Gavin Meredith

High Resolution Detection and Analysis of CpG Dinucleotides Methylation Using MBD-Seq Technology

Methyl-CpG binding domain protein sequencing (MBD-seq) is widely used to survey DNA methylation patterns. However, the optimal experimental parameters for MBD-seq remain unclear and the data analysis remains challenging. In this study, we generated high depth MBD-seq data in MCF-7 cell and developed a bi-asymmetric-Laplace model (BALM) to perform data analysis. We found that optimal efficiency of MBD-seq experiments was achieved by sequencing ∼100 million unique mapped tags from a combination of 500 mM and 1000 mM salt concentration elution in MCF-7 cells. Clonal bisulfite sequencing results showed that the methylation status of each CpG dinucleotides in the tested regions was accurately detected with high resolution using the proposed model. These results demonstrated the combination of MBD-seq and BALM could serve as a useful tool to investigate DNA methylome due to its low cost, high specificity, efficiency and resolution.

Genome-wide methylated CpG island profiles of melanoma cells reveal a melanoma coregulation network

Metastatic melanoma is a malignant cancer with generally poor prognosis, with no targeted chemotherapy. To identify epigenetic changes related to melanoma, we have determined genome-wide methylated CpG island distributions by next-generation sequencing. Melanoma chromosomes tend to be differentially methylated over short CpG island tracts. CpG islands in the upstream regulatory regions of many coding and noncoding RNA genes, including, for example, TERC, which encodes the telomerase RNA, exhibit extensive hypermethylation, whereas several repeated elements, such as LINE 2, and several LTR elements, are hypomethylated in advanced stage melanoma cell lines. By using CpG island demethylation profiles, and by integrating these data with RNA-seq data obtained from melanoma cells, we have identified a co-expression network of differentially methylated genes with significance for cancer related functions. Focused assays of melanoma patient tissue samples for CpG island methylation near the noncoding RNA gene SNORD-10 demonstrated high specificity.

Glycogen synthase kinase-3 (Gsk-3) plays a fundamental role in maintaining DNA methylation at imprinted loci in mouse embryonic stem cells

A genome-wide analysis is given of DNA methylation in mouse embryonic stem cells in which both Gsk-3α and Gsk-3β have been genetically deleted. DNA methylation patterns are compared to those of wild-type cells. More than 75% of known imprinted loci have reduced DNA methylation in the Gsk-3–knockout cells.

Paediatric leukaemia DNA methylation profiling using MBD enrichment and SOLiD sequencing on archival bone marrow smears

BackgroundAcute Lymphoblastic Leukaemia (ALL) is the most common cancer in children. Over the past four decades, research has advanced the treatment of this cancer from a less than 60% chance of survival to over 85% today. The causal molecular mechanisms remain unclear. Here, we performed sequencing-based genomic DNA methylation profiling of eight paediatric ALL patients using archived bone marrow smear microscope slides.FindingsSOLiD™ sequencing data was collected from Methyl-Binding Domain (MBD) enriched fractions of genomic DNA. The primary tumour and remission bone marrow sample was analysed from eight patients. Four patients relapsed and the relapsed tumour was analysed. Input and MBD-enriched DNA from each sample was sequenced, aligned to the hg19 reference genome and analysed for enrichment peaks using MACS (Model-based Analysis for ChIP-Seq) and HOMER (Hypergeometric Optimization of Motif EnRichment). In total, 3.67 gigabases (Gb) were sequenced, 2.74 Gb were aligned to the reference genome (average 74.66% alignment efficiency). This dataset enables the interrogation of differential DNA methylation associated with paediatric ALL. Preliminary results reveal concordant regions of enrichment indicative of a DNA methylation signature.ConclusionOur dataset represents one of the first SOLiD™MBD-Seq studies performed on paediatric ALL and is the first to utilise archival bone marrow smears. Differential DNA methylation between cancer and equivalent disease-free tissue can be identified and correlated with existing and published genomic studies. Given the rarity of paediatric haematopoietic malignancies, relative to adult counterparts, our demonstration of the utility of archived bone marrow smear samples to high-throughput methylation sequencing approaches offers tremendous potential to explore the role of DNA methylation in the aetiology of cancer.

