Maximizing Read Length Output in Next-Generation Sequencing

Case Study

The Project Situation

An agricultural company performed multiplexed next-generation sequencing (NGS) with desalt-treated custom adapters sourced from its current oligonucleotide vendor. Upon reviewing the results, company scientists noticed that the data included a large number of zero length reads. These reads were troubling because they were not only unexplainable but also a waste of reaction media, i.e. the sample chip was not being utilized efficiently. Company scientists ordered a lot of HPLC-purified custom adapters from the current vendor, and again, achieved poor results.

Our Operational Support

The agricultural company turned to us to develop a potential solution to the problem. Our R&D team recognized that adapter performance in multiplexing is sensitive not only to oligonucleotide purity, but often more importantly, cross contamination, which was identified as the culprit in this particular case. We worked closely with the agricultural company’s scientists to design a test plan that offered at least one potential solution. The goal was to maximize read lengths compared to the material from the current oligonucleotide vendor.

The Customized Solution

For this project, constant communication and open dialogue was key between both company’s teams. This allowed for the sharing of data and in-depth NGS analysis in order to maximize read lengths. Due to the large number of adapters required and the requested format, we developed a purification process that minimized cross contamination, yet allowed for high-throughput manufacturing, often conflicting objectives. The new process involved manufacturing adapters in 96-well plates with multiple sequences per well.  We also proposed supplying daughter plates to minimize freeze / thaw cycles and thereby mitigate laboratory mishaps (it is easier and more cost effective to thaw a daughter plate as opposed to reordering a sole plate that was accidentally dropped on the floor). The test lot was shipped to the agricultural company for evaluation in a battery of NGS tests.

Results

Customer-generated data from the test lot demonstrated that our adapters significantly outperformed the material from the agricultural company’s current oligonucleotide vendor by maximizing read lengths. The agricultural company, now our customer, has continued to work with us on the manufacturing of custom adapters for its needs.