Influence of Unequal Amplification of Methylated and Non-Methylated Template on Performance of Pyrosequencing

TY - JOUR

T1 - Influence of Unequal Amplification of Methylated and Non-Methylated Template on Performance of Pyrosequencing

AU - Taryma-Lesniak, Olga

AU - Kjeldsen, Tina E.

AU - Hansen, Lise Lotte

AU - Wojdacz, Tomasz K.

N1 - Publisher Copyright: © 2022 by the authors.

PY - 2022/8

Y1 - 2022/8

N2 - Pyrosequencing is one of the technologies widely used for quantitative methylation assessment. The protocol of pyrosequencing experiment consists of PCR amplification of a locus of interest and subsequent sequencing via synthesis of the amplified PCR product. As the PCR in this protocol utilizes one primer set for the amplification of a template originating from both methylated and non-methylated versions of the analysed locus, the unequal amplification of one of the templates may affect the methylation level assessment by pyrosequencing. We have investigated whether the unequal amplification of one of the templates challenges the quantitative properties of the pyrosequencing technology. Our results show that the sensitivity and dynamic range of pyrosequencing can be significantly affected by unequal amplification of the methylated and non-methylated version of the locus of interest in an assay specific manner. Thus, the assessment of the effect of unequal template amplification on the performances of the specific pyrosequencing assay is necessary before using the assay either in research or especially in diagnostic settings.

AB - Pyrosequencing is one of the technologies widely used for quantitative methylation assessment. The protocol of pyrosequencing experiment consists of PCR amplification of a locus of interest and subsequent sequencing via synthesis of the amplified PCR product. As the PCR in this protocol utilizes one primer set for the amplification of a template originating from both methylated and non-methylated versions of the analysed locus, the unequal amplification of one of the templates may affect the methylation level assessment by pyrosequencing. We have investigated whether the unequal amplification of one of the templates challenges the quantitative properties of the pyrosequencing technology. Our results show that the sensitivity and dynamic range of pyrosequencing can be significantly affected by unequal amplification of the methylated and non-methylated version of the locus of interest in an assay specific manner. Thus, the assessment of the effect of unequal template amplification on the performances of the specific pyrosequencing assay is necessary before using the assay either in research or especially in diagnostic settings.

KW - DNA methylation

KW - in vitro diagnostic

KW - IVD

KW - pyrosequencing

UR - http://www.scopus.com/inward/record.url?scp=85136613421&partnerID=8YFLogxK

U2 - 10.3390/genes13081418

DO - 10.3390/genes13081418

M3 - Journal article

C2 - 36011328

AN - SCOPUS:85136613421

SN - 2073-4425

VL - 13

JO - Genes

JF - Genes

IS - 8

M1 - 1418

ER -