TruSeq Methyl Capture EPIC Library Prep Kit FAQs

The TruSeq Methyl Capture EPIC Sequencing Panel contains content that spans the full human methylome, including CpG islands, shores, shelves, enhancers, promoter regions, sites in open chromatin, and gene bodies.

This panel builds on content included in the Infinium Human Methylation EPIC BeadChip with additional regions of importance identified by ENCODE, FANTOM5, the Epigenomics RoadMap Consortium, and customer requests. 

TruSeq DNA Methylation is used for whole genome bisulfite sequencing and uses different chemistry than TruSeq Methyl Capture EPIC.

A Pearson correlation value of 0.96 was obtained. For more information, see the TruSeq Methyl Capture EPIC data sheet.

The number of samples pooled pre-enrichment depends on the kit:

For more information, see the TruSeq Methyl Capture EPIC Library Prep Reference Guide.

For both the LT and HT configurations, a kit contains 5 boxes.

The oligos are 80 mers.

Currently, Illumina does not offer a custom-designed TruSeq Methyl Capture EPIC panel.

For the majority of target regions, a single strand is captured and sequencing data are highly stranded except in a subset of regions.

Because bisulfite sequencing converts unmethylated cytosines to uracil, which eventually become thymines in the final sequence, C>T SNPs could be mistaken for “unmethylated” cytosines. For regions containing CpGs with common SNPs (MAF >5% in EUR superpopulation 1000 genomes data), probes are designed to capture both strands. As a result, high frequency SNPs at CpGs that would be obscured if only a single strand was sequenced can now be called by looking at the second strand in these regions. If low frequency SNP calls (<5% MAF) at unmethylated CpG sites are desired, other suitable technologies should be used (eg, sequencing an unconverted sample).

Lister, Ecker, and colleagues (Nature 2009) found that 99% of CpG sites on the complementary strand share the same methylation state. Therefore, methylation of the complementary strand can be inferred from single strand methylation data in most cases. 

One year from the date of manufacture. The kit label provides an exact expiration date. Illumina guarantees at least three months from the date of receipt.

Cleanup procedures have been optimized and validated using the magnetic stand specified in the library prep reference guide. Comparable performance is not guaranteed when using other magnets.

If you choose to use a different magnet, test how long samples must sit on the magnet. Times can vary from the protocol.

Two aspects can make a magnet suboptimal for this protocol:

• The magnet is too weak and does not sufficiently pull beads out of solution.
• The geometry of the magnet makes it difficult to remove the entire supernatant cleanly, without removing any beads along with the supernatant.

Illumina recommends using one or more of the following recommended positive control samples. These positive control samples can be used for methylation status control.  

Normal samples: HCC1187 normal (BL) (ATCC, catalog # CRL2323-D), NA12878 (Coriell Institute, catalog # NA12878)

Cancer samples: HCC1187 breast cancer tumor (ATCC, catalog # CRL2322), HeLA (Biochain, catalog # D1255811), Jurkat (Biochain, catalog # D1255815)

All beads required for the protocol are supplied in the kit.

The bisulfite conversion reagents required for the protocol are supplied in the kit.

See the white paper on the kit Documentation & Literature page for more information.

500 ng of input genomic DNA is required.

Illumina has not validated the workflow with less than 500 ng.  When using less than 500 ng of input DNA, a corresponding increase in duplicates were observed. Therefore, libraries may require increased sequencing depth to attain the desired level of coverage.

Illumina has tested up to 1 µg which has produced fewer duplicates and therefore better coverage.

This protocol has not been validated with FFPE samples.

Determining the size of genomic DNA is difficult due to the large size. However, Illumina recommends checking for DNA quality and using only gDNA with 260/280 ratio between 1.8–2.0.

Use fluorometric-based methods for quantification, including Qubit or PicoGreen, to accurately quantify the gDNA.

UV spectrometer-based methods, such as the NanoDrop, measure any nucleotides present in the sample including RNA, dsDNA, ssDNA, and free nucleotides, which provide an inaccurate measurement.

Dilute starting material in RNase/DNase-free water or 10 mM Tris-HCl, pH 8.5. Avoid EDTA containing buffers, such as TE or AE.

DEPC water is not recommended. Contaminants can inhibit parts of the reaction.

