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
- Biotinylated RNA probes according to your approved custom design
- Hybridization and wash reagents
- For myBaits Custom kits, optional custom probe design informatics service (designing and removing non-specific baits; report)
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).
All 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 applicable myBaits manual for detailed protocol instructions for enriching from Standard, High-Sensitivity, Long-Insert, or other specialty target/sample types.
The applicable 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.
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!
For new myBaits Custom 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 myBaits Expert (catalog) kits or reorders of myBaits Custom kits with designs previously manufactured by Daicel Arbor Biosciences, the estimated manufacturing lead time is ~1-2 weeks from the time an order is received.
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 applicable for your chosen kit.
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.
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.
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.
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.
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.
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.
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.
In this context, we use the terms interchangeably. Some fields prefer one term over the other, so we use both terms.
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
- New England Biolabs
- Kapa Biosystems
- PerkinElmer / Bioo Scientific
- Rubicon Genomics / Takara
- Swift Biosciences
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
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.
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.
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 techsupport_at_arbor.daicel.com or reach out to your most recent contact person for assistance.
Generally we recommend probe densities between 3-10 probes per kilobase for target regions larger than 50kb. For target regions between 10-50kb, probe densities should be on the higher end of that range, and we may recommend using multiple fluorophores per probe to boost the signal.
We offer complementary FISH probe design service using our proprietary design algorithm for most types of FISH projects. Please contact us with a brief description of your project, including the name of your study species, genomic coordinates, and any additional information.
We can work with any sequence to design probes. Please contact us with a brief description of your project, including the name of your study species, genomic coordinates, and any additional information.
The number of assays per library depends on a number of factors including the probe density of your library, the size of your target region, the number of probes in the library, and the FISH protocol. Generally we recommend starting with 10pmol of labeled probes per standard FISH slide and then modifying the input amount based on the initial results.
Atto-550 is our most popular dye labeling option, followed by Atto-488, Atto-594, and Atto-647N. Biotin, Digoxigenin and 6FAM are also popular options that work well in multi-color FISH assays.
We may be able to accommodate other labeling options, please contact us for availability.
myTags FISH libraries are compatible with most FISH protocols. Please contact us for recommendations.
We generally ask for up to 4-6 weeks after an order is placed to ship myTags libraries.
Yes, we provide the myTags labeling protocol with all myTags immortal libraries for perfroming the labeling process in your own lab. If you would like to use a different protocol, one of our scientists would be happy to provide assistance to ensure success.
Generally we recommend probe densities between 3-10 probes per kilobase for target regions larger than 50kb. For target regions between 10-50kb, probe densities should be on the higher end of that range, and we may recommend using multiple fluorophores per probe to boost the signal.
We offer complementary FISH probe design service using our proprietary design algorithm for most types of FISH projects. Please contact us for design assistance
We can often accommodate customer-designed probes into the myTags labeling framework. Please contact us for recommendations on the design parameters and other information before designing your probe sequences.
Yes, we can synthesize immortal probe libraries that can be labeled using the Oligopaints labeling method. Please note these probe libraries are not compatible with the myTags labeling protocol due to sequence requirements of the Oligopaints method.
We can work with any sequenced data to design probes. Please contact us with a brief description of your project, including the name of your study species, genomic coordinates, and any additional information.
We generally supply myTags Labeled Libraries in 3-4 weeks and myTags Immortal Libraries in 2-3 weeks, after an order is placed.
Unfortunately, this may lead to considerably decreased performance or even loss of function. To ensure highest kit performance, make sure to store Sigma 70 Master Mix at -80 °C and freeze as soon as possible after usage.
You’re advised to keep the number of freeze-thaw-cycles to a minimum. Nevertheless, we found that up to five freeze-thaw-cycles do not negatively influence protein production efficiency of the Sigma 70 Master Mix.
Yes. The myTXTL® Sigma 70 Master Mix contains tRNAs for seven codons rarely used in E. coli to prevent undesired translation stop.
As the myTXTL® platform completely relies on the endogenous transcription and translation machinery of E. coli making use of the core RNA polymerase and the primary sigma factor 70 (σ70), all genes should be cloned downstream of a σ70-specific promoter, e.g. the promoter found in P70a vectors. For a more general advice on how to construct a functional gene cassette, please refer to the myTXTL® kit manual.
Efficient in vitro protein production is highly dependent on the quality of the template DNA, which should be free of nucleases (DNases, RNases) and inhibitors of the TXTL machinery (e.g. EDTA, ethidium bromide, SDS, Cl- ions, ethanol). Preparation of plasmid DNA with standard commercial kits usually involves sample treatment with RNase, which may not be completely removed during downstream processing. Thus, we strongly recommend subjecting the prepared DNA to either a commercial PCR clean-up kit or standard phenol-chloroform extraction and ethanol precipitation. Ideally, template DNA is suspended in nuclease-free water. Please note, introducing Mg2+and K+ ions can compromise the kit performance, as they are extremely critical for transcription and translation, and are optimized in the systems.
deGFP is a N- and C-terminally truncated version of the reporter eGFP that is more translatable in cell-free systems. The excitation and emission spectra as well as fluorescence properties of deGFP and eGFP are identical.
Yes. Due to the manufacturing process, there might be a small pellet visible. It`s critical that you resuspend the Sigma 70 Master Mix completely before aliquoting it to set up your TXTL reaction(s).
Yes! That only requires the addition of a plasmid coding for T7 RNA polymerase under transcriptional control of a σ70-specific promoter, e.g. P70a-T7rnap. This plasmid – along with hundreds of others – is part of a Toolbox 2.0 Plasmid Collection and can be purchased here (LINK). The optimum concentration of P70a-T7rnap is usually between 0.1 nM and 1 nM. Higher concentration normally does not increase protein yield. The more important parameter for efficient protein expression is the concentration of the plasmid that encodes for your protein of interest downstream of the T7 promoter, which will be most likely in the range of 5-20 nM.
