Q&A With Teodora Orendovici, PhD, Technical Director, High Throughput Sequencing

Teodora Orendovici, PhD

Editor's note: Children's Hospital of Philadelphia Research Institute supports a variety of Core Facilities providing services ranging from high-tech solutions, such as single-cell sequencing and data analysis to filling personnel-based needs for research teams. All the CHOP Cores are designed to enhance the work of our research community, enabling principal investigators and their staff to reach their project goals in partnership with the expertise of the Cores' staff. In each installment of this quarterly series called Tour the Cores, you will meet a different Core Facilities director and get acquainted with how their Core can promote your project's success.

When was the Core established, and how has it grown since its inception and under your leadership?

The High Throughput Sequencing (HTS) Core established in November 2021 at CHOP has provided next-generation sequencing services (NGS), including NGS sample preparation for a variety of DNA and RNA applications. Since our inception, we have grown in terms of both the number of people we serve and the number of services we provide. Our goal is to make the HTS Core accessible to everyone interested in genomic research.

Currently available NGS technologies can be categorized based on read length: short-read (second generation) and long-read (third generation). Until now, we have provided only second-generation sequencing services represented by Illumina technology, but beginning in March 2024, we are also providing third-generation sequencing services represented by Oxford Nanopore Technologies.

What is your professional background and how did your path lead to CHOP?

The HTS Core had to start from scratch, and I found that challenging and exciting. I loved my job as an associate director in the Genomic Sequencing Core at Penn State College of Medicine in Hershey, Pennsylvania, but I was ready to accept more responsibility.

What makes your Core team exceptional?

I know that professionalism and hard work are expected by every employer, but my team is exceptional in this regard. Library preparation and sequencing is a time-consuming process in itself, as we must provide good, reliable data; therefore, we constantly research new methods. We could not be where we are today without hard work — my team goes way above the call of duty.

We are here to listen and create solutions. Although we have many established protocols, the abundance of new applications for sequencing data is constantly growing, driving the need for even more diverse sample preparation protocols. Not all projects are alike; what works for one sample type might not work for a different sample type (e.g., healthy cells versus tumor cells).

Many samples are extracted from a limited number of cells or even a single cell. These samples do not provide enough genetic material alone and need to undergo polymerase chain reaction (PCR), an amplification technique. The amplification step is prone to introducing bias to the sample, therefore we need to determine the number of PCR cycles for each project to limit bias in the final data. When we realize that a protocol suited for many projects does not work for a new project (i.e., the protocol induces bias or creates artificial sequences), we must find a new sample preparation protocol able to deliver better results.

What would you like potential customers to know about working with your Core?

The High Throughput Sequencing Core has provided next-generation sequencing services, including NGS sample preparation for a variety of DNA and RNA applications.

HTS Core turnaround time is a function of multiple interrelated factors, all dependent on the availability of resources such as time, people, equipment, and reagents. All of these factors have to be considered at each moment in time:

• What projects are alike, and can they be prepped at the same time to be able to reduce hands-on time?
• Can we use the automatic liquid handler for this project?
• Who is the person best suited to execute this project?
• How can we schedule the instruments without overlapping and prevent underuse while working with the "first come, first served" concept?
• How can reagents be ready for use knowing that the shelf life of the reagents is short and not being able to stock anything for long?

These considerations all factor into how long the process will take from receiving DNA/RNA samples to providing the requested data.

Most of our clients have their own bioinformatics teams, and analysis of sequenced data is closely related to sample information (e.g., patient, tissue, treatments), not with standard sequencing protocols. For those in need of bioinformatics services, we recommend they reach out to the Bioinformatics group in the Department of Biomedical and Health Informatics; the HTS Core does not provide bioinformatics services.

What equipment does the Core provide that investigators would be interested to learn about?

The HTS Core provides sequencing equipment for short-read sequencing, such as massive parallel sequencing of short reads where millions of individual sequencing reactions occur concurrently. We provide two sequencers, an Illumina Nextseq 1000 and a Novaseq 6000.

We also have the equipment required for any type of bulk library generation. Library generation includes pools of DNA fragments containing adapter sequences compatible with either Illumina or Oxford Nanopore platforms.

However, short-read technology presents challenges with structural variants and/or low-complexity regions. In March 2024, we introduced a new sequencing instrument, a PromethION24 from Oxford Nanopore, to provide a long-read sequencing option. Compared to short-read methods, the third-generation methods include minimal library preparation and generation of >10kb read length directly from native DNA.

Can you provide any additional facts or items of interest to the research community?

Apart from providing full services for sequencing, the HTS Core shares instrumentation. Instruments are scheduled by users, who pay for servicing the instrumentation. Shared instruments are available for sequencing and real-time quantitative PCR. We are excited to now offer long-read sequencing via PromethION24 under the same premises, and everyone acquainted with Oxford Nanopore sequencing is welcome to use the instrument for a small fee. Visit our website for a complete list of available equipment and services.