For important information for your next Arbor Biosciences order, please see our project planning guides.
There are many important considerations when planning a target capture project for next-generation sequencing (NGS). Here we provide some helpful info to guide your myBaits project from start to finish. Also review our FAQ page for additional information about myBaits kits.
Target capture should be performed on DNA libraries that are already prepared and indexed for high-throughput sequencing. This is because (a) post-capture libraries are single-stranded and relatively low molarity, and require amplification to reach sufficient molarity for NGS, (b) working with indexed samples minimizes the risk of cross-contamination, and (c) working with barcoded libraries enables pooling multiple libraries per capture reaction.
myBaits is compatible with all NGS platforms including Illumina, Ion Torrent, PacBio and Oxford Nanopore. We provide blocking oligos with the capture kit specific to the intended NGS platform and will request upon ordering.
We offer affordable pre-designed catalog kits for multiple different types of target capture sequencing: ultraconserved elements (UCEs), whole-genome enrichment (WGE) , mitochondrial DNA, and human cancer exome targets.
For myBaits Custom Kits, baits will target only those loci that you wish to sequence in order to maximize your high-throughput sequencing efficiency. You have full control over the design of your custom baitset and retain full intellectual property rights. If desired, we are happy to suggest reasonable design options based on our vast experience for your particular project.
We accept either FASTA formatted sequences or coordinates from a reference genome.
Eliminating non-specific baits is an important step for most bait designs. Baits that hybridize to multiple locations on the genome are both (a) unlikely to be bioinformatically useful and (b) can necessitate deeper sequencing due to increased relative enrichment coverage of such regions. When a sequenced genome is available for your organism, or from a close relative, we can use our proprietary bioinformatics software to ensure the specificity of the baits in your design, thus reducing the risk of inefficient sequencing due to large amounts of non-specific or off-target reads. Please review our Guidelines for Sequence Submission
myBaits Custom Kits are manufactured in modules of up to 20,000 unique baits. The size of the kit, or number of modules, required for your custom design will be dictated by both the total size of the genomic targets and the specific placement of baits along the targets, such as tiling density, removal non-specific baits, etc. See our Custom Design Calculator to estimate the number of bait sequences required for your design.
The price of a kit with a certain baitset will depend on the number of separate capture reactions required for your project. For custom designs, the smallest kit size we offer is a 16-reaction size. See the myBaits Custom Kit page for more information on pricing.
To produce your unique kit, we first manufacture a completely custom baitset and then perform our robust quality control (QC) procedures that ensure that your baits are present and ready to be used experimentally. As such, there is a minimum production time required for all myBaits Custom Kits which should be taken into account when planning your project. Contact us for the current estimated production time for custom kits.
myBaits kits contain almost all necessary materials for performing the captures, however there are some reagents and equipment that will you need to provide. A complete list of required materials as well as a fully detailed capture protocol can be found in the product manual .
Capturing individual libraries produces the best per-sample results. However, multiple dual-indexed libraries can be pooled into single capture reactions for multiplexing in order to assay more samples in a single reaction. For new baitsets, we strongly recommend first performing trial captures with different pooling schemes to determine optimal conditions for your particular samples and baitset. When pooling libraries that vary in relative target content, such as 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 or rare targets like ancient DNA, or for very large targets such as a WGE baitset targeting an entire genome. When working with ancient DNA specimens and small targets, like 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 libraries should be pooled in order to avoid index dissociation via PCR jumping 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).
A successful capture requires subjecting NGS libraries to a bottleneck, wherein target molecules are captured and therefore enriched, and non-target molecules are therefore removed. In order to have a sufficient abundance of unique molecules for good sequencing coverage of your target regions, successful captures are dependent on having sufficiently complex libraries going into capture.
We recommend a DNA input amount of 100-500ng into each capture reaction. To achieve this, we recommend minimally amplifying your libraries. 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.
More amplification risks skewing the profiles of your libraries through the generation of PCR duplicates, as some molecules will become relatively more abundant while others become rare. The same is true for post-capture libraries: amplify your post-capture libraries the minimum number of cycles necessary to reach the molarity required by your sequencing instrument.
If you need assistance selecting a library preparation kit to pair with your myBaits kit, please see our Frequently Asked Questions, or contact us for more information. myBaits kits are compatible with almost all major NGS library prep options.