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. 2010 Sep;20(9):1279-87.
doi: 10.1101/gr.101212.109. Epub 2010 Jul 13.

Bisulfite Patch PCR enables multiplexed sequencing of promoter methylation across cancer samples

Katherine Elena Varley  1 , Robi David Mitra
Affiliations

Bisulfite Patch PCR enables multiplexed sequencing of promoter methylation across cancer samples

Katherine Elena Varley et al. Genome Res. 2010 Sep.

Abstract

Aberrant DNA methylation frequently occurs at gene promoters during cancer progression. It is important to identify these loci because they are often misregulated and drive tumorigenesis. Bisulfite sequencing is the most direct and highest resolution assay for identifying aberrant promoter methylation. Recently, genomic capture methods have been combined with next-generation sequencing to enable genome-scale surveys of methylation in individual samples. However, it is challenging to validate candidate loci identified by these approaches because an efficient method to bisulfite sequence more than 50 differentially methylated loci across a large number of samples does not exist. To address this problem, we developed Bisulfite Patch PCR, which enables highly multiplexed bisulfite PCR and sequencing across many samples. Using this method, we successfully amplified 100% of 94 targeted gene promoters simultaneously in the same reaction. By incorporating sample-specific DNA barcodes into the amplicons, we analyzed 48 samples in a single run of the 454 Life Sciences (Roche) FLX sequencer. The method requires small amounts of starting DNA (250 ng) and does not require a shotgun library construction. The method was highly specific; 90% of sequencing reads aligned to targeted loci. The targeted promoters were from genes that are frequently mutated in breast and colon cancer, and the samples included breast and colon tumor and adjacent normal tissue. This approach allowed us to identify nine gene promoters that exhibit tumor-specific DNA methylation defects that occur frequently in colon and breast cancer. We also analyzed single nucleotide polymorphisms to observe DNA methylation that accumulated on specific alleles during tumor development. This method is broadly applicable for studying DNA methylation across large numbers of patient samples using next-generation sequencing.

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Figures

Figure 1.
Figure 1.
Bisulfite Patch PCR. (A,B) Genomic DNA restriction digest. (C) Anneal patch oligos and universal primers specifically to the ends of desired fragments. (D) Ligate universal primers (U1 and U2) to targeted fragments. (E) Degrade unselected DNA with exonucleases. Targeted loci are protected from exonuclease by 3′ modification on U2. (F) Treat with sodium bisulfite to convert unmethylated cytosine to uracil, leaving methylated cytosine intact. (G) PCR all loci simultaneously with universal primers tailed with sample-specific DNA barcodes and sequencing machine primers (454A and 454B). Pool PCR products from all samples together for sequencing.
Figure 2.
Figure 2.
Method performance. (A) Number of sequencing reads per promoter for all 94 targeted promoters, order by length in base pairs (bp) on the x-axis. Longer promoter amplicons yield fewer sequencing reads (length bias), but 87 amplicons (93%) have coverage within 10-fold of the median coverage (444 reads). The abundance of each promoter ranged from 10 to 5114 reads. (B) Histogram of the pairwise squared correlation coefficients for the number of reads per promoter for all 48 samples. The mean correlation coefficient is 0.91, indicating that the number of reads per promoter is highly reproducible across patient samples.
Figure 3.
Figure 3.
Methylation at the H19 imprinted locus. Data from four patients who were germline heterozygous for a SNP (rs2251375) in this locus. The sequencing reads are aligned as rows in each panel. Each base in the read is color-coded to indicate the sequence: (yellow) a methylated cytosine; (blue) all other bases. The position of the SNP is indicated by the red and white column: (red base) reads from the G allele; (white base) reads from the T allele. The percentage of reads for each patient that are from the G allele is listed below the patient identifier for each sample. As expected for an imprinted locus, methylation is observed on one allele in both the tumor (left panels) and the adjacent normal tissue (right panels) for each patient. Both alleles and both methylated and unmethylated molecules were amplified and sequenced efficiently from this locus in all samples.
Figure 4.
Figure 4.
Four promoters that exhibit tumor-specific methylation. Sequencing reads from all patients for each type of tissue are grouped together in panels; breast tumors, adjacent normal breast tissues, colon tumors, and adjacent normal colon tissues. The sequencing reads are aligned as rows in each panel and are grouped by patient. Each base in the read is color-coded to indicate the sequence: (yellow) a methylated cytosine; (blue) all other bases. (A,B) ICAM5 and LAMA1 promoters exhibit colon and breast tumor-specific methylation. (C,D) KCNQ5 and CLSTN2 promoters exhibit colon tumor-specific methylation.
Figure 5.
Figure 5.
Allelic tumor-specific methylation. Data from six patients who are germline heterozygous for a SNP (rs2854744) in the IGFBP3 promoter. The sequencing reads are aligned as rows in each panel. Each base in the read is color-coded to indicate the sequence: (yellow) a methylated cytosine; (blue) all other bases. The position of the SNP is indicated by the red and white column: (red) reads from the A allele; (yellow) methylated C allele; (white) C allele, if unmethylated and converted to a T. Patient “breast 8” is unmethylated on both alleles in both the tumor (left column) and normal tissue (right column). Patients “breast 4” and “colon 6” display tumor-specific methylation on only one allele, and the methylated allele differs between them. Patients “colon 7” and “colon 12” display tumor-specific methylation on both alleles. Patient “colon 12” displays different patterns of methylation on each allele in the tumor.
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