The document describes a new method called post-bisulfite next-generation sequencing library construction (PBLC) for whole genome bisulfite sequencing (WGBS) that overcomes challenges with traditional WGBS workflows. PBLC involves bisulfite treatment before library preparation, which fragments the DNA so no additional fragmentation is needed. This reduces workflow time and enables epigenomic studies from lower DNA inputs. Comparison studies show PBLC produces higher library yields than traditional WGBS and comparable sequencing results, making it a more efficient and sensitive method for WGBS.

1. Input DNA
Bisulfite
treatment
Bisulfite
treatment
Final
bisulfite-seq
library
PCR
enrichment
Post-bisultife NGS libraray construction (PBLC)
Classic whole genome bisulfite sequencing (WGBS)
Fragmentation
PCR
enrichment
NGS library
construction
NGS library
construction
Ioanna Andreou, Wolfgang Krebs, Peter Hahn, Silke Huebner, Christin Meerschiff, Annika Piotrowski, Karim Benyaa,
Verena Schramm and Nan Fang
QIAGEN Strasse 1, 40724 Hilden, Germany
110413009/2016
Fast and Efficient Post-Bisulfite-Seq Library Construction
with QIAseq Ultralow Input DNA Library Protocol
Sample to Insight
Introduction
Epigenetic changes play a crucial role in the regulation of important cellular processes such as gene expression and
cellular differentiation, and are also identified as key factors in many diseases.
The methylation of cytosines in the genome, an important epigenetic regulatory mechanism, reduces the transcriptional
activity of adjacent genes. Whole genome bisulfite sequencing (WGBS) – combining bisulfite-mediated conversion of
unmethylated cytosines to uracil – and next-generation sequencing (NGS), allow the genome-wide detection of 5-methyl-
cytosine residues at unprecedented single-base resolution. It also enables the connection between gene activity and the
precise localization of a DNA methylation mark. However, the commonly used approach to treat samples after NGS
library preparation with bisulfite to convert unmethylated cytosines to uracils reveals major challenges, such as significant
bisulfite-induced sample loss due to DNA degradation. Therefore, the traditional library prep method for bisulfite
sequencing demands very high DNA input amounts or requires a large number of PCR cycles during NGS library
construction.
Here, we present a fast and streamlined workflow for bisulfite treatment, double-strand synthesis and NGS library
construction, which overcomes these traditional challenges. We demonstate that this post-bisulfite NGS library construction
(PBLC) approach creates significantly higher library yields than the classic WGBS approach – without any compromise
in data quality. Another advantage is that DNA fragmentation through bisulfite means that no further DNA fragmentation
is needed prior to library prep. This reduces the overall workflow time.
The WGBS method is the treatment of DNA using bisulfite to determine DNA methylation patterns. Treating DNA with
bisulfite leaves 5-methylcytosine residues intact and converts unmethylated cystosine residues to uracil. These specific DNA
sequence changes provide information about the methylation status of a DNA segment. The WGBS method is important for the
detection of epigenetic modifications that impact transcriptional processes. However, this poses some challenges;
importantly, the necessity of larger (microgram) quantities of DNA samples, since WGBS bisulfite treatment of NGS
libraries leads to a degradation of DNA and results in significant sample loss.
Therefore, a new and more sensitive method of DNA methylation is used for smaller DNA sample sizes: post-bisulfite
library construction (PBLC). In the PBLC method, the bisulfite treatment precedes NGS library prep. Then, a subsequent
DNA repair step using random primers allows the generation of NGS libraries from reduced DNA input amounts. During
bisulfite treatment, DNA is fragmented so that no additional DNA fragmentation is required. PBLC enables epigenomic
applications that are not possible with WGBS, especially for a limited amount of starting material.
Comparison of WGBS and PBLC Workflow
Detailed Description of PBLC Workflow
Human leucocyte DNA was used to generate libraries using the PBLC workflow. DNA was bisulfite treated using the EpiTect
Fast Bisulfite Conversion Kit. Then, double-strand synthesis was performed using the Klenow fragment (3'→5' exo-) and
random octamers. Using the double-stranded fragments, libraries were generated using the QIAseq Ultralow Input Library
Kit. Library amplification was performed with the QIAGEN Multiplex PCR Kit using either 6 or 10 cycles of amplification. The
PBLC workflow can create a significantly higher amount of an NGS library than WGBS, independent of the DNA input. For
WGBS, the minimum concentration needed for an Illumina®
MiSeq®
run is 2 nM, but this can be achieved from only 150 ng
of PBLC input DNA.
