2The Department of Medical Genetics, National Taiwan University Hospital, Taipei, Taiwan
3The Department of Oncology Biostatistics and Bioinformatics, Johns Hopkins University School of Medicine, Johns Hopkins Hospital, Baltimore, Maryland
4The Department of Oncology, Johns Hopkins University School of Medicine, Johns Hopkins Hospital, Baltimore, Maryland
*S.W. and J.R.E. contributed equally to this article.
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.
Genetic analysis of human cancers often involves the analysis of xenografted tumors raised in immunodeficient mice (1,2,3). During expansion of human cancer xenografts in mice, human stromal cells are largely replaced by mouse stromal cells, resulting in mixed-species samples (4). Since mouse cell contamination can complicate downstream genetic analysis, an accurate and sensitive estimate of the relative human and mouse composition of xenografted tumors is important. This is especially relevant to international efforts to sequence cancer genomes (3,5). A mixture of human cancer cells and mouse stromal cells could theoretically be sequenced with high depth of coverage in order to discover mutations in the tumor genome, even in the face of some level of mouse cell contamination. The assumptions inherent in this approach are many: (i) the human tumor sample is homogeneous, (ii) the mouse genotype is completely known, (iii) all mutations come from the human tumor sample, (iv) any change in the human genome will clearly be a mismatch to human and not a perfect match to mouse, (v) sequencing coverage is fairly even across both genomes, and (vi) the percentage of mouse contamination is known. While the last point can be known, none of the other assumptions is exactly valid; thus, it is not clear that this approach is entirely tenable.
Length determination assays could potentially provide the required accuracy and sensitivity. Resolving different-length products using capillary electrophoresis and analyzing peak heights typically provides a limit of detection of 1–3% and an accuracy of 97–99% (6). An example is the PCR amplification of the X-Y homologous amelogenin alleles (AMELX, AMELY) to generate amplicons with a 6-bp difference between X and Y alleles (6,7,8,9). This simple PCR assay has been incorporated into commercial multiplex microsatellite identity testing kits for forensic, paternity, and bone marrow transplant analyses.
In this report, we hypothesized that length variation in genes between mouse and human would be common. In a preliminary analysis, 12 loci with length variation were selected from human chromosomes 8, 9, 10, and 12. Surprisingly, only three of them (25%) resulted in accurate standard curves in mixed DNA samples. These three markers were multiplexed into a single PCR reaction to quantify the mouse component in the xenografts of human pancreatic cancer cells. This demonstrated that among 93 xenografts, the contaminating mouse DNA averaged 47%, and could be as high as 73%. In a comprehensive analysis of the two genomes, we further identified 370 loci demonstrating human-mouse length variation. This assay should also be useful in monitoring human cell enrichment—from mouse xenografts of different human cancers—in preparation for next-generation sequencing.Materials and methods Primer design
Initially, 12 loci with length variation between the mouse and human genome were randomly selected from chromosomes 8, 9, 10, and 12 according to the NCBI database. Chromosomes 9 and 12 were included since the cytogenetic studies of the primary pancreatic carcinomas and the comparative genomic hybridization analyses of pancreatic cell lines showed the least amount of chromosomal aberrations within these 2 chromosomes (10,11). The primers were designed to straddle the length variation and placed in regions that were conserved between the human and mouse genomes (Figure 1A). Primers were designed for the three loci that were determined to be accurate for discriminating mouse from human DNA: RNase P/MRP 38kDa subunit gene on chromosome 10p13 [primer pair 5 (F: 5′-TCATTGGCTTAAAATGTGT-3′, R: 5′-FAM-TTTATTTTAAGGGGTTGTAATG-3′)], and two loci downstream ring finger and CCCH-type zinc finger domains 2 (RC3H2) on chromosome 9q34 [primer pair43 (F: 5′-CTATTCCTATAGCACAAAGG-3′, R: 5′-FAM-GATGGTGTACACCCATCATG-3′) and primer pair 45 (F: 5′-HEX-ACTAAGTCAAGGCTACTGTG-3′, R: 5′-TTCTGGTGTCAGTATGGAAG-3′)].
DNA samples were extracted using QIAamp DNA Blood Mini Kit (Qiagen, Valencia, CA, USA) and the concentration was determined using a NanoDrop ND-100 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). PCR reactions were performed in a 10-µL total volume containing 2 pmol forward primer, 2 pmol reverse primer, 2.5 mM MgCl2, 0.2 mM each deoxyribonucleotide, 0.25 units AmpliTaq Gold DNA polymerase and 1.0 µL buffer (Applied Biosystems, Foster City, CA, USA). Samples containing 10 ng DNA were subjected to 35 cycles of denaturation (95°C for 30 s), annealing (52°C for 30 s) and extension (72°C for 60 s). For multiplex PCR, the total amount of forward primers and reverse primers used was 5 pmol each, and the AmpliTaq Gold DNA polymerase was increased to 0.5 units.