To prepare Zymo-Seq WGBS (D5465) libraries, intact genomic DNA is first bisulfite converted. Then, the following library preparation procedures are completed in a single tube: (1) second-strand synthesis, (2) tagmentation with Illumina DNA Prep library prep kit, and (3) library amplification and indexing. After purification, libraries are ready for sequencing on Illumina instruments. (Figure 1).
Figure 1: One Tube Library Prep Workflow using Zymo-Seq WGBS. Enzymatic reactions are consolidated in a single tube to minimize hands-on time.
Due to the necessary output, the libraries were sequenced on the NovaSeq. A 30x coverage per sample was targeted.
Sequencing on the NovaSeq 6000
5 replicates each of matched human brain and spleen were prepared using the Zymo-Seq WGBS Library Kit A 0.5% unmethylated E.coli control was spiked in to assess the conversation.
Libraries were sequenced at 2x151 using NovaSeq 6000 v1.5 chemistry.
Figure 2: the data by cycle for the % base shows an unbalanced library – which is expected with the bisulfite conversion of cytosine. Illumina recommends to spike in at least 1% of PhiX – but it is better to spike in 5% or higher. Despite the unbalanced base composition, high Quality score is maintained on the NovaSeq run.
Sequencing data were downsampled to 350M reads per sample for downstream analysis. Methylation analysis was performed using DRAGEN Methylation. Conversion efficiency was assessed with the unmethylated E. coli control, and was estimated to be higher than 99%.
Reproducible Coverage and unbiased DNA methylation calling are produced. More than 86% of functional genomic region (promoter and gene bodies) are covered with a minimum of 50× which is considered sufficiently deep to characterize a genomic region.
Contact ZYMO Research for any questions regarding the bisulfite conversion, the library prep assay and the analysis results. Contact Illumina Tech support for sequencing and DRAGEN related questions.
For Research Use Only. Not for use in diagnostic procedures.
Special thanks to our colleagues at ZYMO Research for providing ZYMO-seq reagents and controls for this experiment, and hat tip to Illumina Scientist Kevin Thai and the Systems Integration team for sequencing and analyzing these runs for this collaboration.