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 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 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 (available for lower pricing) for a larger number of reactions once you have tested the design. 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 (e.g. “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, 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.

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 library 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, 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 RNA/cDNA, 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 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® and Ion Torrent®. 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 sequencing before performing 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/RNA/cDNA 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

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. This will determine which adapter blocker (Block X) component will be included. There are two options:

Block Xt – for TruSeq-style libraries with single or dual 6-8 bp indexing
Block Xn – for Nextera-style libraries with dual 8bp indexes

For different adapter configurations than those described above, we recommend 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?

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. degraded DNA or RNA). 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 or RNA sources), 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.

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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