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

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Overview

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.

Performance

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.
Resources
FAQs

Frequently Asked Questions – myBaits®

What is hybridization capture for methylation sequencing?


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 is made from bisulfite or enzymatically-converted DNA. A pool of these multiple barcoded NGS libraries is then 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 is included in the myBaits kit?

  • Optional custom probe design informatics service (designing and removing non-specific baits; report)
  • Biotinylated RNA probes according to your approved 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 current myBaits manual to make sure you have everything you need before starting your experiments.

If you are looking to outsource your project to a full-service laboratory and bioinformatics services group, please visit our myReads page for more information about our comprehensive targeted sequencing service options (library preparation, target capture, next-generation sequencing, and optional analysis).

What hybridization protocol should I use?

myBaits Kits include a specific protocol for their use as well as almost all of the materials required to deploy them. In the manual, you will find the complete list of required supplies (reagents and equipment) that you will need in order to perform the captures.

Please see the latest myBaits manual for detailed protocol instructions for enriching from Methyl-Seq target/sample types.

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, high-throughput sequencing, and optional bioinformatics analysis. Visit the myReads page to learn more about our comprehensive laboratory and sequencing service options!
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. Please also remember to accommodate any additional time for your collaborators to approve the final design, if applicable.

For reorders of custom designs previously manufactured by Daicel Arbor Biosciences, 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?

Yes. Specific recommendations for library pooling for co-enrichment with myBaits Custom Methyl-Seq kits can be found in the current myBaits manual.
Can I input less than the recommended library mass per capture reaction?

Specific recommendations for per-library input mass for methyl-seq enrichment projects can be found in the current myBaits manual.

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 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.

How do I submit my sequences for bait design?

myBaits Custom Sequence Submission Guidelines

Please gather your target sequences in FASTA format or as genomic coordinates according to our guidelines, and contact us with details of your project. Our team will provide you with an estimated panel size as soon as possible based on your provided information. Please let us know upfront if there is a specific panel size in which your design should be constrained (e.g. not more than 60,000 probes) so that together we can adjust your design/estimate accordingly. Otherwise, our experts will determine the best size of panel based on your targets and project configuration.

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 reorder for a larger number of reactions once you have tested the design. However please note that any changes to your design (adding or removing baits) would be ordered as a fully new custom kit, which may have a longer delivery time than a reorder of a previous design.

What should I do about unknown or ambiguous bases in my sequences?

Singleton and/or short stretches of N’s will be replaced with T’s to facilitate bait design in these regions. Longer stretches (e.g 10+ N’s) will be skipped over during bait placement.

Ambiguities (e.g. Y/M/R/S/W/K) are allowed, but will be replaced by ONE random candidate base for manufacturing, since we only synthesize A/T/C/G bases (no mixed bases). The hybridization capture system tolerates multiple mismatches between probe:target molecules. However, sequences that contain on average >5-7% ambiguous bases are not recommended. If you are providing consensus sequence(s) generated from a common locus/gene source (e.g. the same gene from multiple genomes, or multiple alleles of a target gene), please provide the original individual sequences. Our informatics experts can remove redundant/similar regions during the design process to ensure all variants are sufficiently represented while minimizing overall unique bait count.

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?

The 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 or mixed bases; all non-A/T/C/G bases will be replaced by a random candidate base during manufacturing.

Can I reorder 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 has been manufactured by Daicel Arbor Biosciences. We can usually provide such re-orders within ~1-2 weeks of ordering.
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?

Use myBaits Custom Methyl-Seq with PCR-amplified and amplifiable NGS libraries generated from bisulfite- or enzymatically-converted nucleic acids in which non-methylated cytosines have been converted to uracils, and following PCR amplification these positions are now thymines. Compatible formats include Illumina® TruSeq®-style, Illumina Nextera® Flex-style, Ion Torrent®, or other libraries with universal adapter priming sites. Do NOT use myBaits with PCR-free libraries; additionally, myBaits are incompatible with libraries made using original Nextera or Nextera XT library preparation kits, or any library type containing biotin. Dual-indexed libraries are strongly recommended to reduce the hazard of mis-indexing induced by PCR jumping events. The current myBaits manual provides detailed protocol instructions for enriching libraries intended for methylation sequencing.

If you are using a never-before-tried library prep protocol to pair with your myBaits kit, we 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 protocol/kit generates libraries of sufficient complexity and minimal bias in your hands, otherwise you may experience poor target capture results. High quality libraries are absolutely essential for achieving a successful target capture project.

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

When ordering your myBaits kit, please indicate the sequencing library configuration you intend to enrich. The standard adapter blocking reagent provided with the kit (Block X) is compatible with Illumina® TruSeq®-style or Nextera®-style libraries with single 6-12 bp or dual 6-12 bp indexing. These options cover the vast majority of currently available commercial library preparation systems intended for sequencing on any Illumina platform.

For different adapter configurations than those described above, we recommend ordering Custom IDT® xGen® Blocking Oligos. At a concentration of 1 μg/μL, custom adapter-blocking oligos can be used in lieu of myBaits Block X.

If you are not certain, or later decide to change your library prep kit, please contact us so we can instruct you on how to obtain the correct blocking oligos.

What sequencing coverage can I expect from myBaits?

myBaits Custom kits have frequently achieved high on-target percentages for a wide range of applications. However since it is not possible to predict the behavior of new baitsets (e.g. on-target percentage, unique read depth, and evenness of coverage) without experimental test data, and knowledge of your experimental parameters, we are unable to provide specific predictions for downstream sequencing performance. 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 will also have significant impacts on post-enrichment outcomes.

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.

Can you help with some technical issues I have after performing the myBaits protocol?

The current myBaits manual covers several troubleshooting topics at the end of the protocol section. Please read through this section first as it may answer your question. If you still have an issue, please contact us via email at techsupport_at_arbor.daicel.com or reach out to your most recent contact person for assistance.

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