Both the LT and HT kits contain enough reagents for the indicated number of samples at only four-plex. You can complete this protocol using various plexities (single-plex, two-plex) with additional PCR cycles, but some reagents run out when using less than the maximum number of samples.

Samples are multiplexed before enrichment. Samples can also be multiplexed before sequencing if loading multiple samples on a single flow cell.

TruSeq LT Set B indexes have not been validated with this workflow and therefore are not supported by Illumina.  

Guidelines can be found in the Index Adapters Pooling Guide.

This kit was designed for 4-plex samples. Certain reagents in the kit can run out if running at less than 4-plex.

When designing low-plexity index pools for single-indexed sequencing, always use at least two unique and compatible barcodes for each index sequenced. The following table describes possible pooling strategies for 2–4 samples generated with the adapter index tubes in each set.

Not all color-balanced pools are listed. Check the color balance using IEM. For more information, see the Illumina Experiment Manager User Guide.

LT Kits

Any 2-plex, 3-plex, or 4-plex combination of AD002, AD004, AD005, and AD006.

HT Kits 

The protocol is optimized for shearing using Covaris fragmentation. Other methods are not supported or tested by Illumina and can result in low yield, unexpected size distributions, or library failure.

There are three safe stopping points in the protocol. The safe stopping points are after the following steps:

• Fragment DNA
• Amplify Enriched Library
• Clean Up Amplified Enriched Library. 

For storage details, see the reference guide.

All reagents that are shipped frozen can withstand the freeze-thaw process. Components such as the magnetic beads and SPM are never frozen.

Illumina’s proprietary method ensures ligation of 2 different adapters in the required orientation to opposing ends of a DNA fragment. PCR selects for these and finalizes the construct ready for hybridizing onto the flow cell surface. Adapter sequences can be determined by sequencing the ligation fragments, but sequence information alone is not sufficient to uncover the method.

To prepare unconverted libraries (not used for methylation calls) with this kit, skip Bisulfite Conversion and go directly to the Amplify Enriched Library step.

Use an Agilent Technologies 2100 Bioanalyzer to check the quality and intended size distribution of the Covaris sheared sample and the final library. For examples of Bioanalyzer traces and library size distributions, see the reference guide.

The expected peak is between 150–170 bp. The expected distribution is > 60% of the DNA between 100–300 bp. The average size of the DNA in that size range is 180–200 bp. See the reference guide for an example Bioanalyzer trace.

The expected size range for final libraries is ~250 bp to ~1 kb. The expected median insert size is 180–200 bp. The reference guide provides an example Bioanalyzer trace.

The expected quantity is 3–12 ng/µL, resulting in 10–40 nM.

A qPCR or fluorometric quantification assay using dsDNA binding dyes (such as Qubit or PicoGreen) can be used to quantify the final libraries.

Illumina does not support running libraries prepared by different library prep kits in the same lane of a flow cell. This practice may impact cluster density, data distribution, and run quality. 

Real-Time Analysis (RTA) v2.7.7 is available to TruSeq Methyl Capture EPIC customers in Q4 of 2016.  It will then be released to all Illumina customers by 2017.

The following table shows the recommended number of libraries per flow cell needed to obtain > 40x mean coverage and > 90% of target bases covered at ≥ 10x. This kit includes index adapters for up to 12 libraries.

55 million reads is recommended to maximize the number of CpGs covered > 10x and to give a mean coverage of > 40x.

The recommended read length is 2 x 101.

This kit is single-index.

If the run is being uploaded to BaseSpace or BaseSpace Onsite, the data can be analyzed using BaseSpace Core App- MethylSeq and BaseSpace Labs App- MethylKit.

MethylSeq provides alignment analysis and MethylKit provides sample to sample comparison analysis.

For more information about MethylSeq, see the MethylSeq BaseSpace App Documentation.

For more information about MethylKit, see the MethylKit BaseSpace Labs page.

Illumina sequence base call output files (*.bcl) can be demultiplexed and converted to FASTQ format using the bcl2fastq converter software. The files can then be used for analysis with other third party software packages, such as Bismark and MethylKit.

Illumina cannot provide support for the use of third party software. Contact the software resources directly with any questions regarding the analysis and use of the software.