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Intra-tumor heterogeneity of MLH1 promoter methylation revealed by deep single molecule bisulfite sequencing.

Varley KE, Mutch DG, Edmonston TB, Goodfellow PJ, Mitra RD - Nucleic Acids Res. (2009)

Bottom Line: A single tumor may contain cells with different somatic mutations.By characterizing this genetic heterogeneity within tumors, advances have been made in the prognosis, treatment and understanding of tumorigenesis.We have characterized epigenetic heterogeneity within individual tumors using next-generation sequencing.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, Center for Genome Sciences, Washington University School of Medicine, St Louis, Missouri, USA.

ABSTRACT
A single tumor may contain cells with different somatic mutations. By characterizing this genetic heterogeneity within tumors, advances have been made in the prognosis, treatment and understanding of tumorigenesis. In contrast, the extent of epigenetic intra-tumor heterogeneity and how it influences tumor biology is under-explored. We have characterized epigenetic heterogeneity within individual tumors using next-generation sequencing. We used deep single molecule bisulfite sequencing and sample-specific DNA barcodes to determine the spectrum of MLH1 promoter methylation across an average of 1000 molecules in each of 33 individual samples in parallel, including endometrial cancer, matched blood and normal endometrium. This first glimpse, deep into each tumor, revealed unexpectedly heterogeneous patterns of methylation at the MLH1 promoter within a subset of endometrial tumors. This high-resolution analysis allowed us to measure the clonality of methylation in individual tumors and gain insight into the accumulation of aberrant promoter methylation on both alleles during tumorigenesis.

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Comparison of sequencing results from conventional cloning and Sanger sequencing and 454 Life Sciences FLX sequencing of MLH1 promoter PCR products from bisulfite treated DNA of an endometrial cancer and normal blood from Patient #1684. (A) Schematic of the MLH1 promoter is presented with arrows indicating the location of PCR primers and vertical lines representing the position of CG dinucleotides. Below the schematic are the results from cloning and bisulfite sequencing 45 molecules from the distal (left) and proximal (right) promoter in each sample (tumor and matched normal blood). Each column represents a CG dinucleotide in the sequence, and corresponds to each vertical line in the promoter schematic. Each row represents a single molecule. The color of the boxes represents the methylation state of each cytosine. Red, methylated; Black, unmethylated. (B) The results from the FLX single molecule sequencing of the same samples. The distance of each CpG from the transcription start site (UCSC Human Genome March 2006) is listed from distal to proximal. The Distal Amplicon: −671, −662, −654, −648, −634, −632, −630, −626, −623, −619, −609, −605, −596, −584, −576, −569, −566, −564, −560, −558, −548, −540, −537, −512, −505, −483, −470, −465, −449, −446, −421, −405, −380, −368. The Proximal Amplicon: −341, −325, −318, −286, −280, −249, −240, −226, −209, −202, −192, −190, −184, −165, −154, −139, −70, −47, −23, −15. As an additional point of reference, the translation start site (ATG) is currently annotated at +60 downstream of the transcription start site (position 0).
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Figure 2: Comparison of sequencing results from conventional cloning and Sanger sequencing and 454 Life Sciences FLX sequencing of MLH1 promoter PCR products from bisulfite treated DNA of an endometrial cancer and normal blood from Patient #1684. (A) Schematic of the MLH1 promoter is presented with arrows indicating the location of PCR primers and vertical lines representing the position of CG dinucleotides. Below the schematic are the results from cloning and bisulfite sequencing 45 molecules from the distal (left) and proximal (right) promoter in each sample (tumor and matched normal blood). Each column represents a CG dinucleotide in the sequence, and corresponds to each vertical line in the promoter schematic. Each row represents a single molecule. The color of the boxes represents the methylation state of each cytosine. Red, methylated; Black, unmethylated. (B) The results from the FLX single molecule sequencing of the same samples. The distance of each CpG from the transcription start site (UCSC Human Genome March 2006) is listed from distal to proximal. The Distal Amplicon: −671, −662, −654, −648, −634, −632, −630, −626, −623, −619, −609, −605, −596, −584, −576, −569, −566, −564, −560, −558, −548, −540, −537, −512, −505, −483, −470, −465, −449, −446, −421, −405, −380, −368. The Proximal Amplicon: −341, −325, −318, −286, −280, −249, −240, −226, −209, −202, −192, −190, −184, −165, −154, −139, −70, −47, −23, −15. As an additional point of reference, the translation start site (ATG) is currently annotated at +60 downstream of the transcription start site (position 0).

