Bisulfite Amplicon Sequencing (BSAS)
For directed DNA methylation analyses (e.g., do the promoter regions of a specific set of genes change with age) or to orthogonally confirm BOCS findings, we have developed BSAS (Masser et al., 2013). This method takes advantage of well-established bisulfite conversion and loci-specific PCR, but then takes the amplicons generated and uses new tagmentation library creation with benchtop NGS to easily and rapidly analyze targeted regions in large numbers of samples with much higher accuracy than existing methods. BSAS uses PCR enrichment of targeted regions from bisulfite-converted DNA and transposome-mediated library construction for rapid generation of sequencing libraries from low (1 ng) sample input. Libraries are sequenced using the Illumina MiSeq benchtop sequencer. Generating high levels of sequencing depth (<1,000 ×) provides for quantitatively precise and accurate assessment of DNA methylation levels with base specificity. Dual indexing of sequencing libraries allows for simultaneous analysis of up to 96 samples. For the targeted regions BSAS provides absolute quantitation of both CG and non-CG sites. During the experiment planning process, the Core will work with investigators to determine the appropriate primer sets to isolate their region of interest. Investigators will send DNA, and the Core will perform BS-PCR, library construction and then sequencing with an Illumina MiSeq. Data analysis is then performed with CLC-Genomics Workbench bisulfite plugin.
Schematic of BSAS method. In this method, genomic DNA is bisulfite converted to modify unmethylation cytosines to uracils. Subsequently, during PCR these uracils are changed to thymines. PCR amplification is directed against regions of interest and highly enriches for just these sequences. The resulting PCR amplicons are made into dual-indexed libraries through a simple tagmentation process. Subsequently the libraries are sequenced on a benchtop next generation sequencer and the sequencing reads are mapped to the in silico converted reference sequence and percent methylation of cytosine’s is determined in a base-specific manner.
Genomic DNA (500ng) is bisulfite converted (EZ DNA Methylation) and targeted genomic loci are amplified using ZymoTaq DNA polymerase, a DNA polymerase capable of amplifying low diversity DNA, specifically bisulfite converted DNA (Zymo Research, Irvine, CA, USA). PCR products are purified by QIAquick PCR columns, to eliminate primers and enzymes and quantified by Picogreen (Invitrogen/Life Technologies, Eugene, OR, USA) fluorometric nucleic acid assay. PCR product sizes were confirmed through polyacrylamide gel electrophoresis on 6% Novex PAGE gels (Invitrogen/Life Technologies, Carlsbad, CA, USA). Dual indexed libraries were generated using Nextera XT library preparation technology according to manufacturer’s protocol (Illumina, San Diego, CA, USA). After the tagmentation reaction, indexing specific PCR primers were added two per well for unique dual indexing of the libraries for multiplex sequencing. Limited cycle-number PCR was performed to amplify the libraries and incorporate the index sequences to the libraries. Amplified libraries were purified using AMPure XP beads. Double-stranded libraries were quality checked on a High Sensitivity DNA Agilent chip run on the Agilent 2100 Bioanalyzer (Agilent Technologies) for size and molarity determination. Denatured and diluted libraries were paired end sequenced on the Illumina MiSeq benchtop sequencer at 151 cycles per read (2 × 151). FASTQ files were imported into CLC Genomics Workbench 8 and analyzed with the bisulfite sequencing plugin.
Total genomic DNA (both nuclear and mitochondrial) should be extracted from tissues using a standard column preparation (e.g., Qiagen AllPrep DNA/RNA) or using solution (Trizol/TriReagent) methods. Core users should prepare their own DNA according their experience as isolation techniques vary according to sample type (tissues, cells, blood). DNA quality should be determined by spectrophotometry with attention to organic contamination (absorbance at 230nm) as high levels of organic contamination can inhibit PCR amplification. At least 1ug of template DNA is required per sample.