Kelly Nudelman and Tae-Hwi Schwantes-An discuss the importance of sample identification and quality control at the Indiana University Genetics Biobank

While troubleshooting a temporary roadblock in one of her own research studies, Kelly Nudelman, PhD, Assistant Research Professor at the Indiana University School of Medicine, discovered a previously unrealized passion for helping other scientists solve problems and ensure the integrity of their research with DNA fingerprinting.

“I had reached an impasse in my study and wondered if the problems I was encountering might be attributed to my research samples. While assessing what was going on, I determined the issue didn’t have to do with the samples I obtained from the biorepository,” she said. “It was a simple calibration issue with some of the instruments I was using. I was able to resume my research, and also realized my fascination with the troubleshooting process.”

Recognizing Nudelman’s talent, Tatiana Foroud, PhD, Scientific Director of the Indiana University Genetics Biobank (IUGB), a notable biorepository run by staff and faculty in the IU School of Medicine, recruited her to join the team.

The IUGB was established in 1990 as a national central repository for research on dementia, funded by the National Institutes of Health, and has since expanded to include a broad range of sample collections with more than 1 million tissue specimens catalogued. The biospecimen types span many indications, including Huntington’s, Parkinson’s and Alzheimer’s diseases. The IUGB’s largest collection is comprised of DNA samples, which are obtained from blood and tissue. More than 240,000 DNA aliquots are distributed annually, supporting hundreds of studies varying in size and scope.

Tae-Hwi (Linus) Schwantes-An, an Assistant Research Professor working alongside Nudelman at the IUGB, has a similar mission. “While my primary research is in liver and kidney disease, what’s really exciting about DNA fingerprinting is working with the people in the lab and collaborating to solve problems.”

Schwantes-An provides scientific support to the biobank, including managing the technical aspects of the DNA fingerprinting process. “This work gives me that additional level of awareness when I look at my own studies. I'm able to ask better questions, and answer questions better.”

Just over one year ago, IUGB transitioned to the DNA fingerprinting methodology enabled by Fluidigm microfluidics technology and systems for genomic analysis, specifically Juno™ and Biomark™ HD. The biobank has a custom-designed panel of 96 SNPs, and it incorporates this panel into a workflow that allows the biobank to simultaneously confirm sample identity and conduct quality checks at key points in the lab’s workstream. Those include before sample accession and again before distribution of samples to requesting researchers.

“One of our goals is to ensure that all of our samples have been quality-controlled to the highest standard,” Nudelman said. “Using DNA fingerprinting to assess samples prior to accession and distribution can save time and resources. Researchers who receive samples from the biorepository need to have confidence that the samples they receive have been correctly identified from the sample source. Additionally, researchers need to have confidence that the samples will yield interpretable data from their planned downstream analyses.”

Because the biobank distributes DNA more than any other type of sample, the team is always looking to improve its distribution and stable storage processes. The Fluidigm workflow was chosen because of its scalability and cost-efficiency and the small quantity of DNA needed to test samples.

“We found that many alternative workflows are either high-throughput/high-cost or low-throughput/low-cost,” said Schwantes-An. “What makes the Fluidigm microfluidics-based workflow unique is that it is high-throughput at a cost that is scalable to our process. It saves our customers and our lab time and resources.”

Ensuring quality control with the Fluidigm DNA fingerprinting workflow

In implementing the Fluidigm microfluidics-based workflow for DNA fingerprinting, Nudelman and Schwantes-An employ a custom-designed SNP panel developed by the IUGB to generate a molecular fingerprint for each sample. The specific assays chosen for the panel enable the team to produce not only a DNA fingerprint for each sample, but also to verify other aspects of sample quality, such as sample swaps, contamination, degradation or discordance in reported versus genetic sex.

The biobank’s customer requests for distribution can vary from two samples to thousands of samples and encompass researchers across segments from academia to clinical research. Bringing DNA fingerprinting in-house enabled the biobank to conduct more streamlined testing before accession and distribution and to better identify and understand potential issues, such as a swapped sample, degraded sample or non-concordance between accession and pre-distribution sample tests. As a result, the biobank can move faster and produce more informative and higher-quality data.

“If a scientist is using a poor-quality sample, the mistake can amplify and cause a cascade of issues as the study moves forward,” Schwantes-An said. “DNA fingerprinting allows us to detect events that might otherwise lead to erroneous results. It helps us spot potential issues before we send samples to the requesting researcher.”

Future implications for DNA fingerprinting

In addition to using the Fluidigm microfluidics workflow, the team at IUGB has been able to collaborate with experts at Fluidigm to design assays for additional genetic variants of interest.

