NGS Delivers Speed, Efficiency, and the Unexpected
Dr. Yan and his team at MDC-NUH are conducting AML and multiple myeloma research.
In light of the increasing number of clinical standard-of-care biomarkers, current molecular testing solutions, such as Sanger sequencing and qPCR, are expensive and time consuming for analyzing AML and other hematologic and lymphoid cancer samples.
Dr. Yan and his team chose the MiSeq System, TruSight Myeloid, and Nextera XT Library Prep Kit as an NGS solution. They are performing correlation studies to gather data, identify Singapore population-specific biomarkers, and become NGS proficient.
Dr. Yan and his team are developing the expertise and resources to perform NGS in the MDC-NUH laboratory and expand its use in other clinical research studies.
Pathologist Dr. Benedict Yan is preparing for the future of molecular diagnostics. Real-time PCR (qPCR) and Sanger sequencing have long been the work horses for his team at the Molecular Diagnosis Centre (MDC) of the National University Hospital (NUH) in Singapore. Yet their workflows are expensive and time-consuming. He’s investigating the use of next-generation sequencing (NGS) as a tool to replace these traditional molecular analysis technologies in his laboratory.
Dr. Yan believes that in the future NGS could streamline diagnoses, particularly for acute myeloid leukemia (AML). “NGS is an efficient, cost-effective alternative to conventional sequencing methods,” Dr. Yan said. He views NGS as the technology of the future in his molecular diagnostics laboratory and wants his team to become NGS proficient.
Choosing the Right Tools
In 2014, Dr. Yan and his team began conducting Sanger/NGS correlation studies with the MiSeq System and TruSight Myeloid Panel. The standard of care today for AML includes analysis of at least seven biomarkers that span more than 10,000 base pairs cumulatively. “We see an increasing number of standard of care markers on the horizon,” Dr. Yan said. “Sanger sequencing is too expensive to analyze all of these genes.”
For their studies, they used more than 150 banked AML samples (peripheral blood and bone marrow) collected at NUH over the last 10 years. “We chose the TruSight Myeloid Panel because there were no other ready-made NGS panel solutions for analyzing myeloid-associated genes,” Dr. Yan said. “Without it, we would have needed to make and develop our own panel. That would have taken quite a bit of time.”
"NGS identified all the mutations in [GC-rich] CEBPA that were identified by Sanger sequencing and detected even more.”
Aiming for Accuracy
Dr. Yan and his team have completed the correlation studies and are validating the findings analytically. “We’re comparing NGS findings with data from Sanger sequencing, which is the current gold standard,” Dr. Yan stated. “We’ve found that NGS is identifying all the mutations that were identified by Sanger sequencing.”
One challenge the MDC team faced was obtaining high coverage of CEBPA, a GC-rich gene that is a standard-of-care AML biomarker. Illumina recommended the Nextera XT Library Prep Kit to obtain higher coverage.1 The combination of the MiSeq System, Nextera XT Library Prep Kit, and TruSight Myeloid Panel was a success. “NGS identified all the mutations in CEBPA that were identified by Sanger sequencing and detected even more.”
Dr. Yan is also using NGS to build a database of novel biomarkers relevant in Singapore’s diverse population. “The body of knowledge for AML biomarkers has been developed with Western populations,” Dr. Yan added. “Even if we obtain a good quality result, we need to determine whether it is a meaningful AML biomarker, particularly in our population.”
"The fusions we found have never been reported in multiple myeloma before. We validated them using wet bench techniques such as reverse transcriptase PCR."
Powered for Discovery
In addition to looking at variants in AML samples, the MDC is investigating fusions in multiple myeloma using the TruSight RNA Pan-Cancer Panel. For the study, researchers used approximately 20 multiple myeloma samples collected over the last five years.
Sequencing was performed at an Illumina laboratory using the NextSeq™ 500 System. While the findings of this study have yet to be published, the panel enabled MDC researchers to identify new fusions. “The fusions we found have never been reported in multiple myeloma before,” Dr. Yan stated. “We validated them using wet bench techniques such as reverse transcriptase PCR.”2,3
Making the Right Choice
Dr. Yan and his team are developing the resources and expertise to expand their use of NGS for other clinical research studies. “The quality of Illumina sequencing is very good and that’s favorable for us,” Dr. Yan said. “Our experience working with Illumina has been fantastic.”
“Even in 2014, Illumina was the dominant market player in NGS,” Dr. Yan added. “There are advantages in using Illumina systems, particularly in gaining acceptance by the medical community. When a technology is used widely, it leads to a greater standardization and common understanding.”
Read more about this study:
Making Discoveries While Becoming Proficient in Next-Generation Sequencing, www.illumina.com/science/customer-stories/icommunity-customer-interviews-case-studies/making-discoveries-while-becoming-proficient-in-ngs.html
Learn more about the Illumina systems and products used in this study:
Ng IK, Ng C, Low JJ, et al. Identifying large indels in targeted next generation sequencing assays for myeloid neoplasms: a cautionary tale of the ZRSR1 pseudogene. J Clin Pathol. 2017; pii:jclinpath-2017-204440. doi: 10.1136/jclinpath-2017-204440. Epub ahead of print.