myBaits – Hyb Capture Kits
myBaits Custom Methyl-Seq
Custom kits for efficient, rapid methylation sequencing of your targets of interest, featuring Daicel Arbor’s optimized probe design and protocol.

myBaits Custom Methyl Sequencing

With this new release, Daicel Arbor Biosciences pairs an innovative methyl-seq probe design algorithm with a highly optimized target capture protocol to efficiently enrich target genes and markers of interest from converted methyl-seq NGS libraries. The hybridization capture technique significantly reduces per-sample sequencing costs compared to whole-genome bisulfite sequencing (WGBS) while preserving the essential strand-specific methylation signals.

myBaits Custom Methyl-Seq kits can be paired with either bisulfite or enzymatic-conversion workflows upstream of NGS library preparation, for seamless integration into your existing methyl-seq pipeline. All kits include the latest “v5” generation of myBaits hybridization and wash reagents, pairing best-ever performance with an easy-to-use protocol.

If a complete solution is needed, from sample preparation to data delivery, our myReads services team is available to handle projects of any size.

  • Unique probe design algorithm for enriching methyl-seq NGS libraries
  • Optimized high-performance hyb capture reagents and protocol
  • Scaleable to any project size
  • Dedicated support from expert scientists
Learn More

Contact our experts today to start your next myBaits Custom Methyl-Seq project.

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What is Hybridization Capture for Methylation Sequencing?

Next-generation sequencing (NGS) is a powerful method which facilitates rapid deep sequencing of DNA samples. However, within a whole genome, the actual sequence regions relevant for a given research project are of low relative abundance, which would necessitate deep shotgun sequencing to accurately detect and reconstruct them. Therefore a targeted approach NGS to increase read coverage just in genomic regions of interest is highly desirable to improve cost-effectiveness and allow for many more samples to be assayed. There are multiple NGS techniques that help to reduce per-sample sequencing costs, but only hybridization capture of NGS library molecules using biotinylated probes provides the best overall balance of ease, efficiency, and cost-effectiveness. By designing a myBaits Custom kit for your next targeted sequencing project, you can bring down the cost per sample AND increase the accuracy and power of your data analysis. Highly effective myBaits hyb capture probes can be designed to enrich a target space as small as a single locus, or as large as many tens of thousands of loci.

How does myBaits Custom Methyl-Seq work?

PREPARATION OF METHYL-SEQ NGS LIBRARY – Genomic dsDNA contains stand-specific patterns of methylated cytosines. In methylation next-generation sequencing, this signal is preserved for NGS by denaturing the two original strands, and separately deaminating any non-methylated cytosines into uracils via bisulfite conversion. This uracils are ultimately transformed into thymines after the original strand is copied by polymerases via amplification. These regenerated dsDNA molecules, with their strand-specific sequences retained, can be converted into NGS libraries, which can be either directly shotgun sequenced, or taken through a hybridization capture procedure.
HYBRIDIZATION CAPTURE – An NGS library is denatured via heat, and allowed to hybridize to a complex mixture of complementary biotinylated RNA baits over the course of several hours. Adapter-specific blocking oligos prevent random annealing of library molecules at the common adapter sites. After the hybridization is complete, the biotin present on each bait is bound to a streptavidin-coated magnetic bead. Wash steps help remove off-target or poorly-hybridized library molecules. The remaining library molecules that are still bound to their complementary baits are denatured via heat, and amplified using universal library primers. This “enriched” library can now be sequenced.

Advantages of Hyb Cap Technology

There are many advantages to the hybridization capture approach, which have made it a workhouse technique for both routine and complex workflows for modern NGS research. While hybridization capture reagents are available from multiple vendors, only myBaits® Custom kits from Daicel Arbor Biosciences offer the optimal balance of flexibility and performance. Our proprietary oligo synthesis platform allows us to manufacture fully customized probes at any scale and level of complexity, making them suitable for any project type and budget. We are experts in the design of hyb capture probes, and our team of scientist project advisers have years of experience with an enormous variety of sample types and project goals, such as ancient DNA, metagenomics, microbiology, phylogenetics, environmental DNA, pathogen sequencing, RNA-seq, and more. And if our free probe design services and easy-to-use kits are not enough, you can even outsource your entire enrichment project to our in-house team to handle for you. Whatever the targeted sequencing need, Daicel Arbor Biosciences has the right solution for you.

