Highlights • Sequencing of STRs reveals substantial variation in addition to length variation. • Sequence variation of 17 STRs was assessed for three globally dispersed populations. • Stutter ratios ...of complex STR alleles with the same length can vary substantially. • Minor mixture contributions of 1% are recovered but are exceeded by stutters reads. • MPS STR analysis of minor mixture contributions down to 5% is routinely feasible.
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.
What's new?
Short tandem repeat (STR) profiling is the recommended approach for authentication testing of human cell lines. To improve its effectiveness, however, a method by which STR can account for genetic drift arising from the passage of malignant cells is needed. Here, algorithms based on a set of match criteria, eight core STR loci, and amelogenin analysis were found to successfully discriminate between related and unrelated samples. The match criteria used here would bring greater rigor to the interpretation of STR profiling results.
Highlights • PowerSeq Auto System and MiSeq can generate concordant result with CE-based methods. • Single source full profiles could be obtained using as little as 62 pg of input DNA. • Partial STR ...profiles of the minor contributor could be detected up to 19:1 mixture. • MPS can improve mixture analysis with the detection of SNPs within STR alleles.
Microsatellite instability (MSI) occurs in over 90% of Lynch syndrome cancers and is considered a hallmark of the disease. MSI is an early event in colon tumor development, but screening polyps for ...MSI remains controversial because of reduced sensitivity compared to more advanced neoplasms. To increase sensitivity, we investigated the use of a novel type of marker consisting of long mononucleotide repeat (LMR) tracts. Adenomas from 160 patients, ranging in age from 29-55 years old, were screened for MSI using the new markers and compared with current marker panels and immunohistochemistry standards. Overall, 15 tumors were scored as MSI-High using the LMRs compared to 9 for the NCI panel and 8 for the MSI Analysis System (Promega). This difference represents at least a 1.7-fold increase in detection of MSI-High lesions over currently available markers. Moreover, the number of MSI-positive markers per sample and the size of allelic changes were significantly greater with the LMRs (p = 0.001), which increased confidence in MSI classification. The overall sensitivity and specificity of the LMR panel for detection of mismatch repair deficient lesions were 100% and 96%, respectively. In comparison, the sensitivity and specificity of the MSI Analysis System were 67% and 100%; and for the NCI panel, 75% and 97%. The difference in sensitivity between the LMR panel and the other panels was statistically significant (p<0.001). The increased sensitivity for detection of MSI-High phenotype in early colorectal lesions with the new LMR markers indicates that MSI screening for the early detection of Lynch syndrome might be feasible.
Research in toxicology relies on in vitro models such as cell lines. These living models are prone to change and may be described in publications with insufficient information or quality control ...testing. This article sets out recommendations to improve the reliability of cell-based research.
Abstract The PowerPlex® Y23 System combines the seventeen Y-STR loci in current commercially available Y-STR kits (DYS19, DYS385a/b, DYS389I/II, DYS390, DYS391, DYS392, DYS393, DYS437, DYS438, ...DYS439, DYS448, DYS456, DYS458, DYS635, and Y-GATA-H4) with six new highly discriminating Y-STR loci (DYS481, DYS533, DYS549, DYS570, DYS576, and DYS643). These six new loci have higher gene diversities than most of the loci in other commercial Y-STR analysis kits, allowing for further distinction between unrelated male individuals. In addition, the inclusion of two rapidly mutating Y-STR loci may allow for the discrimination of related individuals. The PowerPlex® Y23 System is designed to amplify DNA from purified extracts as well as direct amplification from substrates used to collect database samples (e.g. swabs and storage cards). Validation of the PowerPlex® Y23 System includes all of the studies required by the FBI and SWGDAM. The results demonstrate that the PowerPlex® Y23 System is a robust and reliable amplification kit capable of overcoming high concentrations of commonly encountered inhibitors such as hematin, humic acid, and tannic acid. Full profiles are consistently detected with 62.5 pg of male DNA, even in the presence of excessive amounts of female DNA, establishing the PowerPlex® Y23 System as a sensitive method for Y-STR testing. Complete Y-STR profiles are detected from mixed samples with 62.5 pg of male DNA in a background of 400 ng of female DNA or 125 pg of male DNA mixed with 3000 ng of female DNA.
Microsatellite instability (MSI) is an evolving biomarker for cancer detection and treatment. MSI was first used to identify patients with Lynch syndrome, a hereditary form of colorectal cancer ...(CRC), but has recently become indispensable in predicting patient response to immunotherapy. To address the need for pan-cancer MSI detection, a new multiplex assay was developed that uses novel long mononucleotide repeat (LMR) markers to improve sensitivity. A total of 469 tumor samples from 20 different cancer types, including 319 from patients with Lynch syndrome, were tested for MSI using the new LMR MSI Analysis System. Results were validated by using deficient mismatch repair (dMMR) status according to immunohistochemistry as the reference standard and compared versus the Promega pentaplex MSI panel. The sensitivity of the LMR panel for detection of dMMR status by immunohistochemistry was 99% for CRC and 96% for non-CRC. The overall percent agreement between the LMR and Promega pentaplex panels was 99% for CRC and 89% for non-CRC tumors. An increased number of unstable markers and the larger size shifts observed in dMMR tumors using the LMR panel increased confidence in MSI determinations. The LMR MSI Analysis System expands the spectrum of cancer types in which MSI can be accurately detected.
This commentary highlights the article by Murray et al that describes novel probe systems for real-time PCR that provide improvements relative to dual-labeled probes.
Abstract The original CODIS database based on 13 core STR loci has been overwhelmingly successful for matching suspects with evidence. Yet there remain situations that argue for inclusion of more ...loci and increased discrimination. The PowerPlex® Fusion System allows simultaneous amplification of the following loci: Amelogenin, D3S1358, D1S1656, D2S441, D10S1248, D13S317, Penta E, D16S539, D18S51, D2S1338, CSF1PO, Penta D, TH01, vWA, D21S11, D7S820, D5S818, TPOX, DYS391, D8S1179, D12S391, D19S433, FGA, and D22S1045. The comprehensive list of loci amplified by the system generates a profile compatible with databases based on either the expanded CODIS or European Standard Set (ESS) requirements. Developmental validation testing followed SWGDAM guidelines and demonstrated the quality and robustness of the PowerPlex® Fusion System across a number of variables. Consistent and high-quality results were compiled using data from 12 separate forensic and research laboratories. The results verify that the PowerPlex® Fusion System is a robust and reliable STR-typing multiplex suitable for human identification.
Highlights • Multiplex STR typing of 17 STRs and the Amelogenin locus is feasible by MPS. • Sensitivity of detection is comparable with that of CE-based analyses • A size selection method and ...quantity of amplified DNA necessary are described. • The prototype STR multiplex and the illumina MiSeq generated reliable DNA profiles. • Intra-repeat SNPs were observed and this additional variation may be useful.
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