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1.
Int J Cancer ; 132(11): 2510-9, 2013 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-23136038

RESUMO

Continuous human cell lines have been used extensively as models for biomedical research. In working with these cell lines, researchers are often unaware of the risk of cross-contamination and other causes of misidentification. To reduce this risk, there is a pressing need to authenticate cell lines, comparing the sample handled in the laboratory to a previously tested sample. The American Type Culture Collection Standards Development Organization Workgroup ASN-0002 has developed a Standard for human cell line authentication, recommending short tandem repeat (STR) profiling for authentication of human cell lines. However, there are known limitations to the technique when applied to cultured samples, including possible genetic drift with passage. In our study, a dataset of 2,279 STR profiles from four cell banks was used to assess the effectiveness of the match criteria recommended within the Standard. Of these 2,279 STR profiles, 1,157 were grouped into sets of related cell lines-duplicate holdings, legitimately related samples or misidentified cell lines. Eight core STR loci plus amelogenin were used to unequivocally authenticate 98% of these related sets. Two simple match algorithms each clearly discriminated between related and unrelated samples, with separation between related samples at ≥80% match and unrelated samples at <50% match. A small degree of overlap was noted at 50-79% match, mostly from cell lines known to display variable STR profiles. These match criteria are recommended as a simple and effective way to interpret results from STR profiling of human cell lines.


Assuntos
Algoritmos , Perfilação da Expressão Gênica/métodos , Técnicas de Genotipagem/normas , Repetições de Microssatélites/genética , Linhagem Celular , Humanos , Reação em Cadeia da Polimerase
2.
Nat Biotechnol ; 39(9): 1141-1150, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34504346

RESUMO

Clinical applications of precision oncology require accurate tests that can distinguish true cancer-specific mutations from errors introduced at each step of next-generation sequencing (NGS). To date, no bulk sequencing study has addressed the effects of cross-site reproducibility, nor the biological, technical and computational factors that influence variant identification. Here we report a systematic interrogation of somatic mutations in paired tumor-normal cell lines to identify factors affecting detection reproducibility and accuracy at six different centers. Using whole-genome sequencing (WGS) and whole-exome sequencing (WES), we evaluated the reproducibility of different sample types with varying input amount and tumor purity, and multiple library construction protocols, followed by processing with nine bioinformatics pipelines. We found that read coverage and callers affected both WGS and WES reproducibility, but WES performance was influenced by insert fragment size, genomic copy content and the global imbalance score (GIV; G > T/C > A). Finally, taking into account library preparation protocol, tumor content, read coverage and bioinformatics processes concomitantly, we recommend actionable practices to improve the reproducibility and accuracy of NGS experiments for cancer mutation detection.


Assuntos
Benchmarking , Sequenciamento do Exoma/normas , Neoplasias/genética , Análise de Sequência de DNA/normas , Sequenciamento Completo do Genoma/normas , Linhagem Celular , Linhagem Celular Tumoral , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Mutação , Neoplasias/patologia , Reprodutibilidade dos Testes
3.
Nat Biotechnol ; 39(9): 1151-1160, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34504347

RESUMO

The lack of samples for generating standardized DNA datasets for setting up a sequencing pipeline or benchmarking the performance of different algorithms limits the implementation and uptake of cancer genomics. Here, we describe reference call sets obtained from paired tumor-normal genomic DNA (gDNA) samples derived from a breast cancer cell line-which is highly heterogeneous, with an aneuploid genome, and enriched in somatic alterations-and a matched lymphoblastoid cell line. We partially validated both somatic mutations and germline variants in these call sets via whole-exome sequencing (WES) with different sequencing platforms and targeted sequencing with >2,000-fold coverage, spanning 82% of genomic regions with high confidence. Although the gDNA reference samples are not representative of primary cancer cells from a clinical sample, when setting up a sequencing pipeline, they not only minimize potential biases from technologies, assays and informatics but also provide a unique resource for benchmarking 'tumor-only' or 'matched tumor-normal' analyses.