“For example, as the field of neurodegenerative disease grows, new targets of interest will be identified,” Schwantes-An explained. “Being able to customize the assay allows us to adapt to how the field is changing, and this is something we can do using the Fluidigm workflow. Let’s say, for example, a new variant is discovered, and everyone wants to know the status for that particular variant in her or his sample. We’ll be able to easily make a modification to our panel, adding the new variant quickly.”

Nudelman added that they are also interested in adapting this technology to other types of samples. “We feel there's a lot of potential that we could leverage for other things that we're doing,” she said. “For example, our DNA fingerprinting workflow has the potential to be leveraged through our induced pluripotent stem cell workflow.”

For Nudelman, working at the biobank has provided a greater connection to the larger research community.

“Working at the biobank, I get a bird’s-eye view of the research happening in the community,” she said. “Our samples are central to so many research programs, and it’s an amazing feeling to know you’re touching so many people through this process—making things run smoother and ensuring better-quality data.”

Schwantes-An also enjoys working with the biobank to help other investigators, and to fulfill the wishes of individuals who donate their personal samples for future research.

“We’re working together, trying to meet one goal,” said Schwantes-An. “We want to make sure we send out the best-quality samples to the investigators. Oftentimes, patients donate their samples because they're altruistic. It is their desire to help advance clinical research. I feel a sense of duty to make sure that their generosity doesn’t go to waste. Using Fluidigm’s microfluidics workflow for DNA fingerprinting is allowing us to fulfill what these patients wanted, which is sharing their samples for the advancement of medical research.”

For more information on the Fluidigm workflow implemented at the IUGB for DNA fingerprinting, listen to the webinar.

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Jo Vandesompele at Ghent University discusses taking a deep dive into single-cell total RNA sequencing

Jo Vandesompele, PhD, Professor in Functional Cancer Genomics and Applied Bioinformatics at Ghent University, and his team are pioneering new methods in nucleic acid quantification to help understand cancer biology cell by cell. In the past five years, his research has shifted to focus on deciphering long noncoding RNA in the human genome with the goal of informing the development of better cancer diagnostics and therapeutics in the future.

In collaboration with Fluidigm, Vandesompele developed a single-cell total RNA sequencing method that allows for the study of protein-coding and noncoding RNA. It is the first time investigators have been able to study both full-length long noncoding RNA and mRNA at the same time in each single cell in a cost-efficient manner using the C1™ system.

“The transcriptome is much more complex than only polyadenylated RNA, or protein coding RNA,” Vandesompele said. “There are about 60,000 long noncoding RNA genes in the human genome, or about three times more than coding RNA, and this number continues to grow as we learn more about the transcriptome.”

Previous research has demonstrated that noncoding regions of the genome can influence the regulatory components of cells. This single-cell sequencing method can bring a better understanding to transcriptional regulation, specifically concerning how cells transition to disease states.

“Most of the other methods are just scratching the tip of the iceberg,” Vandesompele said. “With this application, we can see what’s below the surface. I think that's important in terms of single cell atlases—that you're not just scratching the surface, but going deep into the cellular identity of these different cell types.”

Programming the Fluidigm C1 platform for noncoding RNA

Vandesompele acquired the C1 system in 2015 because of its simplicity, ease of use and credibility for single-cell genomics studies.

“If you are looking to observe new cell types or cellular heterogeneity, for instance, you don’t need to sequence very deep,” Vandesompele explained. “However, in transcriptional regulation, you have a need for accurate quantification of low-abundant genes. And for these applications, we have found the library prep methods in combination with the C1 platform are best.”

Karen Verboom, a doctoral fellow who works with Vandesompele, optimized the single-cell application. The flexibility of the C1 platform, delivered through Script Builder™, allowed the team to modify the scripts controlling the integrated fluidic circuit (IFC), and enabled the creation of this new workflow.

“Our lab was not satisfied with off-the-shelf solutions,” Vandesompele said. “We knew we wanted to study more than just polyadenylated RNA. The C1 provided us with the flexibility to incorporate our own changes and workflows.”

While designing this method, the team faced a few challenges. Since nearly all single-cell RNA sequencing methods capture only polyadenylated RNAs, its first challenge was enabling quantification of RNA transcripts without polyadenylation tail. However, using random primers in a so-called total RNA sequencing workflow produces an abundance of unwanted ribosomal cDNA, which can dominate the sequenced reads and create bias. So the team introduced Takara Bio USA’s method that uses probes and enzymatic digestion to remove ribosomal cDNA during single-cell library preparation.

A second challenge the team confronted was ensuring that its methods preserved strand orientation. Tracking the RNA strand orientation allows the team to differentiate sense and antisense overlapping genes.

Celine Everaert, another doctoral fellow in the lab, noted, “The deeper we look into the human transcriptome, the more of these overlapping sense and antisense gene pairs we observe. If you want to understand transcriptional regulation, you need to be able to differentiate these, too. Preserving strand orientation allows you to quantify genes with much higher accuracy.”

