Custom RNA target enrichment for cost-effective sequencing

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 applicable myBaits manual provides detailed protocol instructions for enriching libraries for sequencing on short- and/or long-read platforms (e.g. PacBio® or Oxford Nanopore Technologies®).

If you are using a never-before-tried library prep protocol to pair with your myBaits kit, we recommend that you first perform some total library (shotgun) sequencing before doing myBaits enrichment. This is important in order to verify that your chosen library prep protocol/kit generates libraries of sufficient complexity and minimal bias in your hands, otherwise you may experience poor target capture results. High quality libraries are absolutely essential for achieving a successful target capture project.

Provided below are a list of companies that sell NGS library prep kits that are known to be compatible with myBaits. This is NOT an exhaustive list; there are many other unlisted options that are also compatible with myBaits. Also, kits on this list may not necessarily be appropriate for your samples. NGS library prep is not “one size fits all”; different factors such as sample type, DNA input amount, genome complexity, and sequence composition may influence the type of library prep kit that would be best for your application. For example, low input, degraded, and/or damaged DNA templates may require special handling (see below) and/or modifications to commercial kits.

Contact these and other manufacturers to learn about your options and find what works best for your samples and project needs:

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

myBaits hybridization capture kits include:

• Biotinylated RNA probes, with sequences corresponding to your custom design or a predesigned catalog option
• Hybridization and wash reagents
• For myBaits Custom kits, optional custom probe design informatics service (= expert bioinformaticians design and filter bait sequences and provide summary report and recommendations)

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 applicable myBaits manual to make sure you have everything you need before starting your experiments.

We also offer reagents for preparing libraries from DNA samples in advance of performing the myBaits hybridization capture step. Please visit Library Prep Kit for myBaits for more information.

If you are looking to outsource your project to a full-service laboratory and bioinformatics services group, please visit our myReads NGS laboratory and bioinformatics services page for more information about our comprehensive targeted sequencing service options (library preparation, target capture, next-generation sequencing, and optional analysis).

In the context of hybridization capture for targeted next generation sequencing, we use the terms interchangeably. Some fields prefer one term over the other, so we use both terms.

Turnaround time for myBaits targeted next generation sequencing kits varies based on design.

For new myBaits Custom baitset designs, the estimated manufacturing lead time is ~3-4 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 up to ~1-2 weeks from the time an order is received.

All myBaits kits include a specific protocol for their use as well as almost all of the reagents 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.

Hybridization capture is integrated into the overall next generation sequencing 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

Specific recommendations for per-library input mass for different enrichment project types can be found in the applicable 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.

The applicable myBaits manual covers some common technical questions and troubleshooting topics at the end of each protocol. 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.

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.

Yes! Our expert myReads team provides a range of in-house NGS services, including library preparation, target capture with myBaits, high-throughput sequencing, and optional bioinformatics analysis. Visit the Sequencing Services page to learn more about our comprehensive laboratory and sequencing service options!

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. This ensures consistency in your targeted next-generation sequencing workflow.

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.

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.

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 applicable myBaits manual.