Navigating Your Custom NGS Options: Important Considerations to Maximize your Success
In the world of next-generation sequencing (NGS) there are many options for your project. While it may be easy to reach for an off-the shelf product such as whole exome or cancer hotspot, there may be times when such products will not provide the target capture or coverage you are seeking without generating a large amount of data of which you do not plan to take advantage. With the variety of options available, how does one begin to narrow down the best applications for your project? You do not need to navigate these waters alone when you partner with myGenomics.
When selecting any service provider, it is important to clearly communicate your project needs and goals. myGenomics offers a variety of custom sequencing solutions and discusses your project in detail prior to beginning any work to ensure you will obtain the desired data. One of the first questions that should enter your mind is your target of interest. If your interest lies in a select handful of genes, you must weigh the advantages and disadvantages of selecting an off-the-shelf product. While these may be a less expensive option, especially for larger targets, myGenomics recommends you double check the targeted regions, or the .bed file, for the product. Since many of these kits are designed using public annotation databases, there may be the occasional gene that does not include exons not supported by verified transcripts. If you want to look at entire coding regions, exome products may provide some coverage into the introns. However, this coverage typically drops drastically once you fall outside the targeted gene coordinates. Fortunately, custom NGS solutions can overcome many of these obstacles.
Custom solutions often fall under two basic chemistries: amplicon-based and capture-based. With smaller targets capture-based chemistry can prove challenging due to the small percentage of the total sample that is expected to be pulled down by the probes. For example, from a library prepared from human genomic DNA the entire coding region of BRCA1 and BRCA2 would be expected to represent roughly 0.005% of your input (165,388 / 3.3 x 109). At 1 mg of input genomic DNA, this would represent 50 pg of captured material if all steps in library preparation were 100% efficient. Unfortunately, the reactions and clean up steps are not 100% efficient, and capture is more likely to pull down non-specific targets when true target input is very low. Even with post capture PCR amplification, it can be a challenge to obtain sufficient final product to accurately perform quality control testing and have sufficient material for loading on the sequencer. Any off-target library amplified in those PCR steps will also dramatically increase off-target results in the final data. Given these challenges, for those targets less than 1Mb, amplicon-based solutions are recommended.
In contrast, for targets larger than 1Mb, capture-based chemistry becomes a more viable option. Since amplicon-based strategies require PCR primers to be designed to cover the entire target region, the larger the target, the more PCR primers that need to be developed and synthesized. This will result in higher costs for the design. As your target goes beyond 5Mb, it becomes more cost effective to look at capture-based solutions. Depending on the number of samples you wish to process, the cost of amplicon-based library preparation verses capture-based library preparation will need to be carefully compared.
For projects with only a small number of samples, it can be challenging to find a cost-effective solution. With amplicon-based library preparation, many vendors will provide design services. Depending on the chosen vendor, your target may be limitless or may be restricted to a pre-validated set of primer designs. Additionally, vendors place constraints on the kit size, with the smallest order ranging from 48-96 samples. If your project is looking at only 10 samples, it may well be cost prohibitive to order such a kit. In this case, custom homebrew primers may offer a fiscally responsible option. Certain vendors provide more flexibility with their capture reagents, allowing the end user to order reagents for as few as 4 samples. Alternatively homebrew capture probes can be designed by a general oligonucleotide provider or can be generated to target contiguous chromosomal regions using bacterial artificial chromosome (BAC) clones to provide lower cost options for smaller projects. Care should be taken, however, as in larger projects as batch to batch variation in probe preparations could confound your analysis, and blocking reagents to be used during hybridization would need to be provided separately.
Other factors should also weigh in on your NGS planning. Nearly all vendors can provide designs to include or exclude introns and untranslated regions, while BAC-based probes would always include these regions. Samples derived from formalin-fixed paraffin embedded (FFPE) tissue may perform better using amplicon-based strategies since the DNA recovered from such samples is generally significantly fragmented. Several vendors actually allow the end user to specify FFPE as the input sample, and adjusts the design to generate smaller amplicons. Equally important is the total mass available for input, with the suggested input ranging from 10ng to 1ug.
Lastly, any custom design will require lead time to allow your custom products to be prepared. Generally you can anticipate this time to be 4 to 6 weeks. With homebrew options, the lead time can be significantly shorter. For projects where timing will be critical, it will be important that you begin your custom sequencing strategy planning before you begin or early on in your experiment. myGenomics welcomes the opportunity to begin planning your experiment, and offers any custom workflow to help you obtain the highest quality results for your analyses.
*BAC-based probes will capture exons, introns as well as portions of the 5’- and 3’-UTRs. All other methods can be customized to capture exons only or the entire coding region.