  • Straightforward procedure fits in between NGS library prep and sequencing
  • Simple to design custom probes with Daicel Arbor’s complimentary service
  • myBaits Custom kits contain all reagents necessary to perform protocol
  • No special equipment or training necessary to perform

All myBaits kits are for research-use only, and are not validated for diagnostic or therapeutic purposes.


myBaits Custom Methyl-Seq Performance

DNA methylation occurs at cytosines within CpG and other dinucleotides and is a primary epigenetic mechanism controlling a variety of biological processes, especially transcriptional regulation of cell development and various diseases, including cancer. Whole genome bisulfite sequencing (WGBS) has become the gold standard in measuring methylation status genome-wide, as it enables methylation signatures to be discovered in a wide range of sample types. Once regions of specific interest are identified and require characterization for large sample sets, WGBS becomes a relatively expensive option because of its comprehensive nature. Fortunately, hybridization capture, which can target thousands to millions of bases of specific genomic sequence in a single reaction, offers an extremely cost-effective solution that enables deeper sequencing and larger studies using a greater number of samples.

Download App Note
High specificity and orders of magnitude target enrichment. Performance metrics: (A) percentage of on-target reads in enriched and WGBS libraries, (B) average per-site depth of coverage at on-target CpG sites at an even subsample of 950K read-pairs in the enriched libraries, (C) percentage of on-target CpGs with read depth ≥5 at the even subsample in the enriched libraries. Libraries were prepared with the Swift Biosciences Accel-NGS Methyl-Seq Library Kit with varying input amounts and captured with the Daicel Arbor Biosciences myBaits Custom Methyl-Seq system targeting putative promoter regions of 50 cancer-associated genes (total target size ~100Kb).
High sensitivity and fidelity of targeted methylation detection at any methylation levels. (A) target-wide methylation levels in pre- and post-capture libraries. Red lines indicate the simulated level. (B) IGV screenshot showing the uniform distribution of methylation levels along the gene STK12 promoter locus observed with the capture libraries at various methylation levels.
High accuracy and reproducibility of targeted methylation capture. (A) Histograms and scatter plots show the comparison of CpG methylation levels across the target regions between WGBS and capture libraries and among replicates. (B) Circos plot illustrating methylation patterns for the entire target regions in capture and WGBS libraries.

myBaits Custom Methyl-Seq Kit Protocol

Current Version:

myBaits Custom Methyl-Seq Manual v1.52
For kits shipped starting in Sept 2020, with Cat#’s ending in “.v5”

Visit the myBaits Manual archive page to download other manual versions.


Product Sheet

myBaits Custom Methyl-Seq

Application Notes

Accurate, Customizable, Cost-Effective Targeted Methylation Sequencing – with Swift Biosciences

Safety Data Sheet

myBaits Kit SDS – US Version – v5 Chemistry – Sept 2020

myBaits Kit SDS – EU Version – v5 Chemistry – Sept 2020


Frequently Asked Questions – myBaits®

What is hybridization capture?

Hybridization capture is integrated into the overall NGS workflow immediately before sequencing on an NGS platform, such as Illumina. A fully sequenceable, barcoded/indexed NGS library (or pool of multiple libraries) is denatured, and allowed to anneal to complementary target-specific biotinylated probes/baits. These bait:library complexes are then bound to streptavidin-coated magnetic beads via the biotin on the probes, which are washed to remove non-specifically bound molecules. The remaining “enriched” library molecules are then released from the baits and amplified before sequencing.

Note! You may know the “hybridization capture” technique by another name, such as:

  • Target enrichment
  • Target capture
  • Probe capture
  • Exon capture
  • Capture sequencing / sequence capture
  • Hybridization sequencing / hyb-seq
  • Hybridization capture / hyb-cap

What targets should I include in my baitset?

We are pleased to provide as much bait design advice and assistance as possible. However we are unlikely to be sufficiently knowledgeable in your particular field as to help you pick the specific genes/targets for your project. Whether this is your first NGS project and/or you are an experienced genetics researcher, we always recommend that you choose your targets in collaboration with your full research team, especially your bioinformatician(s), so that your kit design is as robust as possible.

Some general suggestions appropriate for many projects would be to exhaustively survey the literature for your organism(s), and consider including neutral and/or control loci in addition to specific targets of interest. You should include enough loci and/or SNPs to draw significant conclusions within the number of specimens that you plan to survey. You should make sure that you have thoroughly evaluated your bait design before proceeding with your kit order.

If you are beginning a completely new project, you may wish to order the smallest number of reactions upfront, and place a re-order for a larger number of reactions once you have tested the design (special reorder pricing available). However just note that any changes (add/drop baits) to your design would need to be manufactured as a new custom kit, which has longer delivery times than a reorder of a previous design.