Using C1 and Script Builder enabled the team to overcome such challenges, and now Script Hub™ houses several C1 Total RNA-Seq workflows.

Single-cell research on long noncoding RNA provides many potential benefits. First, cell specificity is much higher for noncoding RNA, so investigators have a higher chance of finding specific markers for cell states. C1 Total RNA-Seq also allows for an additional layer of information on how transcriptional regulation networks are composed. This can give researchers a better understanding of transcriptional regulation, which is often rewired in diseased cells. Lastly, full-length sequencing allows for uncovering structural information encoded in a transcriptome. Taken together, these total RNA solutions housed on Script Hub enable complete characterization of the transcriptome of single cells.

“A lot of structural information is encoded in a transcriptome, such as alternative splicing (where different exons are fused together), which often brings new functions,” Vandesompele said. “Also, other forms of structural information such as small insertions or deletions or fusing genes are extremely important in studying cancer cells.”

Circular RNAs and long reads: future potential for single-cell total RNA sequencing

Vandesompele and his team are excited about this new method, as are others in the research community. In fact, Vandesompele has started to collaborate with investigators studying other classes of noncoding RNA, such as circular RNAs, which originate from back-splicing of an otherwise linear RNA to form a circular structure.

“Scientists are realizing that the human transcriptome is full of these circular, back-spliced transcripts,” Vandesompele said. “And again, only by sequencing entire genes and not focusing on polyadenylated genes, you're able to exploit the structural information in a rich transcriptome.”

Vandesompele, Verboom and Everaert plan to continue using the flexibility of C1 to optimize their method to encompass a workflow for the investigation of circular RNAs and for full-length single-molecule long-read sequencing. Vandesompele also sees potential for this method in studying microRNAs, which are also crucial regulators of cell identity in health and disease.

“I think the better you understand the deregulated transcriptome, the better you will be able to identify biomarkers and therapeutic targets,” Vandesompele said. “As scientists are developing RNA-targeted therapies, it will be crucial to have a very accurate and detailed view of the transcriptome.”

Download the new C1 total RNA application scripts from Script Hub or click here to learn more.

Fluidigm Products are for Research Use Only. Not for use in diagnostic procedures.

The importance of sample tracking and quality assessment

Scientists involved in human disease research are increasingly mindful of the investments of time and resources required in the design and execution of their studies. Regardless of the experimental objectives or techniques applied, samples obtained for clinical and translational research are precious. Particularly with complex, multiphase studies, where portions of samples may be shared among multiple researchers within a lab or in collaborating laboratories, the importance of ensuring sample integrity and quality from sample acquisition to final data analysis is a shared concern.

Implementing a standard genotyping workflow to confirm the identity and quality of each sample before analysis represents an ideal solution to maximize the integrity of study results.

Learn from the experts

Listen to Kelly Nudelman, PhD
Indiana University School of Medicine
Indiana University Genetics Biobank

The Indiana University Genetics Biobank (IUGB) at the Indiana University School of Medicine is a high-throughput biobanking facility that supports numerous research programs. In this on demand webinar, Kelly Nudelman describes the IUGB biobanking pipeline and how they have implemented sample tracking and quality checks in their workflows.

Listen to Jianhong Hu, PhD
Baylor College of Medicine
Human Genome Sequencing Center

In this on-demand webinar, Jianhong Hu shares the details of how the Human Genome Sequencing Center (HGSC) at the Baylor College of Medicine has implemented SNP Trace™ as part of its NGS workflow. Then in the same webinar learn more about the Advanta™ Sample ID Genotyping Panel from Luke Stewart, Senior Field Applications Manager at Fluidigm.

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“The GO Immuno-Oncology Workbench is a powerful software tool that enables comprehensive analysis of immuno-oncology cohorts, integrating molecular and phenotypic data together with immuno-oncology-specific annotations to power translational studies,” said Manuel Glynias, CEO of GenomOncology. “We are excited to see the new insights uncovered using this comprehensive approach, which we we offer in conjunction with Fluidigm to empower the growing immuno-oncology community.”

“Fluidigm is the partner of choice within the immuno-oncology research community, enabling deep interrogation of the tumor microenvironment and immune response with mass cytometry,” said Chris Linthwaite, President and CEO of Fluidigm. “In partnership with GenomOncology, we are expanding our immuno-oncology gene expression offering to the cancer community with a full microfluidics-based workflow solution from RNA extraction to data analysis. By providing a comprehensive view of tumor immunity, utilizing microfluidics and mass cytometry technologies, we are empowering researchers to uncover unique health insights that could transform the future of cancer care.”