myBaits – Hyb Capture Kits
myBaits Custom RNA-Seq
Custom target capture kits tailored for RNA-seq applications allow rapid, selective target enrichment for highly cost-effective next-generation sequencing (NGS).

myBaits Custom RNA Sequencing

Sequencing of total RNA in a sample (RNA-Seq) is a powerful NGS technique for directly assessing signals of gene expression in a sample. However even with widely-used methods of removing uninformative transcripts prior to total RNA-Seq (e.g. rRNA removal), total RNA-Seq samples are still highly complex, and typically require deep sequencing to fully resolve the signal of relatively rare transcripts of interest. With myBaits Custom RNA-Seq hybridization capture kits, it is easy to design a customized enrichment panel for just your genes of interest, which can then be used to “enrich” those targets from a complex NGS library prior to RNA-Seq. In-solution hybridization capture reduces per-sample sequencing costs by orders of magnitude while still preserving the essential gene expression signals.

Daicel Arbor Biosciences’ proprietary oligo synthesis platform allows flexible probe production with competitive pricing. We offer a broad range of panel sizes that can accommodate any size target from a single locus to tens of thousands of loci, as well as an assortment of kit reaction sizes to accommodate any number of samples. Our easy protocol delivers consistently reproducible results, and our team of expert scientists will assist you with a complimentary bait design and experimental design advice. If a complete solution is needed, from sample preparation to data delivery, our myReads services team is available to handle projects of any size.

  • Cost-effective – Save orders of magnitude in NGS costs per sample by sequencing only genes of interest
  • Affordable and Scalable – Kit sizes available for any size project
  • Scientific Expertise – Free bait and project design assistance from our team of expert scientists
  • Convenient Kits – Each order includes probes and hybridization/washing reagents
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Contact our experts today to start your next targeted NGS project with myBaits Custom RNA-Seq.

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What is Hybridization Capture with Custom Probes?

Next-generation sequencing (NGS) is a powerful method which facilitates rapid deep sequencing of RNA samples. However, within a whole or meta transcriptome sample, the transcripts specifically relevant for a given research project are often of low relative abundance, which would necessitate deep total RNA sequencing to accurately detect and reconstruct them. Therefore a targeted approach NGS to increase read coverage just for transcripts of interest is highly desirable to improve cost-effectiveness and allow for many more samples to be assayed. Hybridization capture of NGS RNA-Seq library molecules using biotinylated probes provides the best overall balance of ease, efficiency, and cost-effectiveness. By designing a myBaits Custom RNA-Seq kit for your next targeted RNA-Seq project, you can bring down the cost per sample AND increase the accuracy and power of your data analysis.

Your genes of interest can themselves be highly complex targets, as it is possible to target anywhere from dozens up to hundreds of thousands of different loci in a single myBaits Custom RNA-Seq kit. For example, enriching for all members of a certain gene family of interest, or enriching all genes from a given pathogen genome out of a complex host+pathogen RNA sample.

Figure 1. How does myBaits work?
(1) HYBRIDIZATION – A barcoded NGS cDNA library (or pool of libraries) 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.
(2) WASHING – 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.
(3) AMPLIFICATION – 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 (or pool of libraries) 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 from one or more reference sequences
  • Probe sequences do not have to be exact match to targets (>20+% divergence can be tolerated)
  • myBaits hyb cap kits contain all reagents necessary to perform protocol
  • No special equipment or training necessary to perform
  • Can use with any NGS platform, including both short- and long-read sequencing

High Accuracy and Reproducibility of myBaits cDNA RenSeq Enrichment
myBaits capture prior to RNA-Seq provides high accuracy and reproducibility of transcript abundance signals.
(A) A scatter plot of log2 read counts of NLR genes shows a high correlation between cDNA RenSeq and deep RNA-Seq libraries. (B) Hierarchical clustering of gene expression in different tissues. Data analyzed from Steuernagel et al (2018, BioRxiv). Read more about this study in our featured Application Note.