Abstract 2422: Simultaneous detection of activating somatic DNA mutations and expressed fusion transcripts from lung tumor FFPE samples

Worldwide, lung cancer is the most commonly diagnosed form of cancer with a survival rate among the lowest. Combined, somatic mutations (in the form of SNVs and InDels) and gene fusions, account for the majority of interpretable and actionable genomic alterations. Importantly, this typically requires the analysis of DNA and RNA from limited amounts of FFPE-preserved specimens. Currently, these analyses typically require complex sample pre-processing for assay on separate platforms or separate complex library preparation methods for assessment by high throughput sequencing. To provide a unified and simpler alternative, NanoString’s molecular barcoding technology has been modularized to permit simultaneous digital measurement of cancer-relevant targets that span these two analyte classes. Novel ‘SNV’ probes enable sensitive and specific identification of DNA mutant allele sequences down to a level of detection of ≤ 5% from 5 ng of FFPE-extracted genomic DNA. Fusion transcripts are detected with 5’/3’ imbalance probes and toehold-mediated junction probes. This dual analyte workflow requires just a single 5-10 micron section of FFPE tissue and provides to sample-to-answer results with approximately 5 minutes of hands-on time per sample after nucleic acid extraction. To demonstrate utility, 37 lung cancer samples were assayed simultaneously with an SNV panel that targets >100 solid tumor somatic mutations and a lung cancer fusion gene panel that provides general evidence of ALK, RET, and ROS1 gene fusion events along with specific detection of 35 unique fusion transcripts that correspond to known break-points. In this particular cohort, 16 samples were positive for activating KRAS SNVs (one of which was also positive for an activating STK11 variant), 3 were positive for activating EGFR mutations including two SNVs and an 18-base InDel and one was positive for an activating KIF5B16:RET12 fusion transcript. Positive mutation calls obtained with the SNV panel could only be confirmed by whole-exome sequencing (average depth of 100X) for 13 of 20 variants detected; however, ultra-deep (average depth of 4400X) targeted sequencing revealed that the 7 additional panel-detected mutations were, in fact, present. Measured against the sequencing datasets, the SNV panel provided 100% sensitivity, specificity, accuracy and precision for all variants present at 5% or greater allele frequency. The KIF5B16:RET12 fusion event was also confirmed by sequencing. Combined, these results show that these two important classes of activating mutations can be readily and efficiently assayed together on a NanoString nCounter® system (for research use only). Citation Format: A. McGarry Houghton, Gavin Meredith, Julia Kargl, Jill McKay-Fleisch, P. Martin Ross, Anisha Kharkia, Afshin Mashadi-Hossein, Dae Kim, Joseph Beechem. Simultaneous detection of activating somatic DNA mutations and expressed fusion transcripts from lung tumor FFPE samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2422. doi:10.1158/1538-7445.AM2017-2422

Abstract 2441: NanoString 3D Biology™ technology: simultaneous digital counting of DNA, RNA and protein

Introduction: Development of improved cancer diagnostics and therapeutics requires detailed understanding of the genomic, transcriptomic, and proteomic profiles in the tumor microenvironment. Current technologies can excel at measuring a single analyte, but it remains challenging to simultaneously collect high-throughput DNA, RNA, and protein data from small samples. We have developed an approach that uses optical barcodes to simultaneously profile DNA, RNA, and protein from as little as 5ng DNA, 25ng RNA, and 250ng protein or just 2 5µm FFPE slides, and simplifies data analysis by generating digital counts for each analyte. Methods: The approach uses paired capture and reporter oligonucleotide probes and optical barcodes to enumerate up to 800 targets. The platform was initially developed to measure RNA, and we have adapted it to measure DNA single nucleotide variants (SNVs), proteins, and phospho-proteins. SNVs are detected by direct hybridization of sequence discriminating probes to the wild-type and mutant sequence of interest. Proteins are detected via binding of oligonucleotide-conjugated antibodies. Results: Combinations of DNA, RNA, and protein in biological and experimental contexts. SNV probes are able to detect variant alleles down to 5% abundance within a wild type population and can discriminate variants within mutation hotspots. It was >96% accurate at identifying variants from samples displaying a range of allele frequencies and DNA integrity when benchmarked against next-generation sequencing. Protein detection has been developed for cell surface, cytosolic, and nuclear proteins, as well as phospho-proteins. It was validated against flow cytometry, western blot, and mass spectrometry using cell lines with ectopic target expression and primary cells. To demonstrate concurrent measurement of DNA, RNA, and protein from a single system, BRAFWT or BRAFV600E cell lines were treated with the BRAFV600E inhibitor vemurafenib and the MEK inhibitor trametinib. We measured the allele usage at the BRAFV600 locus, as well as BRAFV600E dependent changes in mRNA expression, protein expression and protein phosphorylation in a single experiment. Conclusions: 3D Biology has several advantages over other analytical approaches. Direct, single-molecule digital counting allows detection over a broad dynamic range with high reproducibility, often over 98% concordance between technical replicates. The simultaneous interrogation of DNA, RNA, and protein maximizes the amount of data obtained from precious samples and minimizes instrumentation demands by leveraging a single detection platform. The 3D Biology approach allows holistic, digital analysis of biological samples with high specificity and precision. This technology is currently available for research use, but may also have clinical application in the future. Citation Format: Chris Lausted, Yong Zhou, Jinho Lee, Christopher Vellano, Karina A. Eterovic, Ping Song, Lin-ya Tang, Gloria Fawcett, Tae-Beom Kim, Ken Chen, Gary Geiss, Gavin Meredith, Qian Mei, Gokhan Demirkan, Dwayne Dunaway, Dae Kim, P. Martin Ross, Elizabeth Manrao, Nathan Elliott, Sarah Warren, Christina Bailey, Chung-Ying Huang, Joseph Beechem, Gordon Mills, Leroy Hood. NanoString 3D Biology™ technology: simultaneous digital counting of DNA, RNA and protein [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2441. doi:10.1158/1538-7445.AM2017-2441