Mentions: The standard method for determining methylation patterns on single molecules is bisulfite treatment followed by cloning and Sanger sequencing. Single molecule PCR methods (34,35) have been recently introduced as simplified workflow and to mitigate cloning bias. We compared the methylation patterns obtained using the standard method to those obtained using deep single molecule sequencing on second-generation sequencing machines. We cloned bisulfite-treated DNA amplified from the tumor and matched blood of a single patient (Patient #1684) and Sanger sequenced 45 clones from both the distal and proximal region of the promoter (Figure 2A). Results for conventional sequencing analysis were compared to the 454 FLX sequencing of the same samples, comprising 496 reads from the distal region of the MLH1 promoter and 480 reads from the proximal region (Figure 2B). The 454 FLX sequencing and Sanger sequencing results were nearly identical. To quantify the similarity between the results from the two methods we calculated the percent of cytosines methylated at each CG in each sample and determined the correlation coefficient for the two analytic approaches. The methods produce highly similar methylation patterns (R2 = 0.96). Both methods revealed the majority of tumor DNA molecules from the MLH1 promoter were densely methylated. A small percentage of molecules in the tumor were unmethylated. These unmethylated sequences are likely derived from normal non-neoplastic cells in the tumor. The neoplastic cellularity (NPC) of tumor 1684 was estimated to be 70% based on histological assessment of the tissue used for DNA preparation. Both 454 FLX and Sanger sequencing revealed dense methylation of the proximal promoter with heterogeneous methylation of five CpG positions. The normal blood from Patient #1648 was unmethylated in the MLH1 promoter as assessed by both 454 FLX and Sanger sequencing (Figure 2A). We found that >99% of non-CpG cytosines were converted in each sample, indicating the sodium bisulfite conversion was successful. The similarity between the Sanger and FLX sequencing for this patient's samples confirmed that the FLX sequencing strategy can be used to bisulfite sequence single molecules in individual samples in high-throughput.Figure 2.


Intra-tumor heterogeneity of MLH1 promoter methylation revealed by deep single molecule bisulfite sequencing.

Varley KE, Mutch DG, Edmonston TB, Goodfellow PJ, Mitra RD - Nucleic Acids Res. (2009)

Comparison of sequencing results from conventional cloning and Sanger sequencing and 454 Life Sciences FLX sequencing of MLH1 promoter PCR products from bisulfite treated DNA of an endometrial cancer and normal blood from Patient #1684. (A) Schematic of the MLH1 promoter is presented with arrows indicating the location of PCR primers and vertical lines representing the position of CG dinucleotides. Below the schematic are the results from cloning and bisulfite sequencing 45 molecules from the distal (left) and proximal (right) promoter in each sample (tumor and matched normal blood). Each column represents a CG dinucleotide in the sequence, and corresponds to each vertical line in the promoter schematic. Each row represents a single molecule. The color of the boxes represents the methylation state of each cytosine. Red, methylated; Black, unmethylated. (B) The results from the FLX single molecule sequencing of the same samples. The distance of each CpG from the transcription start site (UCSC Human Genome March 2006) is listed from distal to proximal. The Distal Amplicon: −671, −662, −654, −648, −634, −632, −630, −626, −623, −619, −609, −605, −596, −584, −576, −569, −566, −564, −560, −558, −548, −540, −537, −512, −505, −483, −470, −465, −449, −446, −421, −405, −380, −368. The Proximal Amplicon: −341, −325, −318, −286, −280, −249, −240, −226, −209, −202, −192, −190, −184, −165, −154, −139, −70, −47, −23, −15. As an additional point of reference, the translation start site (ATG) is currently annotated at +60 downstream of the transcription start site (position 0).
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Related In: Results  -  Collection

