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Guidelines for DNA Methylation Sequencing Projects

The High Throughput Genomics Core Facility provides support for whole genome bisulfite sequencing in addition to a targeted approach that enables investigators to interrogate regions in which methylation status is known to impact gene expression. Options supported by the core facility include the Epicentre EpiGnome Methyl-Seq kit and the Agilent SureSelect XT Human Methyl-Seq Kit. A description of these products and recommendations for preparing DNA samples for methylation sequencing projects are described below:

  1. Library Preparation kits for Methylation Sequencing projects that are supported by the High Throughput Genomics Core Facility include the following:
    1. The Epicentre EpiGnome Methyl-Seq kit enables the construction of whole genome bisulfite sequencing libraries from 50 ng of DNA (in a volume of 20 ul). The EpiGnome kit utilizes a method in which library construction follows bisulfite treatment which results in a more diverse library with improved coverage of CpG, CHG and CHH regions than conventional library preparation methods in which bisulfite treatment follows the adapter ligation. The average insert size of libraries constructed with the EpiGnome kit average approximately 180 bp. The recommended sequence depth for libraries constructed with the EpiGnome kit is 60-90 Gbp per sample.
    2. The Agilent SureSelect XT Methyl Seq kit enables enrichment of an 84 MB target that represents 3.7 million CpGs. Library construction is initiated with 500-3000 ng of DNA (in a volume of 100 ul) which is hybridized to biotinylated-RNA baits. Following hybridization, the enriched DNA is treated with bisulfite and pcr-amplified. The SureSelect Methyl-Seq kit enables high sensitivity with single base resolution of regions in which methylation status is known to influence gene expression. These regions include CpG islands, CpG island shores, undermethylated regions, promoters, enhancers and differentially methylated regions. The recommended sequence depth for libraries constructed with the SureSelect Methyl Seq kit is 12 Gb of raw sequence data per sample, of which approximately 40% will represent unique reads.
  2. Purification of Genomic DNA: Genomic DNA should be purified using a column-based purification protocol such as the Qiagen DNeasy Blood and Tissue kit (cat#69504), or one of the DNA purification kits from Zymo Research (Quick-gDNA MiniPrep, Quick-gDNA Blood MiniPrep, or ZR Genomic DNA-Tissue MiniPrep).
  3. Purification of DNA from FFPE Tissues: Genomic DNA from FFPE tissues should be purified using a column-based purification protocol such as the Qiagen DNA FFPE Tissue Kit (cat#56404), the Zymo Research ZR FFPE DNA MiniPrep, or the Ambion RecoverAll Total Nucleic Acid Isolation Kit for FFPE (cat#AM1975).
  4. Assessment of DNA Concentration: The concentration of genomic DNA can be determined using a Qubit dsDNA BR assay (Invitrogen cat#Q32850) or the Qubit dsDNA HS assay (Invitrogen cat#32851). These assays use a fluorescent dye that is highly selective for double-stranded DNA over RNA and they are able to measure samples in a concentration range from 10 pg/ul to 1000 ng/ul. In contrast, a NanoDrop measurement often exaggerates the concentration of purified DNA as any RNA that co-purifies with the DNA will contribute to the absorbance measurement at 260 nm.
  5. Assessment of DNA Quality: The quality of genomic DNA can be assessed by running an aliquot of the sample (approximately 10-100 ng) on a 1% agarose gel stained with SYBR Safe DNA Gel Stain (Invitrogen cat#S33102). High quality, intact genomic DNA should appear as a high molecular weight band (>10,000 bp) in the absence of a lower molecular weight smear. Low molecular weight smearing can be indicative of the presence of RNA.
  6. Number of Sequence Reads:
  7. Avoid Organic Extraction Methods: We recommend that you avoid organic extraction methods (such as phenol or Trizol) or other methods that include an alcohol precipitation step. The quality of DNA purified by these protocols tends to be lower due to co-precipitation of contaminants. Furthermore organic carryover can inhibit the enzymatic reactions used in library preparation.
  8. Library QC: Sequencing libraries will be validated by running an aliquot on an Agilent 2200 TapeStation and by defining the molarity of the library using a qPCR assay (Kapa Biosystems Library Quantification Kit for Illumina). After normalizing library concentrations following the completion of these two assays, libraries can be pooled in preparation for sequencing.