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


myBaits Custom RNA-Seq Performance

Hybridization capture with myBaits Custom is an extremely versatile and flexible technology that has been applied to an enormous variety of applications and taxa, such as plants, animals, humans, bacteria, and viruses. Hundreds of studies have been published with myBaits over the years. Across the wide variety of applications, myBaits Custom kits frequently achieve high on-target read percentages and/or high unique read complexity for most applications, creating many orders of magnitude savings in sequencing compared to shotgun approaches. In addition, our research scientists are continually innovating novel improvements to the speed, ease of use, and performance of our myBaits kits. We are always available to provide specialized experimental design advice to maximize success with your next NGS targeted sequencing project.

High Accuracy and Reproducibility of myBaits cDNA RenSeq Enrichment
Figure 1. High Accuracy and Reproducibility of myBaits cDNA RenSeq Enrichment. (A) A scatter plot of log2 read counts of NLR genes shows a high correlation between cDNA RenSeq and deep RNA-Seq libraries. (B) Hierarchical clustering of gene expression in different tissues. Data analyzed from Steuernagel et al (2018, BioRxiv). Read more about this study in our featured Application Note.

myBaits Enrichment of Rare Transcripts Enables High Coverage Sequencing
Figure 2. myBaits Enrichment of Rare Transcripts Enables High Coverage Sequencing. A high level of antisense RNA transcription of the Pcdhα alternate exons was revealed by myBaits Custom RNA-Seq capture with high confidence from increased coverage. Orange bar: myBaits for Pcdh α and γ clusters. Image based on Figure S1.E from Canzio et al (2019, Cell). Read more about this study in our featured Publication Note.

High Correlation and Reproducibility of myBaits Pathogen RNA-Seq Capture
Figure 3. High correlation and reproducibility of myBaits pathogen RNA-Seq capture. Scatter plots of log2 reads counts from three replicates show a high correlation between enriched and non-enriched (shotgun) pathogen transcript read counts. The reads were obtained from a sample of host RNA spiked with 0.3% pathogen RNA.

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 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 Standard, High-Sensitivity, and Long-Insert target/sample types.

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

The latest myBaits manual provides detailed protocol support for “High Sensitivity” type samples, including ancient DNA and other samples that are expected to have degraded/damaged target molecules. Please review this recommended protocol carefully to ensure that you purchase the correct amount of reagents required to perform your chosen protocol. For example, if you wish to do two rounds of enrichment, you may need to purchase additional sets of myBaits hybridization/capture reagents, which are available for purchase in 16, 48, or 96 Reaction sizes.
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?

Capturing individual libraries typically produces the best per-sample results. However, multiple dual-indexed libraries can be pooled into single capture reactions (e.g. “multiplexing”) in order to assay more samples per 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. Optimal pooling parameters (both in terms of number of libraries and total mass per library) will vary between library types and bait sets, and will require trials to identify. However, many configurations should work well.

Specific recommendations for library co-enrichment pooling for different project types 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 different enrichment project types 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.

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 with PCR-amplified and amplifiable NGS libraries, including Illumina TruSeq® -style, Illumina Nextera® Flex-style, Ion Torrent, or other libraries with universal adapter priming sites. It is NOT recommended to 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 for sequencing on short- and/or long-read platforms (e.g. PacBio® or Oxford Nanopore Technologies®).

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.

Provided below are a list of 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 options 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:

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

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 each of the protocol sections (Standard, High-Sensitivity, and Long-Insert). Please read through the relevant section first as it may answer your question. If you still have an issue, please contact us via email at or reach out to your most recent contact person for assistance.


Featured Publications

The publications listed below highlight some recent examples of the use of myBaits® Custom hyb capture kits for RNA-Seq target capture. A more comprehensive searchable database of hundreds of myBaits publications is available on our general Daicel Arbor Biosciences publications page. Thank you to all our new and returning clients for trusting myBaits for your innovative research projects!

For links to hundreds more myBaits publications, visit the publications page!

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