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Figure 2: Comparison of sequencing results from conventional cloning and Sanger sequencing and 454 Life Sciences FLX sequencing of MLH1 promoter PCR products from bisulfite treated DNA of an endometrial cancer and normal blood from Patient #1684. (A) Schematic of the MLH1 promoter is presented with arrows indicating the location of PCR primers and vertical lines representing the position of CG dinucleotides. Below the schematic are the results from cloning and bisulfite sequencing 45 molecules from the distal (left) and proximal (right) promoter in each sample (tumor and matched normal blood). Each column represents a CG dinucleotide in the sequence, and corresponds to each vertical line in the promoter schematic. Each row represents a single molecule. The color of the boxes represents the methylation state of each cytosine. Red, methylated; Black, unmethylated. (B) The results from the FLX single molecule sequencing of the same samples. The distance of each CpG from the transcription start site (UCSC Human Genome March 2006) is listed from distal to proximal. The Distal Amplicon: −671, −662, −654, −648, −634, −632, −630, −626, −623, −619, −609, −605, −596, −584, −576, −569, −566, −564, −560, −558, −548, −540, −537, −512, −505, −483, −470, −465, −449, −446, −421, −405, −380, −368. The Proximal Amplicon: −341, −325, −318, −286, −280, −249, −240, −226, −209, −202, −192, −190, −184, −165, −154, −139, −70, −47, −23, −15. As an additional point of reference, the translation start site (ATG) is currently annotated at +60 downstream of the transcription start site (position 0).
Mentions: The standard method for determining methylation patterns on single molecules is bisulfite treatment followed by cloning and Sanger sequencing. Single molecule PCR methods (34,35) have been recently introduced as simplified workflow and to mitigate cloning bias. We compared the methylation patterns obtained using the standard method to those obtained using deep single molecule sequencing on second-generation sequencing machines. We cloned bisulfite-treated DNA amplified from the tumor and matched blood of a single patient (Patient #1684) and Sanger sequenced 45 clones from both the distal and proximal region of the promoter (Figure 2A). Results for conventional sequencing analysis were compared to the 454 FLX sequencing of the same samples, comprising 496 reads from the distal region of the MLH1 promoter and 480 reads from the proximal region (Figure 2B). The 454 FLX sequencing and Sanger sequencing results were nearly identical. To quantify the similarity between the results from the two methods we calculated the percent of cytosines methylated at each CG in each sample and determined the correlation coefficient for the two analytic approaches. The methods produce highly similar methylation patterns (R2 = 0.96). Both methods revealed the majority of tumor DNA molecules from the MLH1 promoter were densely methylated. A small percentage of molecules in the tumor were unmethylated. These unmethylated sequences are likely derived from normal non-neoplastic cells in the tumor. The neoplastic cellularity (NPC) of tumor 1684 was estimated to be 70% based on histological assessment of the tissue used for DNA preparation. Both 454 FLX and Sanger sequencing revealed dense methylation of the proximal promoter with heterogeneous methylation of five CpG positions. The normal blood from Patient #1648 was unmethylated in the MLH1 promoter as assessed by both 454 FLX and Sanger sequencing (Figure 2A). We found that >99% of non-CpG cytosines were converted in each sample, indicating the sodium bisulfite conversion was successful. The similarity between the Sanger and FLX sequencing for this patient's samples confirmed that the FLX sequencing strategy can be used to bisulfite sequence single molecules in individual samples in high-throughput.Figure 2.

Bottom Line: A single tumor may contain cells with different somatic mutations.By characterizing this genetic heterogeneity within tumors, advances have been made in the prognosis, treatment and understanding of tumorigenesis.We have characterized epigenetic heterogeneity within individual tumors using next-generation sequencing.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, Center for Genome Sciences, Washington University School of Medicine, St Louis, Missouri, USA.

ABSTRACT
A single tumor may contain cells with different somatic mutations. By characterizing this genetic heterogeneity within tumors, advances have been made in the prognosis, treatment and understanding of tumorigenesis. In contrast, the extent of epigenetic intra-tumor heterogeneity and how it influences tumor biology is under-explored. We have characterized epigenetic heterogeneity within individual tumors using next-generation sequencing. We used deep single molecule bisulfite sequencing and sample-specific DNA barcodes to determine the spectrum of MLH1 promoter methylation across an average of 1000 molecules in each of 33 individual samples in parallel, including endometrial cancer, matched blood and normal endometrium. This first glimpse, deep into each tumor, revealed unexpectedly heterogeneous patterns of methylation at the MLH1 promoter within a subset of endometrial tumors. This high-resolution analysis allowed us to measure the clonality of methylation in individual tumors and gain insight into the accumulation of aberrant promoter methylation on both alleles during tumorigenesis.

Show MeSH
Related in: MedlinePlus