p a c b .c o m /e p i g e n e t i c s

Phylogenetic tree of 230 sequenced prokaryotic organisms. Outer bars indicate the number and types of active MTases detected per genome1.

- Characterize bacterial DNA modiications and methyltransferase recognition motifs

- Identify hyper- and hypo-methylated CpG islands to explore eukaryotic gene expression and regulation

- Observe allele-speciic methylation by using long reads to phase SNPs when bisulite sequencing

- Leverage epigenomic data to cluster contigs and associated plasmids in metagenomic communities

- Detect strand-speciic modiication such as hemimethylation

- Uncover DNA modiications and methylation events with hypothesis-free analysis

GENOME-WIDE METHYLATION PATTERNS COMPARED IN 230 DIVERSE PROKARYOTES

N6–ADENINE METHYLATION FOUND IN MAMMALIAN GENOME

Single Molecule, Real-Time (SMRT®) Sequencing directly detects DNA modiications by measuring variation

in the polymerase kinetics of DNA base incorporation during sequencing. With high throughput, long reads,

and the sensitivity to detect epigenetic modiication without ampliication or chemical conversions,

the PacBio® Systems offer scalable solutions for assessing DNA modiications in bacterial and eukaryotic

genomes.

CHARACTERIZE THE EPIGENETIC LANDSCAPE OF YOUR GENOME

(a) SMRT Sequencing worklow used to identify N6-adenine methylation in Alkbh1 knockout embryonic stem cells. (b) The levels of 6mA are controlled by Alkbh1, whereby increasing its levels in ES cells led to gene silencing. (c) A downregulation of the dax1 gene, located 30 kb from 6mA-enriched young full-length L1 repeat element, was also observed2.

PacBio RS II System Sequel System

KEY REFERENCES1. Blow, M. J. et al. (2016) The epigenomic landscape of prokaryotes. PLoS Genetics. 12 (2), e1005854. 2. Wu, T. P. et al. (2016) DNA methylation on N(6)-adenine in mammalian embryonic stem cells. Nature. 532 (7599), 329–333. 3. Yang, Y. et al. (2015) Quantitative and multiplexed DNA methylation analysis using long-read single-molecule real-time bisulite sequencing (SMRT-BS).

BMC Genomics. 16 (1), 350. 4. Leonard, M. T. et al. (2014) The methylome of the gut microbiome: disparate Dam methylation patterns in intestinal Bacteroides dorei. Frontiers in

Microbiology. 5 (361), 1–6. 5. Manso, A. S. et al. (2014) A random six-phase switch regulates pneumococcal virulence via global epigenetic changes. Nature Communications. 5, 5055. 6. Roberts, R. J. et al. (2015) REBASE-a database for DNA restriction and modiication: enzymes, genes and genomes. Nucleic Acids Research. 43 (D1),

D298–D299. 7. Suzuki, Y. et al. (2015) Measuring the landscape of CpG methylation of individual repetitive elements. bioRxiv. Available at http://dx.doi.

org/10.1101/018531.

For Research Use Only. Not for use in diagnostic procedures. © Copyright 2016, Paciic Biosciences of California, Inc. All rights reserved. Information in this document is subject to change without notice. Paciic Biosciences assumes no responsibility for any errors or omissions in this document. Certain notices, terms, conditions and/or use restrictions may pertain to your use of Paciic Biosciences products and/or third party products. Please refer to the applicable Paciic Biosciences Terms and Conditions of Sale and to the applicable license terms at http://www.paciicbiosciences.com/licenses.html.

Paciic Biosciences, the Paciic Biosciences logo, PacBio, SMRT, SMRTbell, Iso-Seq, and Sequel are trademarks of Paciic Biosciences. BluePippin and SageELF are trademarks of Sage Science. NGS-go and NGSengine are trademarks of GenDx. All other trademarks are the sole property of their respective owners. PN: AP102-051216

SMRT Sequencing with PacBio Systems

- Take advantage of the Sequel™ System to reduce project costs and generate 7X more reads compared with the PacBio RS II

- Achieve ~10 kb average read lengths, with some reads as long as 60 kb

- Simultaneously capture epigenetic information

FROM DNA TO EPIGENETIC INFORMATION

Library Preparation

- Prepare long-insert SMRTbell™ library - For SMRT bisulite sequencing, generate SMRTbell libraries

from long amplicons (1–2 kb) following bisulite treatment - Size-selection options enrich for longest inserts - Use recommended gDNA library inputs

- > 5 μg for large genome project - Low-input options (100 ng)

- Library automation supported

Data analysis with SMRT Analysis or PacBio DevNet

- Characterize bacterial methylomes with SMRT Analysis epigenomic tools for modiication detection and motif analysis

- Identify hypo- and hyper-methylated CpG regions in eukaryotic genomes with > 20-fold coverage

- Detect CpG methylation across 1.5 kb regions with SMRT bisulite sequencing