myBaits – Hyb Capture Kits
myBaits Custom
Custom target capture kits provide in-solution biotinylated RNA probes complementary to sequences of interest, which allow rapid, selective target enrichment for highly cost-effective next-generation sequencing (NGS).

In-solution probes allow for specific, yet flexible hybridization to complementary target molecules, which provides the ability to recover variable regions from taxonomically divergent species and strains. This allows capture probes to be successfully employed even on non-model organisms which lack a sequenced genome or transcriptome. myBaits kits have been successfully used in research projects from hundreds of genome types including animals, plants, and microbes and from fresh, degraded, and environmental DNA sources.

myBaits custom sequence capture kits utilization across a wide variety of organisms and applications make them the perfect choice for any targeted NGS project. Arbor Biosciences’ proprietary oligo synthesis platform allows flexible production of high-quality baitsets at extremely competitive pricing for any project size. The kits are perfect for new or expert users as the easy to follow protocol delivers consistently reproducible results. Our team of expert scientists will assist with a streamlined complimentary bait design process and provide experimental design advice as needed in order to deliver a successful project. If a complete solution is needed, from sample preparation to data delivery, our myReads services team is available to handle projects of any size.

  • Affordable and Scaleable -Kit sizes available for any size project
  • Easy Design – Design your probes from any nucleic acid source – genomes, transcripts, SNPs, etc.
  • Scientific Expertise – Free bait and project design assistance from our team of expert scientists
  • Convenient Kits – Each order includes probes and hybridization/washing reagents
Ordering Information
Size
Cat#
Qty
Price
myBaits Custom 1-20K
Kit for enrichment of custom target sequences, for designs with up to 20K unique probes.
300116
$3,600
myBaits Custom 20-40K
Kit for enrichment of custom target sequences, for designs with up to 40K unique probes.
300216
$4,850
myBaits Custom 40-60K
Kit for enrichment of custom target sequences, for designs with up to 60K unique probes.
300316
$6,100
myBaits Custom 60-80K
Kit for enrichment of custom target sequences, for designs with up to 80K unique probes.
300416
$7,350
myBaits Custom 80-100K
Kit for enrichment of custom target sequences, for designs with up to 100K unique probes.
300516
$8,600
myBaits Custom 100-120K
Kit for enrichment of custom target sequences, for designs with up to 120K unique probes.
300616
$10,100
myBaits Custom 120-140K
Kit for enrichment of custom target sequences, for designs with up to 140K unique probes.
300716
$11,600
myBaits Custom 140-160K
Kit for enrichment of custom target sequences, for designs with up to 160K unique probes.
300816
$13,100
myBaits Custom 160-180K
Kit for enrichment of custom target sequences, for designs with up to 180K unique probes.
300916
$14,600
myBaits Custom 180-200K
Kit for enrichment of custom target sequences, for designs with up to 200K unique probes.
301016
$16,100
New custom panels typically take 4-6 weeks minimum to produce, in addition to the upfront design consultation.
Larger panels available upon request.


Overview

What is Hybridization Capture with Custom Probes?

Next-generation sequencing (NGS) is a powerful method which facilitates rapid deep sequencing of DNA or RNA samples. However, within a whole genome or metagenome, the actual sequence regions relevant for a given research project are typically 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 many NGS techniques that help to reduce per-sample sequencing costs (such as single- or multi-plex PCR amplicons, GBS/RAD-seq, etc), 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 as small single locus for a single organism, or as broad as tens of thousands of loci from an entire taxonomic group.


Figure 1. How does myBaits work?
(1) HYBRIDIZATION – 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.
(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 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 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, 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

Figure 2. Hybridization capture tolerates both rearrangements and sequence variation.
Both hyb cap and amplicon sequencing can effectively enrich for known sequence regions. However, only hyb cap can retrieve target sequences that have significant rearrangements and/or mutations relative to the reference used for probe design, for example when capturing genomic content from novel microbial genomes.


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

Performance

Understanding myBaits Custom 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 across a variety of applications. We are always available to provide specialized experimental design advice to maximize success with your next NGS targeted sequencing project.


Figure 1. Typical example visualizing NGS reads from enriched human gDNA library. Single-end NGS reads are shown aligned to the hg38 genomic reference sequence. Positions of the four 80nt myBaits probes in this region are indicated by blue bars. Unique read coverage is highest across the baited region, and tapers off upstream and downstream of the baited region. If sequencing further into the known or unknown flanking regions is desired, simply increase the length of your NGS library molecules (compatible with any myBaits kit).

Resources

myBaits Kit Protocol

Current Version: v4
myBaits Manual v4
Visit the myBaits Manual archive page to download past versions.

 

Safety Data Sheet

myBaits Kit SDS
FAQs

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. 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 new kits? Are rush orders available?

The bait manufacture turnaround time varies depending on the specific baitset and the number of other kits in our queue, but it is typically ~4-8 weeks minimum for your baits to pass our QC process. Our turnaround time is necessary so that we can carefully manufacture your custom probes according to our high-quality standards. Please do NOT wait until the last minute to place your order, as we cannot rush orders. We are continually working to reduce manufacturing turnaround times.

Also, please consider that if you choose to utilize our complementary bait design services, we will typically be in correspondence for an additional upfront period (up to several weeks) regarding a design before we can proceed to the bait manufacturing (see above). This can be skipped if you provide us with pre-designed probes that do not require any additional design work. Please remember to accommodate additional time for your collaborators to approve the final design.

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 (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 <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), you can re-order any past design that we have manufactured. We can usually provide such re-orders within ~2 weeks of ordering. Please no rush orders.
Can I purchase a discounted kit for <20K probes and/or <16 reactions?

Any design requiring from 1 to 20,000 probes falls into our smallest “myBaits-1” 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, https://peerj.com/articles/7755/
  • 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.
Publications

Featured Publications

The publications listed below highlight some recent examples of the use of myBaits® Custom hyb capture kits in various applications, including ancient DNA, virology, phylogenetics, agricultural genomics, RNA-seq, and more. A more comprehensive searchable database of hundreds of myBaits publications is available on our general 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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