Higher yields with PBLC. 250 ng DNA was used to generate bisulfite-
treated libraries using classic WGBS and PBLC workflows. Higher amounts
were obtained with PBLC, as bisulfite treatment after library construction
led to high losses of the generated DNA library.
Generated library yields. Final NGS library concentrations and molarities
are shown, as calculated from Agilent Bioanalyzer traces. PBLC DNA input
150–1000 ng; 6x amplification cycles.
PBLC Delivers Higher Yields of NGS Libraries
Conclusions
The combination of highly specific bisulfite conversion reagents of the EpiTect Fast Bisulfite Conversion Kit and the ultra-
efficient end-polishing and adapter ligation chemistries of the QIAseq Ultralow Input Library Kit allow fast and efficient
bisulfite conversion and NGS library preparation to accurately interrogate the methylome. Efficient amplification of library
is performed using the QIAGEN Multiplex PCR Kit.
The PBLC method enables:
• Epigenetic studies even from low DNA input amounts
• A low number of cycles for library amplification, reducing the number of PCR duplicates
• A very fast protocol
• No extra fragmentation of DNA
• The use of non-methylated Illumina adapters that are commonly used in library construction for NGS libraries.
For up-to-date licensing information and product-specific disclaimers, see the respective QIAGEN kit handbook or user
manual. QIAGEN kit handbooks and user manuals are available at www.qiagen.com or can be requested from QIAGEN
Technical Services or your local distributor.
Trademarks: QIAGEN®
, Sample to Insight®
, EpiTect®
, GeneRead™
(QIAGEN Group); Accel-NGS®
(Swift Biosciences Inc.); Adaptive Focused Acoustics™
(Covaris, Inc.); AMPure®
(Beckman Coulter, Inc.); EZ DNA Methylation™
(Zymo Research); MiSeq®
(Illumina). Registered names, trademarks, etc. used in this
document, even when not specifically marked as such, are not to be considered unprotected by law.
© 2016 QIAGEN, all rights reserved. PROM-9997-001
Comparable Results to Existing WGBS Workflows
Bisulfite-treated libraries were generated from:
• 250 ng human leucocyte DNA using the PBLC workflow (<4.5 h).
• 2 µg human leucocyte DNA using the WGBS workflow: fragmentation using Adaptive Focused Acoustics™
(AFA)
technology (Covaris), EZ DNA Methylation™
Kit (Zymo) and the Accel-NGS®
Methyl-Seq DNA Library Kit (Swift) (5 h).
• Libraries were sequenced at depths of 0.3–0.4X and analyzed with the QIAGEN®
CLC Genomics Workbench (v8.01).
Two replicate libraries generated using each method were sequenced. Sequencing of the PBLC workflow resulted in a high
percentage of mapped reads and comparable methylation degree to the WGBS workflow.
Percentage of mapped reads to the reference genome hg19. Percentage of methylated cytosines in CpG sites.
WGBS and PBLC workflows.
PBLC workflow.
Perform desulfonation reaction and cleanup using EpiTect Fast Columns
2x: Incubate 5 min 95°C,15 min 60°C, hold 20°C
Sequencing
Add Bisulfite-Protect Mix to purified DNA
EpiTect®
Fast Bisulfite
Conversion Kit protocol
Double strand synthesis
End-polishing
Adapter ligation
Size selection
Library amplifiaction
30 min at 25°C and 15 min at 65°C, hold at 4°C
Add Ligation Mix and adapters from Ultralow input Kit
Add EP Mix from QIAseq Ultralow Input Library Kit
Add Klenow 15U random primer 7 µM and 100 µM dNTP
30 min at 37°C, stop at 68°C 10 min, cool at 4°C
Perform column purification using GeneRead™
Size Selection Kit
AMPure®
beads or GeneRead size selection
6 cycles with QIAGEN Multiplex PCR Kit
Purify with 1x AMPure beads or with GeneRead Size Selection Kit
10 min at 25°C, hold at 4°C
Concentration (pM)
70,000
60,000
50,000
40,000
30,000
20,000
10,000
0
Classic WGBS PBLC
Library concentration (nM)
14
12
10
8
6
4
2
0
PBLC 150 ng PBLC 250 ng PBLC 500 ng PBLC 1000 ng
Mapped reads, %
120
100
80
60
40
20
0
Zymo/Swift workflow PBLC
Methylated cytosines, %
100
80
60
40
20
0
Zymo/Swift workflow PBLC