How do I submit my sequences for bait design?

Please gather your target sequences in FASTA format or as genomic coordinates according to our guidelines, and contact us with the details of your project to obtain a Quote. Please let us know upfront if there is a specific kit size in which your design should be constrained (e.g. a myBaits-3 kit with up to 60,000 probes) so that together we can adjust your bait design accordingly. Otherwise, we can let you know the estimated price of your design after processing your sequences.

What is the estimated turnaround time for kits?

For new baitset designs, the estimated manufacturing lead time is ~4-8 weeks minimum, starting from when your order is received and you have approved the final design. In addition, please consider that if you utilize our included bait design services, we will typically be in correspondence for an additional upfront period (up to several weeks) regarding a design before manufacturing can begin. In addition, please remember to accommodate any additional time for your collaborators to approve the final design, if applicable.

For reorders of previously manufactured bait designs, the estimated manufacturing lead time is ~1-2 weeks from the time an order is received.

Can I pool multiple samples in a single reaction?

Capturing individual libraries produces the best per-sample results. However, multiple dual-indexed libraries can be pooled into single capture reactions (i.e. ‘multiplexing’) in order to assay more samples with a smaller kit. For new baitsets, we strongly recommend first performing trial captures with different pooling schemes to determine what works best for your particular samples and bait set. When pooling libraries that vary in relative target content (e.g., ancient, forensic, or environmental samples), try to equilibrate by observed or expected target molarity, rather than by total library molarity.

We generally do NOT recommend pooling multiple samples per capture reaction for very degraded and/or rare targets (e.g. ancient DNA), or for very large targets (e.g. a WGE baitset targeting an entire genome). When working with ancient DNA specimens and small targets (e.g. mitochondrial DNA), consider diluting your probes and performing separate captures, rather than pooling multiple samples into a single capture reaction.

Can I input less than 100-500ng per capture reaction?

Target capture necessarily requires subjecting your libraries to a bottleneck, wherein target molecules are captured and therefore enriched, and non-target molecules are therefore removed. To have sufficient unique molecules for good sequencing coverage of your targets, successful captures DEPEND on the input of sufficiently complex libraries. For this reason, we recommend an input DNA amount of 100-500ng into each capture reaction, as amounts in this range typically perform very well. However myBaits can be used with as few as 1 ng and as many as 2 µg of library. For libraries with a significant non-target component (e.g., ancient, forensic, or environmental samples), maximize the target component in each capture by using as much library as possible up to 2 µg, and consider two rounds of capture for higher percentage of reads on-target.

For best results, it is recommended that only amplified (non-PCR-free) NGS libraries are used for target capture. This provides multiple copies of each starting template molecule, increasing the chance of each individual molecule getting enriched. However if you need more starting material to reach the recommended amount, it is generally preferable to generate more library from fresh genomic DNA or a new batch of indexed library, rather than through extra amplification. This is because while some amplification is good, over-amplification risks reducing the observable complexity of your libraries through the uneven action of PCR bias, as some molecules will become relatively more abundant while others become rare. This is also true for manipulating your libraries after capture: amplify your post-capture libraries the minimum number of cycles necessary to reach the molarity required by your sequencing facility.

What should I do about ambiguous bases in my sequences?

We cannot synthesize mixed bases, only A/T/C/G bases. If there are ambiguous bases (except for ‘N’) in your sequences, we will replace them with a single candidate base (e.g. C or T for ‘Y’) before bait synthesis.

If there are ‘N’ bases in your sequences, we will skip over these ambiguities by default when we design baits. If you do not want this, you should replace them with putative sequence according to your best knowledge — for example, fill in those positions with ‘best guess’ sequence from another allelic or species variant, if possible. If this is not possible, then you can instruct us to replace them with a filler base (T) prior to bait design. Using filler bases is appropriate for singleton or a couple of Ns, however long stretches of unknown sequence should just be omitted outright because they do not make any useful contribution to your bait design.

How should I handle known/unknown exon boundaries in my transcriptome sequences?

If you are using transcriptome sequences for your bait design, you may or may not know the location of the exon boundaries. However, this is not necessarily a problem for bait design, since we will typically design overlapping baits tiled across the full sequence. Any baits spanning across exon boundaries may not work well, but they will have neighboring baits which will still function. However any short exons (below the bait length) may not be recoverable unless they can be “padded” with true genomic (intronic) sequence.
Should I include more than one variant for a given candidate locus in my design?

