Frequently Asked Questions (FAQ)
Please review this FAQ list to see if your question has been addressed. If you cannot find an answer, please e-mail us or reach out to your current MYbaits contact directly.
How do I begin a project?
You should review the Project Planning page to review the parameters that you will need to choose for your design (namely, bait length & tiling density), and the Custom Kits page to understand the pricing structure of our kits. 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 parameters accordingly (use our Calculator to estimate the number of baits your design will require). Otherwise, we can let you know the estimated price of your design after processing your sequences.Guidelines for Sequence Submission
What is included in the kit price?
- Bait design assistance: dividing sequences into probes & removing non-specific baits (if you wish)
- Biotinylated RNA baits according to your custom design
- Hybridization reagents
You will receive enough probes & reagents for performing the stated number of individual capture reactions of your kit size (e.g., 12 reactions) according to our current protocol.
There are some additional reagents & equipment you will need to supply in order to perform a MYbaits capture. Please review the list of required materials in the 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, & NGS sequencing), please visit our MYreads page for more information.
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 <12 reactions?
Any design requiring from 1 to 20,000 probes is a MYbaits-1 tier baitset. Therefore, there is no reduced price for ordering smaller probesets. This is because, at this time, our manufacturing & production commitment is the same for any design for up to 20,000 baits. 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.
12 reactions is now our minimum kit size. We cannot provide discounted kits for fewer reactions.
What is the estimated turnaround time for new kits? Can I rush this order?
The bait manufacture turnaround time varies depending on the specific baitset and the number of other kits in our queue, but it is typically less than ~4-6 weeks for your baits to pass our QC process (though you can opt to receive early delivery under some circumstances). 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 under most circumstances we cannot rush orders. We are continually working to reduce manufacturing turnaround times.
Also, please do not forget to account for upfront bait design in your planning timeline. Typically we will be in correspondence for up to ~1-2 weeks regarding a design before it is finalized (unless you send us pre-designed probes that do not require any processing or assistance). Please accommodate some 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.
Note that for Illumina libraries, ONLY dual-indexed/dual-barcoded libraries should be pooled, in order to avoid index dissociation via jumping PCR during post-capture library amplification (see Kircher et al. 2012; doi: 10.1093/nar/gkr771; also see Rohland & Reich 2012; doi: 10.1101/gr.128124.111 for an alternative approach)
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. To achieve this, we recommend only MINIMAL amplification of your libraries, if possible. More 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. If you need more starting material, it is generally preferable to generate more library from fresh genomic DNA or a new batch of indexed library, rather than through re-amplification.
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.
These principles are 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 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 & 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. Any user-supplied hybridization protocol should be appropriate for biotinylated RNA oligos of your chosen length.
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 other types of NGS platforms.
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.
Modified protocols for lower-cost library preps:
- TC Glenn et al. 2016. "Adapterama I: Universal stubs and primers for thousands of dual-indexed Illumina libraries (iTru & iNext)". bioRxiv, http://dx.doi.org/10.1101/049114
- 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
My targets are short and/or rare (e.g. ancient DNA). Should I modify the protocol?
Hybridization & 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.
Are the accuracy and quality of MYbaits probes guaranteed?
Our high-fidelity synthesis and bait production process delivers high-quality RNA probes of your intended design. In addition to our robust oligo production, we subject your probes to a rigorous hybridization-based QC process in order to assess presence of individual probe sequences in the kit. We can guarantee that all the probes which ultimately pass our QC are present the baitset, and we will not ship until >95% of your probes pass our QC (unless you request early shipment).
What sequencing coverage can I expect from MYbaits?
We do not 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 & targets, the quality of your 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 >>80%. But the outcome of your experiment will be necessarily dependent on the properties of the baitset, your samples, and your chosen experimental conditions.
The 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 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.
Should I include >1 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 theoretically possible for a bait 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.
What about known/unknown exon boundaries in my transcriptome sequences?
If you are starting your bait design from transcriptome sequences, you may or may not know 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.
An example of using baits derived from expressed sequence tag cDNA sequences to define intron-exon boundaries was presented at PAG XXI
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 kit upfront (12 reactions), 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.
Can I hire you to do my target captures?
Yes! We offer our expertise in performing a range of in-house services for custom projects, including library preparation, target capture with MYbaits, and high-throughput sequencing. Read more about our service options and pricing or contact us with details about your project for more information.
Help! My capture results are poor and/or confusing. Can you help me troubleshoot?
Yes. Please contact technical support or your MYbaits contact for assistance. We will need to know at least the following information, among other things, to help us better assist you:
- What manual version are you using?
- What library prep kit are you using?
- How many cycles of library amplification did you run (pre- and post-capture)?
- How much library (total ng) did you input into each capture?
- How many sample(s) did you pool per capture reaction, if applicable?
- What type of samples are you working with (e.g. fresh, museum/archival, "ancient DNA", etc)?
What is the difference between a "probe" and "bait"?
There is no difference, and we use the terms interchangeably. Some fields prefer one term over the other, so we use both terms.