Explore the expertise of IAB’s specialists in a curated collection of posters and publications based on the most intricate customized NGS projects. Hundreds of unique projects enable us to build a knowledge base and enrich our expertise. Don’t hesitate to contact us, even with the most complex scientific problems. Our technologies are at your service.
Exosome-derived microRNA NGS library preparation
Exosomes are lipid vesicles (30-150 nm in diameter) released by cells into the blood, urine, breast milk and many other body fluids. Various miRNA species were proposed to play an important role in the distant cell-to-cell signalling and regulation of gene/cell function, including tumorigenesis. Therefore, analysis of exosome-derived miRNA species constitutes a quickly evolving field with huge implications in research and diagnostics (Huang T. et al. 2019). Next-generation sequencing (NGS) proved to play a crucial role in the discovery of both known and novel small RNA species, however, the specifics of small RNA NGS library preparation present obstacles usually associated with limited RNA amount, so typical for the isolates from exosomes or body fluids. Since the miRNA represents a tiny fraction of total RNA, the limited amount primarily leads to the abundance of high adaptor-dimer content in the resulting library. Due to its size (18-34 nt), removing dimer sequences from the small RNA library might be limited or impossible. The presence of unwanted dimer molecules leads to a partial or complete loss of library during additional clean-ups and lower or even ruin the descriptive potential of sequencing data. Also, the limited input is more prone to bias the abundance of different miRNA species as more amplification steps in the library preparation protocol are needed. Here we present our experience with the QIAseq miRNA library kit (QIAGEN) applied on isolates from body fluid-derived exosomes.
Novel long-read sequencing technology in practise
Whole genome short-read sequencing is currently commonly used in number of diagnostic applications. The limitations are mostly related to difficulties in mapping of short reads to the challenging regions of the reference genome such as highly homologous regions and repetitive regions. Long-read sequencing is gaining ground in clinical applications as it overcomes some of these issues. However, many long-read sequencing solutions have been plagued by high DNA input requirements, complex workflows with low throughput and highly variable results and these have limited their utility and adoption. Illumina Complete Long Reads (ILCR) makes long-read sequencing accessible and streamlined for genomic labs. Additionally, this protocol enables generation of comprehensive human genomic data with combination of both long and short reads sequencing.
Published in BMC Genomics (IAB in collaboration with research partners)
Blood-derived genomic DNA (gDNA) is routinely used in the clinical environment. Numerous POPGEN studies and commercial tests benefit from easy saliva sampling. In our validation study we proved, that quality pattern of called variants obtained from genomic-reference-based technical replicates correlates with data calls of paired blood–saliva-derived samples in all levels of tested examinations. When considering non-human-mappable reads, we found that they are present in relatively large proportions in the saliva-derived gDNA when compared with blood-derived gDNA, despite a strict sampling protocol. Although microbiome-to-human misalignment cannot be equivocally ruled out, the results suggest that the end effect does not deviate sequencing accuracy from values typically obtained using blood-derived gDNA.
According to our protocol the saliva can be considered an equivalent material to blood for genetic data analysis.
Kvapilova, K., Misenko, P., Radvanszky, J. et al. Validated WGS and WES protocols proved saliva-derived gDNA as an equivalent to blood-derived gDNA for clinical and population genomic analyses. BMC Genomics 25, 187 (2024).
Adoption of small custom-designed NGS panel for rare FFPE samples of macaque monkey
Whole exome sequencing (WES) is a targeted next-generation sequencing method that identifies protein-coding genes (exons) in the genome. Targeted sequencing using custom-designed NGS panels offers a cost-effective method for detecting rare variants. Important advantage of custom target sequencing is the possibility to personalize the panel (i.e., the inclusion of certain genes and the possibility to sequence exons, specific intronic regions, promoter regions, or the 3′ untranslated regions). On the other hand customized NGS panel designs have to be updated and optimized with regard to gene content or inclusion of known intronic splice variants. It was the optimization of the NGS library preparation from DNA samples using a custom panel that IAB dealt with. The data presented here originate from project of SOTIO Biotech a.s.
Evaluation of WGS and WES protocols based on dried bood spot sample-derived gDNA
Whole exome sequencing (WES) and whole genome sequencing (WGS) have become a standard method in human clinical genetics. Blood-derived gDNA is routinely used in the clinical environment, presenting difficulties associated with invasive sampling. Dried venous blood spots (DBS) represent a convenient gDNA source in respect to low amount of collected material and simple sample transport and storage. However, the use of DBS-derived gDNA for NGS applications has not been analyzed in detail. To address this point, IAB validated both WGS and WES NGS protocols based on DBS-derived isolates.
Validated WGS and WES protocols proved salivaderived gDNA as an equivalent to blood-derived gDNA for clinical and population genomic analyses
Whole exome sequencing (WES) and whole genome sequencing (WGS) have become standard methods in human clinical diagnostics as well as in population genomics (POPGEN). Blood-derived genomic DNA (gDNA) is routinely used in the clinical environment. Conversely, many POPGEN studies and commercial tests benefit from easy saliva sampling. Here, we evaluated the quality of variant call sets, the level of genotype concordance of single nucleotide variants (SNVs), and small insertions and deletions (indels) for WES and WGS using paired blood- and salivaderived gDNA isolates employing genomic reference-based validated protocols.