Your decision whether to include >1 bait variant to represent additional diversity for a given region should depend on (1) the amount of diversity you want to have the ability to capture and (2) the maximum number of unique probe sequences that you want to purchase.

The ability of a given bait to hybridize to a target sequence will necessarily be dependent on the hybridization & washing conditions that you choose. Under the standard capture conditions, it is generally expected that a bait should be able to capture sequences of at least 5-10% local nucleotide divergence. Therefore, for example, it is normally NOT considered necessary to include probes for both allelic variants of a singleton SNP in a bait design, since a single bait should be able to capture both. However if you have many SNPs within a small window, you may wish to include >1 representative haplotype within your baitset. Please note that we cannot synthesize ambiguities.

That being said, the specific performance outcome of YOUR custom baitset, samples, and experimental conditions cannot be directly predicted. Target capture parameters such as sequence content of your custom baitset (e.g. GC% range, secondary structure), bait-to-bait interactions, and hybridization & washing performance will all impact capture outcomes. Additional experimental factors such as library quality, amplification parameters, multiplexing level, and sequencing depth will also be of critical relevance.

Can I re-order a previous custom kit design?

Yes! As long as we receive written permission from the original designer(s) (if it is not your kit and the bait sequences are not publicly available), you can re-order any past design that we have manufactured. We can usually provide such re-orders within ~1-2 weeks of ordering.
Can I purchase a discounted kit for less than 20K probes and/or less than 16 reactions?

Any design requiring from 1 to 20,000 probes falls into our smallest myBaits Custom tier baitset scale, therefore there is no reduced price for ordering smaller probesets. However please note that probes for all custom designs are provided at the same concentration, regardless of the number of individual baits in the design.
16 reactions is now our minimum custom kit size (for both new and repeat orders). We cannot provide discounted kits for fewer reactions.
What is included in the myBaits kit?

  • Probe design assistance (optional): dividing sequences into probes and removing non-specific baits (if you wish)
  • Biotinylated RNA probes according to your custom design
  • Hybridization and wash reagents

You will receive enough probes and reagents for performing the stated number of individual capture reactions of your kit size (e.g., 16 reactions) according to our current protocol. Please note that there are some additional reagents and equipment you will need to supply in order to perform a myBaits capture. Please review the list of required materials in the myBaits Manual to make sure you have everything you need before using your kit.

If you are looking for prices for in-house laboratory services (library preparation, target capture, and NGS sequencing), please visit our myReads page for more information.

What is the difference between a probe and bait?

In this context, we use the terms interchangeably. Some fields prefer one term over the other, so we use both terms.
What NGS library prep kit should I use?

myBaits kits are compatible with almost all major NGS library preparation kits on the market for most major types of sequencing platforms, such as Illumina®, Ion Torrent®, and Roche 454®. Contact us to ask about how to perform myBaits capture with other types of NGS platforms (e.g. PacBio®, Oxford Nanopore®).

If you are using a never-before-tried library prep protocol with your myBaits kit (especially if you are new to NGS), we strongly recommend that you first perform some total library (shotgun) sequencing before doing myBaits enrichment. This is important in order to verify that your chosen library prep kit generates libraries of sufficient complexity and minimal bias in your hands, otherwise you will experience poor target capture results. High quality libraries are absolutely essential for achieving a successful target capture project.

Provided below are links to companies that sell NGS library prep kits that are known to be compatible with myBaits. This is NOT an exhaustive list; there are many other unlisted kits that are also compatible with myBaits. Also, kits on this list may not necessarily be appropriate for your samples. NGS library prep is not “one size fits all”; different factors such as sample type, DNA input amount, genome complexity, and sequence composition may influence the type of library prep kit that would be best for your application. For example, low input, degraded, and/or damaged DNA templates may require special handling (see below) and/or modifications to commercial kits.

Contact these and other manufacturers to learn about your options and find what works best for your samples and project needs:

  • Illumina
  • New England Biolabs
  • Kapa Biosystems
  • PerkinElmer / Bioo Scientific
  • Rubicon Genomics / Takara
  • Swift Biosciences
  • Biosearch / Lucigen

Modified protocols for lower-cost library preps:

  • TC Glenn et al. 2016. “Adapterama I: universal stubs and primers for 384 unique dual-indexed or 147,456 combinatorially-indexed Illumina libraries (iTru & iNext)”. PeerJ,
  • N Rohland, D Reich. 2012. “Cost-effective, high-throughput DNA sequencing libraries for multiplexed target capture”. Genome Research, doi: 10.1101/gr.128124.111
  • Recommended especially for degraded/ancient DNA (blunt-ended library prep):
    • M Meyer, M Kircher. 2010. “Illumina Sequencing Library Preparation for Highly Multiplexed Target Capture and Sequencing”. Cold Spring Harbor Protocols, doi:10.1101/pdb.prot5448
    • M Kircher, S Sawyer, M Meyer. 2012. “Double indexing overcomes inaccuracies in multiplex sequencing on the Illumina platform.” Nucleic Acids Research 40(1): e3, doi: 10.1093/nar/gkr771

Do blocking oligos provided with the kit work with any NGS library?

