifty-five laboratories used both single nucleotide variants and indels for TMB calculation; 20 centers included only non-synonymous variants. In conclusion, the data from this survey indicate that multiple global laboratories were capable of rapidly introducing routine clinical TMB testing. However, the variability of testing methods raises concerns about the reproducibility of results among centers.

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