Epigenetic regulation of tau gene expression

Background: The formation of neurofibrillary tangles is one of the characteristic features in the brains of patients with Alzheimer’s disease. Hyperphosphorylation of tau protein by kinases such as Gsk-3 have been implicated in neurodegeneration. As such, small molecule inhibitors of Gsk-3 have been pursued as a potential therapeutics in the effort to slow or prevent neurodegeneration. Methods: Taq Man qPCR was performed on RNA isolated from wild-type and Gsk-3-deficient mouse embryonic stem cells. A role for Gsk-3 in DNA methylation at the Mapt locus was revealed by isolation of total genomic methylated DNA from wild-type and Gsk-3-deficient mouse embryonic stem cells using the Methyl Miner kit. Libraries of methylated DNAwere created, and whole-genome sequencing was performed on a SOLiD machine. Bioinformatic analysis of data identified regions of the mouse genome that were differentially methylated in the Gsk-3-deficient cells.Results:Here we present data showing that Gsk-3 activity also regulates the expression of tau mRNA. Tau mRNA expression is reduced by more than 80% in Gsk-3 deficient cells. This reduction in gene expression is accompanied by changes in DNA methylation at the Mapt locus. Conclusions: These data suggest that inhibition of Gsk-3 activity not only affects tau phosphorylation, but also provides a second layer of possible beneficial effects by reducing the expression of tau mRNA. Finally, the role of DNA methylation in regulating tau gene expression affords a novel therapeutic approach to preventing tangle-related neurodegeneration.

Abstract LB-133: Genome-wide differences in DNA methylation between tumor and normal adjacent tissue mapped by high-throughput sequencing of methyl-CPG binding domain based affinity-purified genomic fragments

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Aberrant DNA methylation is characteristic of many cancers and differences in methylation have been observed in a wide variety of genomic contexts; for example, both within “classic” promoter-associated CpG islands and also in distal, non-CpG island regions. Establishing a method to broadly and efficiently survey the methylation patterns genome-wide in matched tumor and normal genomic DNA and to elucidate differences in these “methylomes” is the objective of the work presented. The method combines the power of methyl-CpG binding domain (MBD) proteins to sensitively and selectively bind methylated DNA sequences with the coverage, precision, and accuracy provided by high-throughput sequencing. Notably, MBD-affinity capture can also be used to sub-fractionate genomic DNA based on its average methyl-CpG content. In parallel, patient-derived matched samples of fragmented genomic DNA from a breast tumor and normal adjacent tissue were enriched and fractionated based on their methyl-CpG content with a commercial MBD-based affinity reagent (see figure) and high-throughput sequencing libraries were prepared from each fraction. The libraries were sequenced at 50 bp read-length on a SOLiD 3 Plus system and mapped to the human reference genome. Peak analysis of the distribution of mapped reads permitted the discovery of hundreds of putative differentially methylation regions (DMRs). Differential methylation at a number of these positions was confirmed by bisulfite-sequencing. We conclude that such enrichment and fractionation, when coupled to high-throughput sequencing, can be used to efficiently survey the majority of DNA methylation marks within samples of genomic DNA and to discover genomic loci of differential methylation. We conclude that such enrichment and fractionation, when coupled to high-throughput sequencing, can be used to efficiently survey a large fraction of all DMRs between samples of genomic DNA and to discover genomic loci of differential methylation. These datasets can then be compared with ChIP-seq and/or RNA-seq data sets to begin to decipher the functional genomics and epigenomics of cancer. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-133.