NEXTFLEX® Methyl Sequencing 1 Kit

  • Methylome-level assessment with broad genome coverage
  • Make methyl rich libraries using methylated DNA immunoprecipitation (MeDIP) or MBD capture (MeCAP)
  • Quantify absolute DNA methylation levels
  • Identify differentially methylated regions (DMRs)
  • Enhanced adapter ligation technology with NEXTFLEX® Ligation
  • Flexible barcode options– 6, 12, 24, 48, 96 and 384 unique single-index adapters
  • Gel-free, bead-based cleanup protocols
  • Automation-friendly workflow is compatible with liquid handlers
  • Barcoded adapters for multiplexing contain embedded index sequence
  • Functionally validated with Illumina® sequencing platforms

  • NOVA-5118-02


    48 RXNS
Download Manual
For research use only. Not for use in diagnostic procedures.


  • NOVA-514101


    48 RXNS

  • NOVA-514102


    96 RXNS

  • NOVA-514103


    192 RXNS

  • NOVA-514104


    384 RXNS

  • NOVA-514105


    96 BARCODES (in 96-well plate)
    768 RXNS

  • NOVA-514106


    96 BARCODES (in tubes)
    768 RXNS

Methylated DNA Libraries for Illumina® Sequencing

The NEXTFLEX® Methyl-Seq 1 Kit is designed to enrich and prepare single, paired-end and multiplexed methylated DNA libraries for sequencing using Illumina® sequencing platforms. NEXTFLEX® Methyl-Seq utilizes a versatile immune-capture (MeDIP) or methyl-CpG-binding domain (MeCAP) for detection of methylated DNA allowing the user to easily assess the methylation state of the genome.

This kit features Enhanced Adapter Ligation Technology, resulting in library preps with a larger number of unique sequencing reads. This specially designed NEXTFLEX® ligation enzymatic mix allows users to perform ligations with longer adapters and better ligation efficiencies. The NEXTFLEX® Methyl-Seq 1 Kit simplifies workflow by using master mixed reagents and magnetic bead based cleanup, reducing pipetting and eliminating time consuming steps in library preparation.

Flexible Multiplexing Options

NEXTFLEX® DNA Barcodes, NEXTFLEX-96 DNA Barcodes and NEXTFLEX-HT Barcodes can be used for multiplexing libraries prepared with the NEXTFLEX® Methyl-Seq 1 Kit. These kits include adapters containing indexed sequences that offer an improved multiplexing workflow and flexible setup. These barcodes are available in sets of 6, 12, 24, 48, 96 and 384 Illumina-compatible adapters. The ability to pool samples in an efficient way significantly decreases hands on time while providing robust data quality. These barcodes can be used with single, paired-end and multiplex reads.


  • NEXTFLEX® Methyl End Repair Buffer Mix
  • NEXTFLEX® Methyl End Repair Enzyme Mix
  • NEXTFLEX® Methyl Adenylation Mix
  • NEXTFLEX® Methyl Ligation Mix
  • NEXTFLEX® DNA Adapter (25 µM)
  • NEXTFLEX® Primer Mix (12.5 µM)
  • NEXTFLEX® Methyl PCR Master Mix
  • Nuclease-free Water
  • Resuspension Buffer


  • 1 µg of fragmented genomic DNA in up to 40 µL nuclease-free water.
  • NEXTFLEX® DNA Barcodes – 6 / 12 / 24 / 48 (Cat # 514101, 514102, 514103, 514104) or NEXTFLEX-96 DNA Barcodes (Cat # 514106)
  • Methylated DNA Enrichment Kit
  • Methylated DNA IP Kit (Zymo Research Corp., Cat. # D5101) or
  • MethylMiner Methylated DNA Enrichment Kit (Life Technologies, Cat. # ME10025)
  • Ethanol 100% (room temperature)
  • Ethanol 80% (room temperature)
  • 96 well PCR Plate Non-skirted (Phenix Research, Cat # MPS-499) / or / similar
  • 96 well Library Storage and Pooling Plate (Fisher Scientific, Cat # AB-0765) / or / similar
  • Adhesive PCR Plate Seal (BioRad, Cat # MSB1001)
  • Agencourt AMPure XP 5 mL (Beckman Coulter Genomics, Cat # A63880)
  • Magnetic Stand -96 (Ambion, Cat # AM10027) / or / similar
  • Heat block
  • Thermocycler
  • 2, 10, 20, 200 and 1000 L pipettes / multichannel pipettes
  • Nuclease-free barrier pipette tips
  • Microcentrifuge
  • 1.5 mL nuclease-free microcentrifuge tubes
  • Low melt agarose such as Low Gelling Temperature Agarose with a melt point of 65ºC (Boston Bioproducts, Cat # P-730)
  • 1X TAE buffer
  • Clean razor or scalpel
  • SYBR Gold (Invitrogen, Cat # S11494)
  • UV transilluminator or gel documentation instrument
  • Gel electrophoresis apparatus
  • Electrophoresis power supply
  • Vortex

NEXTFLEX® Methyl-Seq 1 Kit Protocol – Option 1
Option 1 is for users who are interested in size selecting a specific range of DNA fragments post ligation with an agarose gel. Proceed to Option 2 for the gel-free protocol.

