Customer Publications Throughout July 2020

         July 30, 2020

In this month’s Customer Publications blog post, our VarSeq software is taking center stage! From whole exome sequencing to copy number variant calling, VarSeq can be used for a range of scientific investigations. Although this blog features several examples of cancer investigations in human patients, it’s interesting to see how this platform can be utilized in a variety of investigational situations. As you read further, you will see it is not only a powerful tool for human medicine but can also apply to other species too! 

Analytical Validation of an Error-corrected Ultra-sensitive ctDNA Next-generation Sequencing Assay

Australian researchers sought to develop a custom ultra-sensitive NGS assay for detecting plasma circulating tumor DNA (ctDNA). Using molecular barcoding technology and off-the-shelf reagents combined with bioinformatics tools, the team set out to create an inexpensive, highly sensitive assay that has the potential to significantly improve the field of precision oncology. ctDNA is a minimally invasive approach to investigating a tumor landscape. However, ctDNA can be highly diluted with normal cell-free DNA. The development of hybrid capture-based targeted molecular barcoding assays has led to the ability to confidently detect variants at variant allelic fractions (VAF )below 2%. Unfortunately,  they also produce a large number of unusable reads that do not map to regions of interest, thus reducing the economic viability of these assays in clinical settings. Integrating VarSeq’s CNV caller (VS-CNV) into their protocol for generating molecularly barcoded libraries with targeted hybrid capture, the team was able to develop a highly sensitive, end-to-end targeted sequencing assay which can reliably detect ultra-rare variants (under 1% VAF) with a low false-positive rate. The team hopes their assay will help to further technological advancements that will allow the increased use of liquid biopsies in early stage diagnosis and more comprehensive profiling of tumor landscapes in both research and clinical settings.

Tu Nguyen-Dumont, Jason Steen & Colleagues, Monash Health / Published in BioTechniques

Neuronal Ceroid Lipofuscinosis in a Domestic Cat Associated with a DNA Sequence Variant that Creates a Premature Stop Codon in CLN6

A team of investigators from the University of Missouri were presented with a unique case involving a relatively young domestic cat displaying a range of symptoms including visual impairment, focal myoclonus and frequent severe seizures which were not receptive to treatment with standard seizure medications. Diagnostic imaging revealed diffuse global brain atrophy. Upon humane euthanasia due to the severity of the seizures, the team took a deeper dive into the root cause of the animal’s ultimately fatal condition. The clinical signs and neuropathology strongly suggested he was suffering from neuronal ceroid lipofuscinosis (NCL). NCL’s are hereditary progressive neurological diseases that also occur in several species, including humans. In humans, NCL-causing sequence variants have been identified in 12 genes so far. However, there has only been one feline NCL-causing variant previously identified. The team performed whole exome sequencing (WES) on the animal and with the help of VarSeq software, they compared it to the WES data of 40 healthy cats from a previous study that were assumed to be unaffected. The team identified a variant in the CLN6 gene of the affected cat that represented the only loss of function variant detected. This variant was not present in the other cats that were included in the study. Their discovery was only the second case of feline NCL in which the underlying molecular genetic cause has been determined. The team’s investigation demonstrated the success of WES analyses in identifying the molecular genetic bases of two feline NCL’s, which they hope will open the door to discovering other sequence variants that cause NCL in domestic cats. This discovery may also lead to the establishment of a feline model of CLN6 disease that could be used in therapeutic intervention studies.

Leslie Lyons & Colleagues, University of Missouri / Published in Genes, Genomes & Genetics

Assessment of Somatic Mutations in Urine and Plasma of Wilms Tumor Patients

Researchers in Brazil studied the potential of tracking somatic mutations in free DNA in plasma and urine collected from Wilms tumor (WT) patients for the purpose of monitoring treatment response. Wilms tumor is a rare kidney cancer that primarily affects children and its mutational contributors are not completely understood. The team identified somatic mutations from tumor tissue DNA by screening matched tumor/leukocyte DNAs using either a panel of 16 WT-associated genes or whole-exome sequencing (WES). They employed VarSeq software for the tumor WES portion of their study. The identified somatic tumor mutations were then tracked in urine and plasma DNA collected before, during and after treatment. The team discovered that in all patients, there was a decrease of the variant allele fraction of somatic mutations in body fluids during neoadjuvant chemotherapy. Five patients were tested in all, with one patient (who progressed to death) exhibiting persistently high levels in serial bodily fluid samples during treatment. For three patients without disease progression, the mutations were not consistently detected throughout the study and for one patient with both kidneys involved, a low level of somatic mutations were detected. The results of this investigation made a case for the tracking of somatic mutations in urine and plasma DNA as a minimally invasive tool for monitoring WT patients. The team is hopeful this study will lead to more studies of its kind to validate the clinical value of their monitoring strategy.

Dirce Carraro & Colleagues, A C Camarago Cancer Research Center / Published in Cancer Medicine

Association of Histologic Subtypes with Genetic Alteration and PD-L1 Expression in Pulmonary Adenocarcinoma

Lung cancer is the leading cause of carcinoma-related death worldwide. In the case of adenocarcinoma (ADC), the most common classification of non-small cell lung cancers, genetic alterations and programmed death-ligand 1 (PD-L1) expression have been associated with various subtypes of this form of pulmonary cancer. A team of researchers from Thailand set out to explore the association between histological subtypes and genetic alterations, as well as PD-L1 expression. The aim of the study was to identify the association between histological subtypes of pulmonary ADC and genetic alterations identified by next-generation sequencing in a Thai population. Using VarSeq for their variant discovery analysis, the data revealed that genetic mutations and the PD-L1 expression level may serve as representative predictive factors for lung ADC, and it may be useful in developing targeted immunotherapeutic strategies. The results of this study may pave the way to selecting appropriate areas of the heterogeneous tumor feature for the patients who require personalized therapy and may also be of use when predicting their prognosis.

Ramathibodi Hospital, Thailand / Published in Molecular and Clinical Oncology

I hope you enjoyed this collection of featured publications. To find the complete list of publications citing Golden Helix products, please visit our Published Articles page on our website.

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