One important component of your myBaits kit are the adapter-specific blocking oligos (reagent “Block A”). The type of Block A that is provided to you varies depending on the specific adapter and indexing structure of NGS library that you are using (e.g. Illumina TruSeq-style libraries with dual 8bp barcodes vs. single 6bp barcodes vs. Nextera-style with dual 8bp barcodes). In order for us to provide the correct Block A option(s), it is critical that you accurately report which NGS library preparation system you plan to use with your myBaits kits. If you are not certain, or later decide to change your library prep kit, please contact us ASAP so we can instruct you on how to obtain the correct blocking oligos.
What sequencing coverage can I expect from myBaits?

We are unable to offer any guarantees for downstream sequencing performance. This is because it is not possible to predict the actual sequencing behavior of new baitsets (including parameters such as on-target percentage, unique read depth, and evenness of coverage) without experimental test data, and knowledge of your experimental parameters. Factors such as the overall size and GC content of the bait sequences, the sequence divergence between baits and targets, the quality of your NGS libraries, and the sequencing depth that you use will have a major impact on your post-enrichment outcomes.

In practice, myBaits kits have frequently achieved high on-target percentages. But the outcome of your experiment will be necessarily dependent on the properties of the baitset, your samples, and your chosen experimental conditions.

If sequencing efficiency is critical to your project, best practice for optimizing new target capture designs is to perform a pilot test to determine the behavior of the baitset under your chosen conditions and with your samples, and adjust parameters such as sequencing depth, hybridization stringency, or number of capture rounds accordingly. For example, to maximize your on-target percentage, you could consider making upfront protocol adjustments such as performing two consecutive rounds of capture, as long as you are working with sufficiently high-quality, complex libraries.

What hybridization protocol should I use?

We provide most of the hybridization reagents and a specific protocol to use with the kits. Please see the latest myBaits Manual for the current protocol. Here you will find the list of materials (reagents and equipment) that you will need to supply in order to perform the captures.

Alternatively, experienced users may wish to supply their own reagents in order to use the myBaits probes in a custom hybridization protocol. Please verify that your user-supplied hybridization protocol is appropriate for biotinylated RNA probe oligos. Please be aware that we cannot offer technical support for experiments that deviate from our recommended protocol and/or supplied reagents.

My target molecules are short or rare (e.g. ancient DNA). Should I modify the protocol?

Hybridization and Wash Temperature – For most applications, we recommend 65°C for hybridization, bead-bait binding, and wash temperatures. However for samples where a majority of targets are shorter than the baits (i.e., from degraded DNA sources such as aDNA specimens), we recommend ~55°C for all three steps for improved captured target complexity, or ~60°C-65°C if higher on-target percentage is the priority. However, please note that optimal temperatures WILL vary by bait set and library, and will require trials to identify.

Hybridization Time – For most applications, hybridize for 16 to 24 hours. For very rare targets (e.g., those in ancient, forensic, or environmental samples) hybridize for 24 to 40 hours. Shorter and longer times can be tolerated, though will require trials to identify optimal performance. Ensure that the chosen combination of tubes and thermal cycler allows no more than 15% volume evaporation over the chosen time and temperature.

One or Two Rounds of Capture – For rare targets, the improvement in specificity (raw reads mapping to your targets) may not be high enough after a single round of target capture for sufficient unique coverage at your chosen sequencing depth. For example, even a 100× enrichment rate will only increase the specificity of a 0.1% pre-enrichment target (for example, 1K target reads among 1M total shotgun reads) to 10% post-enrichment. In such cases, performing two consecutive rounds of target capture might be the most efficient route to boost your sequencing specificity. However, there will always be a loss in sensitivity (unique read complexity) following a second round of capture; depending on your starting complexity, this sensitivity loss may or may not be tolerable in your experimental design.

Do you offer services for target capture projects?

Yes! Our expert myReads team provides a range of in-house NGS services for custom projects, including library preparation, target capture with myBaits, and high-throughput sequencing. Visit the myReads product page to learn more about our service options and pricing.
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