Methyl-Seq Flowchart Option 1

NEXTFLEX® Methyl-Seq 1 Kit Protocol – Option 2
Option 2 is a completely gel-free protocol that utilizes a magnetic bead based cleanup to size select DNA insert fragments between 300 – 400 bp.

Methyl-Seq Flowchart Option 1

Sequence Comparison between Differentiated and Non-differentiated Cells

Methyl-Seq Sequence Comparison

DNA Methylome, Genome Scale

Methyl-Seq dna methylome genome scale

This figure shows that on a genome scale, DNA methylomes of human embryonic stem cells and iPSCs transformed from a human dermal fibroblast cell are similar to one another and highly distinct from the primary somatic cell lines.

Frequency of DNA Methylation at Both CG and Non-CG sites Suggest iPSCs Resemble ES and are Distinct from Somatic Cells

Methyl-Seq graph

This bar graph shows that high sequence coverage of four DNA methylomes allowed interrogation of 87-94% of genomic methylated cytosines using both MeDIP and MeCAP enrichment. Pluripotent cell coverage is significantly different from somatic cell lines using both enrichment methods.

Papers that Cite the Use of the NEXTFLEX® Methyl-Seq Kit

  • Martínez Pinto, J. (2014) Neonatal exposure to estradiol reprograms the expression of androgen receptor and anti-müllerian hormone [recurso electrónico]: short and long term effects and their relation to the polycystic ovary phenotype. Disponible en
  • Winans, B., et al. (2015) Linking the Aryl Hydrocarbon Receptor with Altered DNA Methylation Patterns and Developmentally Induced Aberrant Antiviral CD8+ T Cell Responses. The Journal of Immunology. 194:9, 4446-4457.



What applications can the NEXTFLEX® Methyl-Seq Kit be used for?
The NEXTFLEX® Methyl-Seq Kit can be used for either methylated DNA immunoprecipitation (MeDIP) sequencing or affinity purification using a methyl specific capture protein (MeCap) sequencing on any Illumina® sequencing platform. These applications are designed to assess the methylation status of CpG rich regions in the genome (CpG islands). They offer global profiling of the methylome and are used to compare methylation patterns in different cell types. As only the rich methylated regions of the genome are sequenced, the amount of sequencing and coverage required is reduced in comparison to bisulfite sequencing applications.

How much input DNA can be used with the NEXTFLEX® Methyl-Seq Kit?
1 µg of fragmented genomic DNA can be used in up to 40 µL nuclease-free water.

How many samples can be multiplexed using the NEXTFLEX® Methyl-Seq Kit? Are the indexed adapters available separately?
Up to 192 libraries can be multiplexed using the NEXTFLEX® Methyl-Seq Kit and barcodes, which are provided separately. The NEXTFLEX® DNA Barcodes are recommended for use with the NEXTFLEX® Methyl-Seq Kit. These barcodes are not methylated. When performing MeDIP or MeCap library preparation, it is essential to use un-methylated barcodes. To offer greater flexibility, these indexed adapters are sold separately from the library preparation kits, in sets of 6, 12, 24 or 48 NEXTFLEX® DNA Barcodes. For additional multiplexing capabilities, the NEXTFLEX-96 DNA Barcodes or the NEXTFLEX-HT Barcodes™ can be used, which allow up to 384 samples to be multiplexed.

What kits do you recommend for methylated DNA enrichment?
We recommend either the Methylated DNA IP Kit (Cat. # D5101) from Zymo Research or the MethylMiner Methylated DNA Enrichment Kit (Cat. # ME10025) from Life Technologies.

The NEXTFLEX® Methyl-Seq 1 Kits contain enough material to prepare 8 or 48 genomic DNA samples for Illumina® sequencing. The shelf life of all reagents is 12 months when stored properly. DNA Binding Buffer and DNA Wash Buffer should be stored at room temperature. All of the other components can be safely stored at -20°C.

  • NEXTFLEX® DNA Barcodes
  • NEXTFLEX-96 DNA Barcodes
  • NEXTFLEX-HT Barcodes


  • NEXTFLEX® Bisulfite-Seq Kit