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

Human cancer cell lines and xenografts are valuable samples for whole-genome sequencing of human cancer. Tumors can be maintained by serial xenografting in athymic (nude) or severe combined immunodeficient (SCID) mice. In the current study, we developed a molecular assay to quantify the relative contributions of human and mouse in mixed DNA samples. The assay was designed based on deletion/insertion variation between human and mouse genomes. The percentage of mouse DNA was calculated according to the relative peak heights of PCR products analyzed by capillary electrophoresis. Three markers from chromosomes 9 and 10 accurately predicted the mouse genome ratio and were combined into a multiplex PCR reaction. We used the assay to quantify the relative DNA amounts of 93 mouse xenografts used for a recently reported integrated genomic analysis of human pancreatic cancer. Of the 93 xenografts, the mean percentage of contaminating mouse DNA was 47%, ranging from 17% to 73%, with 43% of samples having >50% mouse DNA. We then comprehensively compared the human and mouse genomes to identify 370 additional candidate gene loci demonstrating human-mouse length variation. With increasing whole-genome sequencing of human cancers, this assay should be useful to monitor strategies to enrich human cancer cells from mixed human-mouse cell xenografts. Finally, we discuss how contaminating mouse DNA affects next-generation DNA sequencing.

[1]  D. Eberhard,et al.  A tumor sorting protocol that enables enrichment of pancreatic adenocarcinoma cells and facilitation of genetic analyses. , 2009, The Journal of molecular diagnostics : JMD.

[2]  G. Parmigiani,et al.  Core Signaling Pathways in Human Pancreatic Cancers Revealed by Global Genomic Analyses , 2008, Science.

[3]  Joshua M. Korn,et al.  Comprehensive genomic characterization defines human glioblastoma genes and core pathways , 2008, Nature.

[4]  Richard K. Wilson,et al.  Aspects of coverage in medical DNA sequencing , 2008, BMC Bioinformatics.

[5]  E. Jaffee,et al.  Molecular cytogenetic characterization of pancreas cancer cell lines reveals high complexity chromosomal alterations , 2007, Cytogenetic and Genome Research.

[6]  R. Hruban,et al.  Chromosomal abnormalities of adenocarcinoma of the pancreas: identifying early and late changes. , 2007, Cancer genetics and cytogenetics.

[7]  Eleazar Eskin,et al.  A sequence-based variation map of 8.27 million SNPs in inbred mouse strains , 2007, Nature.

[8]  K. Murphy,et al.  Constitutional duplication of a region of chromosome Yp encoding AMELY, PRKY, and TBL1Y: implications for sex chromosome analysis and bone marrow engraftment analysis. , 2007, The Journal of molecular diagnostics : JMD.

[9]  Amit U. Sinha,et al.  Cinteny: flexible analysis and visualization of synteny and genome rearrangements in multiple organisms , 2007, BMC Bioinformatics.

[10]  E. Sausville,et al.  Contributions of human tumor xenografts to anticancer drug development. , 2006, Cancer research.

[11]  Jane Fridlyand,et al.  High-resolution analysis of DNA copy number alterations in colorectal cancer by array-based comparative genomic hybridization. , 2004, Carcinogenesis.

[12]  Jorma Isola,et al.  Patterns of chromosomal imbalances defines subgroups of breast cancer with distinct clinical features and prognosis. A study of 305 tumors by comparative genomic hybridization. , 2003, Cancer research.

[13]  J. Testa,et al.  Chromosomal imbalances in human lung cancer , 2002, Oncogene.

[14]  P. Suess,et al.  Comparison of short tandem repeat and variable number tandem repeat genetic markers for quantitative determination of allogeneic bone marrow transplant engraftment , 2002, Bone Marrow Transplantation.

[15]  A. Killeen,et al.  Chromosomal aneuploidy in leukemic blast crisis: a potential source of error in interpretation of bone marrow engraftment analysis by VNTR amplification. , 1999, Molecular diagnosis : a journal devoted to the understanding of human disease through the clinical application of molecular biology.

[16]  R. Hruban,et al.  Development and characterization of a cytokine-secreting pancreatic adenocarcinoma vaccine from primary tumors for use in clinical trials. , 1998, The cancer journal from Scientific American.

[17]  R. Hruban,et al.  Allelotype of pancreatic adenocarcinoma using xenograft enrichment. , 1995, Cancer research.

[18]  C. McFarland,et al.  Quantitative determination of bone marrow transplant engraftment using fluorescent polymerase chain reaction primers for human identity markers. , 1995, Blood.

[19]  P. Gill,et al.  A rapid and quantitative DNA sex test: fluorescence-based PCR analysis of X-Y homologous gene amelogenin. , 1993, BioTechniques.

[20]  O. Takenaka,et al.  A human X-Y homologous region encodes "amelogenin". , 1991, Genomics.

[21]  S. Hirohashi,et al.  Transplantation of human tumors in nude mice. , 1976, Journal of the National Cancer Institute.