Targeted : An Efficient, Streamlined Approach to Maximize Throughput and Minimize Cost

Ioanna Andreou1, Brian Dugan2, Jonathan Schaffer2, Mohammad Nezami Ranjbar2, Sascha Strauss1, Quan Peng2, Silke Huebner1, Christin Meerschiff1, Katja Heitz1, Christopher Wach1, Eric Lader2 1 QIAGEN GmbH, QIAGEN Straße 1, 40724 Hilden, Germany 2 QIAGEN Sciences Inc., 6951 Executive Way, Frederick, MD 21703, USA

Introduction QIAseq Targeted Methyl Panel Workflow

Epigenetic changes have a significant impact on human health and disease susceptibility since they play a crucial role in the DNA regulation of important cellular processes. In recent years, significant progress has been made in this rapidly advancing field. DNA bisulte conversion: EpiTect Fast DNA Bisulte Kit EpiTect Fast Bisulfite Conversion One of the most stable forms of epigenetic regulation is DNA . Bisulfite sequencing (BS) is considered the best (cat. nos. 59802, 59824, 59826) chemistry method for detecting DNA methylation with single resolution. However, the high cost of whole BS and UUCCU

the low throughput of BS are challenges that must be overcome. DNA end repair

Here, we present a streamlined workflow combining optimal bisulfite treatment and targeted bisulfite sequencing. This U CCUU X method utilizes single-primer extension (SPE) and unique molecular identifier (UMI) barcoding technology. This enables Ligation of rst index adapters with UMI highly efficient, flexible and scalable targeted analysis of DNA methylation from genomic DNA (gDNA), formalin-fixed QIAseq Targeted Methyl Panel chemistry for target enrichment UMI U CCUU paraffin-embedded (FFPE) and circulating cell-free DNA (ccfDNA) samples. 7.5 h and generation of NGS library 6 h suitable for Illumina platforms Target enrichment by single primer extension Sample to Insight

5' U CCUU

GSP Library FP Sample Bisulfite construction NGS run Data analysis Repeated sampling preparation conversion with target enrichment Library amplication and sample indexing = Methylated C = Non-methylated C ® ® ® ® DNeasy , QIAamp , EpiTect Fast Bisulfite • Primer design tool Illumina platforms • Bisulfite DNA 5' TCTTC UMI: Unique molecular index Gentra®, or GeneRead™ Conversion Kits for: • QIAseq® Targeted analysis pipeline SIP FP: Forward primer technologies for isolation of: Methyl Panel of the GeneGlobe® UP • Purified gDNA GSP Gene-specic primer • QIAseq Methyl DNA Data Analysis Center : • gDNA • FFPE DNA UP Index Kits • CLC Genomics : Universal primer • ccfDNA Library SIP: Sample index primer • FFPE DNA Workbench for Methylation

Streamlined workflow for DNA methylation analysis. QIAseq Targeted Methyl Panel workflow.

Performance: gDNA Performance: ccfDNA

Control gDNA (0% and 100% methylated, with inputs of 1 ng, 10 ng, 50 ng and 100 ng) was bisulfite treated using the ccfDNA was purified using QIAamp chemistry and subsequently bisulfite treated using EpiTect Fast chemistry. 10 ng ccfDNA EpiTect Fast DNA Bisulfite Kit and libraries were generated using the using the Colorectal Panel (MHS-002Z) and was processed using three different QIAseq Targeted Methyl Panels. Library amplification with 19 cycles resulted in high QIAseq Targeted Methyl Panel chemistry. The libraries were sequenced using a MiSeq® instrument with sequencing depth of yields of the generated library. The expected 350 bp library size represents the combination of 170 bp (typical size of approx. 150x. Data were analyzed using the Bisulfite DNA analysis pipeline of the GeneGlobe Data Analysis Center. The ccfDNA with 180 bp adapters). This shows that no DNA degradation occurred during bisulfite treatment with EpiTect Fast results show high library quality, a high mapping rate – even starting from 10 ng input gDNA and high (C) coverage. chemistry. The libraries where sequenced on MiSeq 2x150 bp paired end run and a sequencing depth of approx. 200x. The high-quality libraries resulted in high number of unique mapped reads which led to a high cytosine coverage to allow accurate methylation calls even from low input of the strong fragmented ccfDNA. Colorectal carcinoma panel Colorectal carcinoma panel ccfDNA library characteristics ccfDNA 20 ng input % of total reads Covered , % Average fragment Total reads, % 100 100 Yields, nM size, bp 35 400 150 355 343 350 80 80 30 350 300 100 25 60 60 250 20 50 13.1 15.4 200 40 40 15 150 9.0 0 10 748 primer 1900 primer 1400 primer 20 20 100 5 50 Methyl panel 0 0 0 0 1 ng 10 ng 50 ng 100 ng 1 ng 10 ng 50 ng 100 ng 1 ng 10 ng 50 ng 100 ng 1 ng 10 ng 50 ng 100 ng Methyl panel (748) Methyl panel (1900) Methyl panel (1400) Reads after trimming, % Reads matched a primer, % Reads unique mapped 0% methylated 100% methylated 0% methylated 100% methylated Input 10 ng ccfDNA On-target, % Off-target, % C covered, % CpG covered >30 UMI, %

