Figures
Fig. 1. Schematic of PARE approach. The method is based on next-generation mate-paired analysis of resected tumor DNA to identify individualized tumor-specific rearrangements. Such alterations are used to develop PCR-based quantitative analyses for personalized tumor monitoring of plasma samples or other bodily fluids.
Fig. 2. Detection of tumor-specific rearrangements in breast and colorectal cancers. Two representative rearrangements are shown for each tumor sample. (A) PCR amplification across breakpoint regions. MW, molecular weight; T, tumor; N, normal. (B) Genomic coordinates for a representative mate pair of each rearrangement.
Fig. 3. Detection of tumor-specific rearrangements in mixtures of tumor and normal DNA. Decreasing amounts of tumor DNA were mixed with increasing amounts of normal tissue DNA (300 ng total) and were used as template molecules for PCR using chromosome 4:8 translocation-specific primers or chromosome 3 control primers (see Materials and Methods for additional information).
Fig. 4. Detection of tumor-specific rearrangements in plasma of cancer patients. (A) The identified chromosome 4:8 and 16 rearrangements were used to design PCR primers spanning breakpoints and to amplify rearranged DNA from tumor tissue and plasma from patients Hx402 and Hx403, respectively. A plasma sample from an unrelated healthy individual was used as a control for both rearrangements. (B) Plasma samples from patient Hx402 were analyzed at different time points using digital PCR to determine the fraction of genomic equivalents of plasma DNA containing the chromosome 4:8 rearrangement. The fraction of rearranged DNA at day 137 was 0.3%, consistent with residual metastatic lesions present in the remaining lobe of the liver.
Tables
- Table 1.
Summary of mate-paired tag libraries.
Samples Single tag analyses Mate-paired tag analyses Number of
beads*Number
of tags
matching human
genomeTotal
bases
sequenced
(bp)Expected
coverage
per
3-kb binNumber of
mate-paired
tags matching
human genomeDistance
between
mate-paired
tags (bp)Total
physical
coverage by
mate-paired
tags (bp)Expected
genome
coverageColon cancer Co108
tumor526,209,780 121,527,707 3,038,192,675 122 21,899,809 1371 30,024,693,714 10.0 Co108
normal328,599,033 86,032,253 2,150,806,325 86 11,694,361 1254 14,665,530,804 4.9 Co84
tumor677,137,128 256,065,437 6,401,635,925 256 58,678,410 1488 87,292,060,006 29.1 Co84
normal486,663,520 218,280,146 5,457,003,650 218 59,019,031 1384 81,690,396,379 27.2 Hx402
tumor523,745,015 198,342,749 4,958,568,725 198 43,457,431 1629 70,789,547,653 23.6 Hx403
tumor475,658,760 164,061,938 4,101,548,450 164 37,123,395 1705 63,295,388,475 21.1 Breast cancer B7
tumor840,979,999 281,027,274 7,025,681,850 281 27,548,989 1220 33,604,662,404 11.2 B7
normal705,704,265 253,482,262 6,337,056,550 253 57,878,644 1404 81,271,654,770 27.1 B5
tumor444,249,217 147,612,941 3,690,323,525 148 29,961,045 1193 35,730,144,651 11.9 B5
normal549,237,156 220,669,795 5,516,744,875 221 53,611,974 1205 64,591,276,025 21.5 *Number of beads corresponds to the number of magnetic beads containing clonally amplified DNA fragments and represents the maximal number of raw sequence reads for each run.
- Table 2.
Summary of rearrangements identified in tumor samples.
Sample Rearrangement type Total
rearrangementsTested
rearrangementsConfirmed somatic
rearrangementsIntrachromosomal Interchromosomal Tumor and normal libraries B5 7 4 11 7 5 (71%) B7 17 4 21 16 15 (94%) Co84 0 7 7 6 4 (67%) Co108 6 12 18 13 11 (85%) Tumor libraries Hx402 7 2 9 9 4 (44%) Hx403 17 0 17 12 7 (58%)
- Supplementary Material for:
Development of Personalized Tumor Biomarkers Using Massively
Parallel SequencingRebecca J. Leary, Isaac Kinde, Frank Diehl, Kerstin Schmidt, Chris Clouser, Cisilya Duncan, Alena Antipova, Clarence Lee, Kevin McKernan, Francisco M. De La Vega, Kenneth W. Kinzler, Bert Vogelstein, Luis A. Diaz Jr., Victor E. Velculescu*
*To whom correspondence should be addressed. E-mail: velculescu{at}jhmi.edu
Published 24 February 2010, Sci. Transl. Med. 2, 20ra14 (2010)
DOI: 10.1126/scitranslmed.3000702This PDF file includes:
- Table S1. Comparison of SOLiD sequencing, Illumina SNP arrays, and Digital Karyotyping for analysis of copy number alterations.
- Table S2. Putative copy number alterations identified by SOLiD sequencing in Co84 that were not identified by Illumina SNP arrays or Digital Karyotyping.
- Table S3. Confirmed somatic rearrangements in breast and colorectal cancer samples.
- Fig. S1. Flow chart of approach used to identify rearranged sequences.
- Fig. S2. Comparison of Digital Karyotyping, Illumina SNP array, and SOLiD sequencing results on chromosome 8.
