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

• Optional custom probe design informatics service (designing and removing non-specific baits; report)
• Biotinylated RNA probes according to your approved 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 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).

Please see the latest myBaits manual for detailed protocol instructions for enriching from Standard, High-Sensitivity, and Long-Insert target/sample types.

For reorders of custom designs previously manufactured by Daicel Arbor Biosciences, the estimated manufacturing lead time is ~1-2 weeks from the time an order is received.

Specific recommendations for library co-enrichment pooling for different 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.

myBaits Custom Sequence Submission Guidelines

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.

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.

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.

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.

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

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

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.

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.