Pyrosequencing is a DNA sequencing technology that has many applications including rapid genotyping of single nucleotide polymorphisms (SNP) and other sequence variations between different cells, individuals and species. Identifying the specific composition of microorganisms inside a microbial population may help choose the most specific treatment in case of infectious disease. Likewise, pyro-sequencing can also apply to the screening and identification of oncogenic mutations(s) in a heterogeneous sample containing tumour cells of interest in a background of wild type cells. Converting a pyrosequencing signal into a nucleotide sequence appears highly challenging when signal intensities are low (unitary peak heights <5) or when complex signals are produced by several target amplicons. In these cases, the pyrosequencing software fails to provide correct nucleotide sequences. At the CTMA (Center for Applied Molecular Technologies), researchers developed the AdvISER-PYRO, an algorithm based on a machine learning method (i.e., a sparse representation) to perform an automated, fast and reliable analysis of pyrosequencing signals which circumvents above limitation (Figure 1). Analysis was carried out on pyrosequencing signals generated in various clinical applications including bacterial and human genetics. For exemple, AdvISER-PYRO was applied to genotype the human thiopurine S-methyltransferase (TPMT), which is an enzyme responsible for the detoxification of widely used thiopurine drugs such as azathioprine (Aza). Its activity is inversely related to the risk of developing severe hematopoietic toxicity in certain patients treated with standard doses of thiopurines. Multiplex pyrosequencing assay using AdvISER-PYRO algorithm proved to be a rapid, reliable and cost-effective way to genotype the most common (i.e. TPMT*2, *3A, *3C) as well as less common TPMT alleles (i.e., TPMT*3B, *9, *12, *16, * 19, *20, *21, *25, *33). These findings improved the knowledge of the genetic basis of inter-individual variability in TPMT activity and enhanced the efficiency of genotyping methods to predict patients at risk of inadequate responses to thiopurine therapy.
Researchers:
- Jérôme Ambroise, Master of Science in Chemical Engineering and Master of Advanced Studies in Statistics, Biostatistics and Epidemiology, PhD Engineering, researcher logistician
- Jean-Luc Gala, MD, PhD, Full Professor, Chief resident at Cliniques universitaires St Luc, retired Colonel, and head of the CTMA and of its military Biological CBRN research branch, Defence Laboratories Department (DLD-Bio).
Publications:
- Ambroise, J., Piette, A. S., Delcorps, C., Rigouts, L., de Jong, B. C., Irenge, L., … & Gala, J. L. (2013). Adviser-pyro: amplicon identification using sparse representation of pyrosequencing signal. Bioinformatics, 29(16), 1963-1969.
- Ambroise, J., Deccache, Y., Irenge, L., Savov, E., Robert, A., & Gala, J. L. (2014). Amplicon identification using SparsE representation of multiplex PYROsequencing signal (AdvISER-M-PYRO): application to bacterial resistance genotyping. Bioinformatics, 30(24), 3590-3597.
- Ambroise, J., Butoescu, V., Robert, A., Tombal, B., & Gala, J. L. (2015). Multiplex pyrosequencing assay using AdvISER-MH-PYRO algorithm: a case for rapid and cost-effective genotyping analysis of prostate cancer risk-associated SNPs. BMC medical genetics, 16(1), 42.
Link with the laboratory: The AdvISER-MH-PYRO algorithm is used every day for our clinical analytical work aiming to identify TPMPT gene mutaiotns in patient samples referred to Cliniques Universitaires Saint Luc as wll as on smaples sent by external laboratories. on TPTMT and it has proved to be reliable and accurate (blind test / proficiency tests controlled yearly by an external certified organisms: 100% matching results over the last four years)
CTMA Website: https://uclouvain.be/fr/instituts-recherche/irec/ctma
Figure 1: Examples of signal identification with AdvISER-PYRO. The pyrosequencing signal is represented by vertical black lines. The deconvolution of the signal enables to genotype several SNPs with a single pyrosequencing reaction.