Accel-NGS® Methyl-Seq DNA Library Kit Library Preparation for Bisulfite-Converted DNA

Accel-NGS® Methyl-Seq DNA Library Kit Applications Whole Genome Bisulfite Sequencing (WGBS) Reduced Representation Bisulfite Sequencing (RRBS) Bisulfite-converted DNA enriched by MeDIP, ChIP, or other methods Hybridization capture using NimbleGen™ SeqCap™ Epi Enrichment System Ancient DNA samples that may contain uracil nucleotides as a result of damage Sample Types Genomic DNA Formalin-fixed, paraffin-embedded (FFPE) Circulating, cell-free DNA (cfDNA) Fresh and frozen tissue NimbleGen and SeqCap are trademarks of Roche NimbleGen, Inc.

Comparison of Methyl-Seq Workflows

Methyl-Seq Performance: Human WGBS ACCEL-NGS METHYL-SEQ TRADITIONAL WORKFLOW RANDOM PRIMER Bisulfite-Converted Library Input (ng) 1 1000 50 PCR Cycles 11 10 Yield (nM) > 4 GC Bias Low High Post-bisulfite library construction enables high recovery of input DNA Minimize the number of amplification cycles required Create libraries from as little as 100 pg of DNA Sequence-independent adapter ligation avoids bias Cover AT-rich sequences produced by bisulfite conversion

Methyl-Seq Performance: Human WGBS SAMPLE METHOD % READS ALIGNED GENOME COVERAGE % DUPLICATE READS EST. LIBRARY SIZE (MILLIONS) 10 ng Human Accel-NGS Methyl-Seq 86.4 8.9X 7.9 1,393

Methyl-Seq Performance: Arabidopsis WGBS Arabidopsis thalania Small genome model organism for methylation analysis Methyl-Seq constructs higher complexity libraries and provides comprehensive coverage of CpX (CpG + CpH) sites SAMPLE % READS ALIGNED GENOME COVERAGE % DUPLICATE READS EST. LIBRARY SIZE (MILLIONS) RELATIVE LIBRARY SIZE % CPX MISSING % CPX COVERED > 10X 100 ng Arabidopsis Accel-NGS Methyl-Seq 89.6 22X 1.9 714 1 0.56 92.2 Traditional 80.2 21X 2.7 604 0.85 0.57 88.1 Random Primer 71.4 16X 22.1 48 0.07 7.70 39.4 10 ng Arabidopsis 87.8 406 0.58 90.4 76.7 19X 11.9 70 0.17 83.9 71.9 22.2 45 0.11 5.2 45.2 1 ng Arabidopsis 83.3 18X 18.2 38 0.59 77.1 80.7 10X 62.3 6 0.16 2.00 17.0 73.4 12X 46.1 12 0.31 6.60 31.3

Methyl-Seq Performance: Arabidopsis WGBS CpX Sites Uncovered CpX Sites Covered ≥ 10X (A) At all three inputs (100 ng, 10 ng, and 1 ng), the Accel-NGS® Methyl-Seq Kit and the Traditional Method leave a minimal amount of of CpX (CpG + CpH) sites uncovered. However, the Random Primer method exhibits at least 4% of CpX sites missing at every input tested. (B) The percentage of CpX sites covered at least 10X is greater than 90% for Methyl-Seq at 100 ng, but decreases to approximately 75% at 1 ng. The Traditional Method covers greater than 80% of CpX sites at least 10X for 100 ng and 10 ng inputs, but this coverage diminishes greatly at 1 ng. The Random Primer Method exhibits poor coverage of CpX sites at each input, with the percentage of CpX sites covered at least 10X at about 40% for all inputs.

Methyl-Seq: cfDNA and Cancer Burden PNAS, vol 110, no 47 (2013) pp18761-18768

Methyl-Seq: cfDNA and Cancer Burden 5 ng of cfDNA for bisulfite-converted WGBS libraries 10 million mapped reads to detect hypomethylation levels Experimental design considerations Acquire positive cfDNA samples Need to be less than 3 days old From patients with diagnosed cancer Identifying known positive controls “Healthy” cfDNA control samples Cancer-derived samples are compared to an average of “healthy” controls Not all healthy controls are fit Does obesity, alcoholism, age, inflammation….change methylation levels?

