Effects of Improved DNA Integrity by Punch From Tissue Blocks as Compared to Pinpoint Extraction From Unstained Slides on Next-Generation Sequencing Quality Metrics

Morlote, Diana; Janowski, Karen M.; Siniard, Rance C.; Guo, Ruiyun; Winokur, Thomas S.; DeFrank, Gina; Harada, Shuko

doi:10.1093/ajcp/aqz014

Cited by 19 publications

(16 citation statements)

References 19 publications

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“…Consistent with a previous study, we observed a positive correlation between the percentages of on-target aligned reads and DIN [ 14 ]. DIN is a useful indicator of the ability to capture target areas.…”

Section: Discussionsupporting

confidence: 92%

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Areas of Crush Nuclear Streaming Should Be Included as Tumor Content in the Era of Molecular Diagnostics

Noda

Yamaka

Atsumi

et al. 2023

Cancers

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Degenerated tissues are frequently observed in malignant tumors, but are not analyzed. We investigated whether nuclear streaming and necrosis samples could be used for genetic analysis to expand the sample pool. A total of 81 samples were extracted from small cell carcinoma and lymphoma FFPE tissue blocks and classified into three histological cohorts: 33 materials with well-preserved tumor morphology, 31 nuclear streaming samples, and 17 necrosis samples. DNA and RNA integrity numbers, percentage of RNA fragments with >200 nucleotides, and next-generation sequencing quality metrics were compared among the cohorts. DNA quality did not significantly differ between nuclear streaming materials and materials with well-preserved morphology, whereas that of the necrosis samples was inferior. RNA quality decreased in the following order: materials with well-preserved morphology > nuclear streaming > necrosis. The sequencing metrics did not differ significantly between the nuclear streaming samples and materials with well-preserved morphology, and reliable variants were detected. The necrosis samples extracted from resections exhibited sequencing failure and showed significantly fewer on-target aligned reads and variants. However, variant allele frequency did not differ among the cohorts. We revelated that DNA in nuclear streaming samples, especially within biopsies, could be used for genetic analysis. Moreover, degenerated non-tumor cells should be counted when evaluating tumor content to avoid misinterpreting the variant allele frequency.

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Section: Discussionsupporting

confidence: 92%

“…DIN is a scale used to assess the quality of DNA, and it ranges from 1 (completely degraded) to 10 (completely intact), based on gDNA degradation [ 7 , 10 , 14 ]. Samples with DIN > 3 are recommended for NGS analysis [ 7 ].…”

Section: Discussionmentioning

confidence: 99%

Areas of Crush Nuclear Streaming Should Be Included as Tumor Content in the Era of Molecular Diagnostics

Noda

Yamaka

Atsumi

et al. 2023

Cancers

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“…The DIN calculated by the TapeStation is a useful tool for quantitative analysis of DNA integrity and determining the fitness of a DNA specimen for sequencing. 40 There has been variation in prior studies as to the acceptable cutoff, ranging from 4.0 41 to 7.0 42 or 8.0, 43 when analyzing DNA specimens for next generation sequencing, though sequencing data can still be obtained from DNA specimens with DIN < 4.0. 44 Though a weak inverse correlation was found between DIN and processing interval, all DNA specimens processed during the pilot and early phases of the DAP, had a DIN ⩾ 7.2 and 98 out of the 109 (89.9%) had a DIN ⩾ 9.0.…”

Section: Discussionmentioning

confidence: 99%

Purpose, Partnership, and Possibilities: The Implementation of the Dog Aging Project Biobank

Mouttham

Castelhano

2022

Biomark�Insights

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Background: Biobanks have been supporting longitudinal prospective and retrospective studies by providing standardized services for the acquisition, transport, processing, storage, and distribution of high-quality biological material and associated data. Here, we describe how the Dog Aging Project (DAP), a large-scale longitudinal study of the domestic dog ( Canis familiaris) with translational applications for humans, developed a biobank of canine biospecimens and associated data. Design and methods: This was accomplished by working with the Cornell Veterinary Biobank, the first biobank in the world to receive accreditation to ISO 20387:2018—General Requirements for Biobanking. The biobank research team was involved in the early collection stages of the DAP, contributing to the development of appropriate workflows and processing fit-for-purpose biospecimens. In support of a dynamic strategy for real-time adjustment of processes, a pilot phase was implemented to develop, test, and optimize the biospecimen workflows, followed by an early phase of collection, processing, and banking of specimens from DAP participants. Results: During the pilot and early phases of collection, the DAP Biobank stored 164 aliquots of whole blood, 273 aliquots of peripheral blood mononuclear cells, 130 aliquots of plasma, and 70 aliquots of serum, and extracted high molecular weight genomic DNA suitable for whole-genome sequencing from 109 whole blood specimens. These specimens, along with their associated preanalytical data, have been made available for distribution to researchers. Conclusion: We discuss the challenges and opportunities encountered during the implementation of the DAP Biobank, along with novel strategies for promoting biobanking sustainability such as partnering with a DAP quality assurance manager and a DAP marketing and communication specialist and developing a pilot grant structure to fund small innovative research projects.

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“…A critical initial step of NGS is adequate pathological examination, as a properly selected and dissected tissue sample is a necessity for any further investigation [11]. Samples should contain a sufficient proportion of tumor cells to differentiate germline and somatic mutations.…”

Section: First Steps Of Whole Exome Sequencingmentioning

confidence: 99%

Comprehensive Outline of Whole Exome Sequencing Data Analysis Tools Available in Clinical Oncology

Bartha

Győrffy

2019

Cancers

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Whole exome sequencing (WES) enables the analysis of all protein coding sequences in the human genome. This technology enables the investigation of cancer-related genetic aberrations that are predominantly located in the exonic regions. WES delivers high-throughput results at a reasonable price. Here, we review analysis tools enabling utilization of WES data in clinical and research settings. Technically, WES initially allows the detection of single nucleotide variants (SNVs) and copy number variations (CNVs), and data obtained through these methods can be combined and further utilized. Variant calling algorithms for SNVs range from standalone tools to machine learning-based combined pipelines. Tools for CNV detection compare the number of reads aligned to a dedicated segment. Both SNVs and CNVs help to identify mutations resulting in pharmacologically druggable alterations. The identification of homologous recombination deficiency enables the use of PARP inhibitors. Determining microsatellite instability and tumor mutation burden helps to select patients eligible for immunotherapy. To pave the way for clinical applications, we have to recognize some limitations of WES, including its restricted ability to detect CNVs, low coverage compared to targeted sequencing, and the missing consensus regarding references and minimal application requirements. Recently, Galaxy became the leading platform in non-command line-based WES data processing. The maturation of next-generation sequencing is reinforced by Food and Drug Administration (FDA)-approved methods for cancer screening, detection, and follow-up. WES is on the verge of becoming an affordable and sufficiently evolved technology for everyday clinical use.

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Effects of Improved DNA Integrity by Punch From Tissue Blocks as Compared to Pinpoint Extraction From Unstained Slides on Next-Generation Sequencing Quality Metrics

Cited by 19 publications

References 19 publications

Areas of Crush Nuclear Streaming Should Be Included as Tumor Content in the Era of Molecular Diagnostics

Areas of Crush Nuclear Streaming Should Be Included as Tumor Content in the Era of Molecular Diagnostics

Purpose, Partnership, and Possibilities: The Implementation of the Dog Aging Project Biobank

Comprehensive Outline of Whole Exome Sequencing Data Analysis Tools Available in Clinical Oncology

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