Planning your capture project
There are many important considerations when planning a capture project. 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.
I. Sequencing platform
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 major NGS platforms (e.g. Illumina, Ion Torrent)! We will ship your kit with blocking oligos depending on the platform of your choice, so please provide this information upon request.
II. Bait design
For our 100% custom kits, your baits will target only those loci that you wish to sequence, for maximizing your high-throughput sequencing efficiency. You have full control over the parameters of your custom baitset, and you retain full intellectual property on your baitset. You can choose the customized bait length and tiling density that is appropriate for your project goals. Predesigned kits have already been optimized for these parameters. We can work with you to determine the best parameters for your particular project, based on our experience. Read more about these important design aspects below:
We offer bait lengths of 80mer, 100mer, and 120mer. However you can also choose intermediate sizes.
The minimum size for a MYbaits probe is 50-60mer, depending on the sequence composition and application. However for most applications, we recommend a minimum size of 80mer.
Tiling density refers to the degree of overlap between the baits that target a given sequence. For example, 100mer baits tiled at 2x density will have 50 bases overlapping between baits. Overlapping baits are advantageous because poor performance of a given bait can potentially be compensated for by better performance of neighboring baits. 2x tiling density is the typical default recommendation for most applications.
For many applications, 1x or 2x tiling density is probably sufficient for effective capture. However some baitsets should be designed with higher tiling densities when appropriate or necessary, according to your experimental design. A 4x tiling density is recommended for applications such as ancient DNA enrichment, where DNA sequences are highly fragmented, and more closely positioned baits can increase the likelihood that a given unique fragment will be captured.
If your budget dictates that your design remain within the constraints of a particular kit size (e.g. MYbaits-2 with up to 40,000 probes), we can easily adjust your tiling density in accommodation.
III. Providing your custom sequences for bait design
You can provide us either FASTA formatted sequences or coordinates on 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 reads.Guidelines for Sequence Submission
IV. Custom kit size
Our kits come in modules of up to 20,000 unique baits. The size of the kit (number of modules) needed for your custom design will be dictated by (1) the total size of the genomic target(s), (2) bait length, and (3) tiling density that you choose. See our Custom Baitset Calculator to help 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 12-reaction size. See the Custom Kit page for more information on pricing.
V. Delivery timeline
Please note that there is a minimum production time required for all custom MYbaits kits, which should be taken into account when you are planning your project. Contact us for the current estimated production time for custom kits. To produce your unique kit, we first manufacture your 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.
VI. Performing captures
Our MYbaits kits contain almost all materials necessary for performing the captures, but there are some reagents and equipment that you will need to provide yourself. You can find the full detailed capture protocol as well as the list of required materials in the latest Manual.
VII. Pooling multiple samples
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)
VIII. Your libraries
A successful 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 a sufficient abundance of unique molecules for good sequencing coverage of your target regions, successful captures are necessarily dependent on having sufficiently complex libraries going into capture.
We recommend an input DNA amount of 100-500ng into each capture reaction. To achieve this, we recommend minimally amplifying your libraries, if possible. 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 facility.
If you need to select a library preparation kit option to use 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.