Detecting Genome-Wide Hypomethylation with 10 Million Reads Circos plot represents hypomethylation status on chromosomes 1-22 between 5 healthy controls and sample 8 (Metastatic colorectal adenocarcinoma with liver metastasis, 2 cm primary).

Methylation Density: Tumor cfDNA Samples PATHOLOGY % HYPOMETHYLATION 1 Fallopian tube high grade papillary serous carcinoma pT3c N1 with 2 nodes involved by micrometasasis 0.4% 2 5 cm ovarian ‘borderline’ serous content (cancer-like) 1.1% 3 Recurrent pT2, pN0 mammary carcinoma, 2.15 cm 2.4% 4 pT1/pN1 pancreatic adenocarcinoma with neoadjuvant therapy 3.6% 5 Metastatic colon cancer to the liver (previously treated) 4.4% 6 14 cm ovarian ‘borderline’ serous content (cancer-like) 18.0% 7 Colon-cancer, non-resectable Adenocarcinoma T4a by imaging 8 Metastatic colorectal adenocarcinoma with liver metastasis, 2 cm primary 43.4% Percent hypomethylation was calculated by comparing the tumor samples’ methylation density (MD) by 1 Mb genome windows/bins to average of 5 healthy controls. Bins were assigned as hypomethylated if the MD was >3, SD lower than the average MD.

Reduced Representation Bisulfite Sequencing RRBS is a form of targeted BS-Seq MspI digest followed by selection of a specific range of fragment sizes enriches for CpG islands Alternative workflow for RRBS with Accel-NGS® Methyl-Seq Traditional method requires dsDNA for library preparation, so size selection (orange box) selects for 40-220 bp inserts (166-346 bp library molecules). Methyl-Seq library construction is compatible with ssDNA, so size selection selects for 100-220 bp inserts (100-220 bp fragments). Size selection must occur before bisulfite conversion because conversion will fragment DNA, affecting fragment size selection. Improper size selection may affect CpG island coverage.

Targeted Methylation Sequencing from 1 ng INPUT METHOD % ALIGNED % ON TARGET % DUPLICATION MEAN COVERAGE % COVERED > 2X % COVERED > 20x NONE COVERED 100 ng SWIFT 90 73 6.5 49X 98.6 78.6 0.8 1 µg Kapa 80 9.4 51X 81.1 10 ng 91 77 26.0 35X 98.5 71.0 87 78 1X 24.7 0.2 47.7 1 ng 62.0 8X 93.6 2.3 1.0 Coverage metrics were analyzed for inputs of 1 µg and 100 ng, quantities that are within specification for Kapa and Swift library preparation, respectively. Additionally, lower inputs of 10 ng (Kapa and Swift) and 1 ng (Swift only) were also analyzed. A substantial increase in duplicate reads and decrease in genome coverage can be observed for Kapa libraries at 10 ng. However, the Swift kit performs well at 10 ng, with the performance metrics at 1 ng comparable to the 10 ng metrics from the Kapa kit.

Differentially Methylated Region (DMR) Analysis from 10 ng DMR analysis was performed for 10 ng libraries from both Kapa and Swift, comparing DNA from an H1 ES cell line and a B-lymphocyte cell line (NA12878). This figure illustrates the 294,130 DMRs called from the Swift Methyl-Seq kit (37,799 hypomethylated and 256,331 hypermethylated). In contrast, the 10 ng Kapa libraries resulted in only 464 total DMR calls (not shown).

Accel-NGS® Methyl-Seq DNA Library Kit Ordering Information PRODUCT NAME REACTIONS CATALOG NO. Accel-NGS Methyl-Seq DNA Library Kit 12 DL-ILMMS-12 48 DL-ILMMS-48 Visit www.swiftbiosci.com for easy ordering.

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