Assuntos
Benchmarking , Neoplasias da Mama/genética , Análise Mutacional de DNA/normas , Sequenciamento de Nucleotídeos em Larga Escala/normas , Sequenciamento Completo do Genoma/normas , Linhagem Celular Tumoral , Conjuntos de Dados como Assunto , Células Germinativas , Humanos , Mutação , Padrões de Referência , Reprodutibilidade dos Testes
5.
Regen Med ; 6(2): 255-60, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21391858

RESUMO

Authentication of human tissues, cell lines and primary cell cultures (including stem cell preparations) used as therapeutic modalities is often performed using phenotyping and technologies capable of assessing identity to the species level (e.g., isoenzyme analysis and/or karyotyping). This authentication paradigm alone cannot provide assurance that the correct human cell preparation is administered, so careful labeling and tracking of cells from the donor, during manufacture and as part of the final product are also employed. Precise, accurate identification of human cells to the individual donor level could, however, significantly reduce the risks of exposing human subjects to misidentified cells. The availability of a standardized method for achieving this will provide a way to improve the safety profile of human cell-based products by providing assurance that a given lot of cells originated from the intended donor and were not inadvertently mixed or replaced with cells from other donors. In support of this goal, an international team of scientists has prepared a consensus standard on authentication of human cells using short tandem repeat profiling. Associated with the standard itself will be the establishment and maintenance of a public database of short tandem repeat profiles for commonly used cell lines.


Assuntos
Produtos Biológicos/normas , Identificação Biométrica/métodos , Identificação Biométrica/normas , Células , Consenso , Técnicas de Cultura de Células/métodos , Técnicas de Cultura de Células/normas , Células/química , Células/citologia , Células/metabolismo , Conferências de Consenso como Assunto , Contaminação de Equipamentos , Perfilação da Expressão Gênica/normas , Perfilação da Expressão Gênica/estatística & dados numéricos , Humanos , Repetições de Microssatélites/genética , Padrões de Referência , Análise de Sequência de DNA/normas , Análise de Sequência de DNA/estatística & dados numéricos , Células-Tronco/química , Células-Tronco/citologia , Células-Tronco/metabolismo
7.
In Vitro Cell Dev Biol Anim ; 46(9): 727-32, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-20614197

RESUMO

Cell misidentification and cross-contamination have plagued biomedical research for as long as cells have been employed as research tools. Examples of misidentified cell lines continue to surface to this day. Efforts to eradicate the problem by raising awareness of the issue and by asking scientists voluntarily to take appropriate actions have not been successful. Unambiguous cell authentication is an essential step in the scientific process and should be an inherent consideration during peer review of papers submitted for publication or during review of grants submitted for funding. In order to facilitate proper identity testing, accurate, reliable, inexpensive, and standardized methods for authentication of cells and cell lines must be made available. To this end, an international team of scientists is, at this time, preparing a consensus standard on the authentication of human cells using short tandem repeat (STR) profiling. This standard, which will be submitted for review and approval as an American National Standard by the American National Standards Institute, will provide investigators guidance on the use of STR profiling for authenticating human cell lines. Such guidance will include methodological detail on the preparation of the DNA sample, the appropriate numbers and types of loci to be evaluated, and the interpretation and quality control of the results. Associated with the standard itself will be the establishment and maintenance of a public STR profile database under the auspices of the National Center for Biotechnology Information. The consensus standard is anticipated to be adopted by granting agencies and scientific journals as appropriate methodology for authenticating human cell lines, stem cells, and tissues.


Assuntos
Biologia Celular/normas , Perfilação da Expressão Gênica/métodos , Repetições de Microssatélites/genética , Manejo de Espécimes/métodos , Bancos de Tecidos/normas , Linhagem Celular , Humanos , Células-Tronco , Estados Unidos
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