Quantifying the relative amount of mouse and human DNA in cancer xenografts using species-specific variation in gene length

Lin, Ming Tseh; Tseng, Li Hui; Kamiyama, Hirohiko; Kamiyama, Mihoko; Lim, Phillip; Hidalgo, Manuel; Wheelan, Sarah J.; Eshleman, James R.

doi:10.2144/000113363

Cited by 32 publications

(31 citation statements)

References 18 publications

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“…DNA/RNA from xenografts is always contaminated, and although an assay has been published (10) to quantify the proportion of human/mouse DNA in the samples from pancreatic cancer, a generalized method is still lacking. Various studies have reported the need to preprocess xenograft data before performing downstream analysis (9,13).…”

Section: Methodsmentioning

confidence: 99%

“…Although efforts can be made to mitigate these effects experimentally, high levels of infiltrating stromal cells often render this impractical. Consequently, levels of contamination as high as 73% have been observed in pancreatic cancer patient-derived xenograft (PDX) models (10), and data are often variable (11). Instead, studies have typically addressed readheterogeneity in silico (9,12).…”

Section: Introductionmentioning

confidence: 99%

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Next-Generation Sequencing Analysis and Algorithms for PDX and CDX Models

Khandelwal

Girotti

Smowton

et al. 2017

Molecular Cancer Research

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Patient-derived xenograft (PDX) and circulating tumor cellderived explant (CDX) models are powerful methods for the study of human disease. In cancer research, these methods have been applied to multiple questions, including the study of metastatic progression, genetic evolution, and therapeutic drug responses. As PDX and CDX models can recapitulate the highly heterogeneous characteristics of a patient tumor, as well as their response to chemotherapy, there is considerable interest in combining them with next-generation sequencing to monitor the genomic, transcriptional, and epigenetic changes that accompany oncogenesis. When used for this purpose, their reliability is highly dependent on being able to accurately distinguish between sequencing reads that originate from the host, and those that arise from the xenograft itself. Here, we demonstrate that failure to correctly identify contaminating host reads when analyzing DNAand RNA-sequencing (DNA-Seq and RNA-Seq) data from PDX and CDX models is a major confounding factor that can lead to incorrect mutation calls and a failure to identify canonical mutation signatures associated with tumorigenicity. In addition, a highly sensitive algorithm and open source software tool for identifying and removing contaminating host sequences is described. Importantly, when applied to PDX and CDX models of melanoma, these data demonstrate its utility as a sensitive and selective tool for the correction of PDX-and CDX-derived wholeexome and RNA-Seq data.

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Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Next-Generation Sequencing Analysis and Algorithms for PDX and CDX Models

Khandelwal

Girotti

Smowton

et al. 2017

Molecular Cancer Research

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“…The error rates and read length challenges also place limitations on species differentiation. Many experimental systems and samples used for drug discovery contain sequences from multiple species, such as human cancer xenografts in mouse and bacterial or other cell types in human specimens [22]. SGST capabilities impose limitations on the separation of true human signal from other species.…”

Section: Second Generation Sequencing Technologies (Sgsts) and Appmentioning

confidence: 99%

Third-generation sequencing techniques and applications to drug discovery

Ozsolak

2012

Expert Opinion on Drug Discovery

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Introduction There is an immediate need for functional and molecular studies to decipher differences between disease and “normal” settings to identify large quantities of validated targets with the highest therapeutic utilities. Furthermore, drug mechanism of action and biomarkers to predict drug efficacy and safety need to be identified for effective design of clinical trials, decreasing attrition rates, regulatory agency approval process and drug repositioning. By expanding the power of genetics and pharmacogenetics studies, next generation nucleic acid sequencing technologies have started to play an important role in all stages of drug discovery. Areas covered This article reviews the first and second generation sequencing technologies (SGSTs) and challenges they pose to biomedicine. The article then focuses on the emerging third generation sequencing technologies (TGSTs), their technological foundations and potential contributions to drug discovery. Expert Opinion Despite the scientific and commercial success of SGSTs, the goal of rapid, comprehensive and unbiased sequencing of nucleic acids has not been achieved. TGSTs promise to increase sequencing throughput and read lengths, decrease costs, run times and error rates, eliminate biases inherent in SGSTs, and offer capabilities beyond nucleic acid sequencing. Such changes will have positive impact in all sequencing applications to drug discovery.

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“…Samples were subjected to 40 cycles of denaturation (95oC, 30 sec), annealing (57°C, 30 sec) and extension (72oC, 60 sec). Capillary electrophoresis was performed and analyzed as described previously [21]. …”

Section: Methodsmentioning

confidence: 99%

A Novel Tandem Duplication Assay to Detect Minimal Residual Disease in FLT3/ITD AML

et al. 2015

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Background Internal tandem duplication (ITD) of the FLT3 gene is associated with poor prognosis in acute myeloid leukemia (AML) patients with a normal karyotype. The current standard PCR assay for FLT3/ITD detection is not sufficiently sensitive to monitor minimal residual disease (MRD). Clone-specific assays may have sufficient sensitivity but are not practical to implement, since each clone-specific primer/probe requires clinical validation. Objective To develop an assay for clinical molecular diagnostics laboratories to monitor MRD in FLT3/ITD AMLs. Methods We designed a simple novel assay, tandem duplication PCR (TD-PCR), and tested its sensitivity, specificity and clinical utility in FLT3/ITD AML patients. Results TD-PCR was capable of detecting a single ITD molecule and was applicable to 75% of ITD mutants tested. TD-PCR detected MRD in bone marrow prior to patient relapse. TD-PCR also identified low level ITD mutants not only in FLT3/ITD AMLs but also in initial diagnostic specimens reportedly negative by the standard assay in patients who progressed with the same ITDs detected by the TD-PCR assay. Conclusion Detection of MRD by TD-PCR may guide patient selection for early clinical intervention. In contrast to clone-specific approaches, TD-PCR assay can be more easily validated for MRD detection in clinical laboratories due to standardized primers and a universal positive control. In addition, our results on multi-clonality and low-level ITDs suggest that further studies are warranted to elucidate their clinical/biological significance.

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Quantifying the relative amount of mouse and human DNA in cancer xenografts using species-specific variation in gene length

Cited by 32 publications

References 18 publications

Next-Generation Sequencing Analysis and Algorithms for PDX and CDX Models

Next-Generation Sequencing Analysis and Algorithms for PDX and CDX Models

Third-generation sequencing techniques and applications to drug discovery

A Novel Tandem Duplication Assay to Detect Minimal Residual Disease in FLT3/ITD AML

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