Caden White

Helios Scholar

The latest next generation sequencing (NGS) platforms allow for greater sequencing reads which leads to a researcher needing to multiplex samples on the same flow cell to increase cost efficiency. To achieve this, a barcode specific index is added to samples during library prep. Samples are typically normalized by performing quantitative PCR (qPCR) which is a cumbersome process that takes significant time, cost, and has a high chance of user error. This leads to risks such as samples being over- or under- represented in downstream analysis. The main objective of my project was to test a new bead-based normalization kit to normalize libraries for amplicon-based sequencing. The purpose of testing this technology is to streamline the standard qPCR protocol to a faster and more cost-efficient method of uniformly pooling libraries. The project included normalizing and pooling six PCR amplicon samples by the qPCR method and the normalization kit. The samples were then sequenced and bioinformatically compared to evaluate which method achieved more uniform sequencing and conserved the original library diversity. The results for sequencing uniformity demonstrated that the the normalization kit had a lower coefficient of variation (%CV) when compared to the qPCR protocol. The normalization kit had similar % diversity for each sample when compared to qPCR, meaning it conserved the quality of the original library. In conclusion, the bead-based normalization kit had improved uniform sample sequencing, conserved the original library diversity, reduced hands-on time, and increased throughput while being easier to use and more cost-effective.