Yes, although it`s not optimized for linear DNA templates. Considerably enhanced protein yields can be achieved by supplementing the Sigma 70 Master Mix with our nuclease inhibitor GamS (LINK).
Due to the small reaction volume of 12 μL, it is very important to avoid condensation of water on lid of the reaction tube, as it considerably increases the concentration of myTXTL® components. This can lead to an unreproducible kit performance. In general, water facilitates a faster heat transfer than air and a water bath shows low temperature fluctuation, which should – combined with a closed environment with constant temperature surrounding the entire tube – lead to higher reproducibility and yield.
Component inactivation due to improper storage. Sigma 70 Master Mix must be stored at -80 °C and number of freeze-thaw cycles should be minimized.
Contamination of myTXTL® reaction with nuclease. To avoid nuclease contamination, wear gloves and use nuclease-free water, sterilized tips and tubes.
Please review the recommendations to set up a myTXTL® reaction in the kit manual (LINK).
Yes! Parameters that influence protein production efficiency are:
Gene cassette construction (promoter strength, position of affinity tag, TXTL elements)
Incubation temperature and time
Presence of folding helpers, chaperones, oxidizing agents
and should therefore be evaluated for optimization. Please also see our recommendations on Template Design in the kit manual (LINK).
Consider if your recombinant protein requires co-factors like heavy metal ions or coenzymes to be functionally active. Those should be present during protein synthesis. Additionally, a low concentration of mild detergent (e.g. Triton-X-100, sodium dodecyl maltoside, or CHAPS) can be added to the reaction as well as molecular chaperones. Please note that the myTXTL® platform cannot introduce post-translational modifications like glycosylation or phosphorylation to your protein. Reducing the incubation temperature might help to prevent aggregation of the nascent polypeptide chain and to promote proper protein folding.
Unfortunately, not. However, studies have shown that supplementing cell-free systems with mixtures of reduced (GSH) and oxidized glutathione (GSSG), disulfide bond isomerase C (DsbC), protein disulfide isomerase (PDI) and/or chaperones (e.g. DnaK, DnaJ, GroEL, GroES) can promote the formation of disulfide bridges. In addition, pretreatment with iodoacetamide (IAM) to inactivate endogenous reductases which are present in the cell extract might also help (Review Article: Stech M & Kubick S, Antibodies 2015, 4, 12-33).
Batch-to-batch variation can be caused varying levels TXTL inhibitor contamination present in the plasmid solution. Please follow our recommendations on how prepare plasmid DNA as template for TXTL reactions which can be find in the kit manual (LINK).
Sample handling and storage is mainly determined by the stability of your molecule of interest (protein, DNA, RNA) and thus optimal conditions may need to be evaluated. But to ensure sample integrity, we would recommend to either process the myTXTL® reaction immediately after performing the incubation or store it at ≤ -20 °C.
Apart from standard biochemical methods like Coomassie-stained SDS-PAGE and western blot analysis, the great advantage of cell-free protein production is the open-system environment which allows the direct quantification and/or analysis of its functionality in an activity assay or the downstream processing via affinity purification (if an affinity tag is present). If you choose SDS-PAGE analysis, you can either take a small sample (1-3 µL) directly from your TXTL reaction, or – to reduce background signal – precipitate proteins with TCA/acetone or ammonium-acetate/methanol following a standard protocol.
Most importantly, the excitation and emission wavelength should match the fluorescence properties of deGFP/eGFP (e.g. λEm 488 nm, λEx 535 nm). Other reader settings such as reading mode, integration time and gain value should be chosen under consideration of high well-to-well fluorescence reading reproducibility.
No. All our Toolbox 2.0 plasmids (except the positive control plasmid P70a-deGFP that comes with the myTXTL® kit) are meant for plasmid amplification in E. coli only. The degree of purity is NOT sufficient for efficient in vitro production. Please refer to the current myTXTL® manual (LINK) for recommendations on preparation of plasmid templates for TXTL reactions.
Yes. For all plasmids containing the lambda phage promoter (P70a, P70b, P70c, P70d) it is extremely crucial to use E. coli KL740 as transformation strain. When cultivated below 30 °C, this strain over-expresses the lambda phage repressor protein Cl857, thus ensuring high transformation efficiency and plasmid stability. KL740 can be purchased from Coli Genetic Stock Center (Yale) [CGSC#: 4382]. For all other plasmids, a standard laboratory E. coli cloning strain like JM109 or DH5alpha is sufficient.
A standard protocol for chemical transformation usually produces E. coli cells with a sufficient competency for plasmid intake. We recommend following the procedure described in Sambrook et al. 1989.
Transformation efficiency extremely depends on the quality of the competent cells. Make sure that cells were immediately frozen after preparation and stored at ≤ 80 °C. Please also note that for some cells, transformation efficiency drops drastically over time. Additionally, we advise to use E. coli strain KL740 for amplification of any plasmids containing σ70-specific promoter like P70a.
All P70 promoters originate from the lambda phage promoter for the repressor Cro with its two operator sites and are specific to the E. coli sigma factor 70. They differ in strength (P70a > P70d > P70b > P70c) due to mutations that were introduced at -35 and/or -10 regions.
We do offer a comprehensive gene synthesis and cloning service (myDNA®) (LINK) that serves you with error-free DNA molecules at your convenience. Because we work with a modular part system, the cloning procedure will be straight forward with almost no limitations in combinatorial combinations at a very competitive pricing.
Yes. Please note, that every gene circuit should start with a σ70-specific promoter like P70a.