TruSeq Methyl Capture EPIC Library Prep Kit FAQs

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  • Protocol


  • 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 

    Plexity

    Option

    Indexes

    2

    1

    AD006, AD012

     

    2

    AD005, AD019

    3

    1

    AD002, AD007, AD019

     

    2

    AD005, AD006, AD015

     

    3

    2-plex options with any other index

    4

    1

    AD005, AD004, AD007, AD016

     

    2

    AD002, AD004, AD007, AD016

     

    3

    3-plex options with any other index

    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.

  • Analysis


  • 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.