Molarity, nM Average size After trimming, % Map to primer, % Unique maping, % On-target, % Off-target, % % C covered % CpG covered

Yields and mean size of generated libraries using three different panels Quality of generated libraries. Libraries and panels used as a percentage consisting of different primer numbers. of the reads after trimming; reads matched to primer sequence; unique mapped reads on target and off-target reads to total number of reads. Read statistics as percentage of all reads obtained. A high unique mapping Cytosine coverage. Total cytosine coverage and coverage of cytosines in The figure also shows the high coverage of cytosines and cytosines in CpG and on target rate can be achieved even from 10 ng gDNA input in the CpG content as percentage of the theoretical C expected to be covered by content as percentage of the theoretical C expected to be covered by the complete workflow. the corresponding panel. corresponding panels.

Performance: FFPE DNA Conclusions

A QIAseq Targeted Methyl Panel consisting of 93 primers was used to generated libraries with a starting input of 40 ng We present a workflow and chemistry that enables methylation sequencing in a targeted mode, which in turn, allows FFPE DNA. The DNA was first bisulfite converted using EpiTect Fast chemistry. The generated libraries were sequenced on testing of multiple samples and regions of interest in a cost-efficient manner. a MiSeq instrument with a sequencing depth of approx. 200x, generating a mean of 65 UMI per cytosine. Our method:

® The Methylation degree of 7 CpG sites of the MGMT gene was previously validated with . The evaluated • Combines highly efficient EpiTect Fast bisulfite conversion chemistry and the QIAseq Methyl Targeted Panels to reduce methylation degree of the MGMT gene using the QIAseq Targeted Methyl Panel highly correlate with Pyrosequencing data. turnaround time over probe-capture-based enrichment Comparable high C coverage was obtained with all FFPE DNA having different methylation status. • Shows high enrichment specificity with low bias for bisulfite-converted DNA, using an optimized SPE methodology including UMI

MGMT methylation degree MGMT methylation degree • Allows accurate detection of CpG methylation at different levels C covered, % Methylation, % • Is compatible with gDNA, FFPE DNA and ccfDNA 100 100

77,8 • Allows lower DNA input 80 80 75 • Increases flexibility of methylation analysis through highly efficient panel design to enrich only the regions of interest 60 60 • Integrates analysis tools to standardize the method of methylation status evaluation 40 40

20 20 13 9,4 For up-to-date licensing information and product-specific disclaimers, see the respective QIAGEN kit handbook or user 2 2,6 0 0 DNA1 DNA5 DNA9 DNA1 DNA5 DNA9 manual. QIAGEN kit handbooks and user manuals are available at www.qiagen.com or can be requested from QIAGEN 250 bp run Technical Services or your local distributor. C covered Pyro QIAseq Targeted Methyl Panel 9 1 ® ® ® ® ® ® ® ™ ® ® Cytosine coverage obtained in the region of interest. Comparable high Comparison of evaluated methylation degree with QIAseq Targeted Methyl Trademarks: QIAGEN , Sample to Insight , QIAamp , QIAseq , DNeasy EpiTect , GeneGlobe , GeneRead , Gentra , Pyrosequencing (QIAGEN Group);

0/2 0 ® C coverage was obtained with all FFPE having different methylation Panel and Pyrosequencing. The evaluated methylation degree of the MiSeq (Illumina, Inc.). Registered names, trademarks, etc. used in this document, even when not specifically marked as such, are not to be considered status. MGMT gene highly correlates with the methylation degree of the 7 CpG unprotected by law. sites previously validated with Pyrosequencing. © 2019 QIAGEN, all rights reserved. PROM-15023-001 1119112 1 Sample to Insight