False negatives in GBA1 sequencing due to polymerase dependent allelic imbalance

Abstract A variant in the GBA1 gene is one of the most common genetic risk factors to develop Parkinson’s disease (PD). Here the serendipitous finding is reported of a polymerase dependent allelic imbalance when using next generation sequencing, potentially resulting in false-negative results when t...

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Autores principales:	Jonas M. den Heijer, Arnoud Schmitz, Peter Lansbury, Valerie C. Cullen, Dana C. Hilt, Vincenzo Bonifati, Geert Jan Groeneveld
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2021
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/db9c836049f440f78b8e0d48151c2188
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spelling	oai:doaj.org-article:db9c836049f440f78b8e0d48151c21882021-12-02T15:12:51ZFalse negatives in GBA1 sequencing due to polymerase dependent allelic imbalance10.1038/s41598-020-80564-y2045-2322https://doaj.org/article/db9c836049f440f78b8e0d48151c21882021-01-01T00:00:00Zhttps://doi.org/10.1038/s41598-020-80564-yhttps://doaj.org/toc/2045-2322Abstract A variant in the GBA1 gene is one of the most common genetic risk factors to develop Parkinson’s disease (PD). Here the serendipitous finding is reported of a polymerase dependent allelic imbalance when using next generation sequencing, potentially resulting in false-negative results when the allele frequency falls below the variant calling threshold (by default commonly at 30%). The full GBA1 gene was sequenced using next generation sequencing on saliva derived DNA from PD patients. Four polymerase chain reaction conditions were varied in twelve samples, to investigate the effect on allelic imbalance: (1) the primers (n = 4); (2) the polymerase enzymes (n = 2); (3) the primer annealing temperature (Ta) specified for the used polymerase; and (4) the amount of DNA input. Initially, 1295 samples were sequenced using Q5 High-Fidelity DNA Polymerase. 112 samples (8.6%) had an exonic variant and an additional 104 samples (8.0%) had an exonic variant that did not pass the variant frequency calling threshold of 30%. After changing the polymerase to TaKaRa LA Taq DNA Polymerase Hot-Start Version: RR042B, all samples had an allele frequency passing the calling threshold. Allele frequency was unaffected by a change in primer, annealing temperature or amount of DNA input. Sequencing of the GBA1 gene using next generation sequencing might be susceptible to a polymerase specific allelic imbalance, which can result in a large amount of flase-negative results. This was resolved in our case by changing the polymerase. Regions displaying low variant calling frequencies in GBA1 sequencing output in previous and future studies might warrant additional scrutiny.Jonas M. den HeijerArnoud SchmitzPeter LansburyValerie C. CullenDana C. HiltVincenzo BonifatiGeert Jan GroeneveldNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 11, Iss 1, Pp 1-8 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Jonas M. den Heijer Arnoud Schmitz Peter Lansbury Valerie C. Cullen Dana C. Hilt Vincenzo Bonifati Geert Jan Groeneveld False negatives in GBA1 sequencing due to polymerase dependent allelic imbalance
description	Abstract A variant in the GBA1 gene is one of the most common genetic risk factors to develop Parkinson’s disease (PD). Here the serendipitous finding is reported of a polymerase dependent allelic imbalance when using next generation sequencing, potentially resulting in false-negative results when the allele frequency falls below the variant calling threshold (by default commonly at 30%). The full GBA1 gene was sequenced using next generation sequencing on saliva derived DNA from PD patients. Four polymerase chain reaction conditions were varied in twelve samples, to investigate the effect on allelic imbalance: (1) the primers (n = 4); (2) the polymerase enzymes (n = 2); (3) the primer annealing temperature (Ta) specified for the used polymerase; and (4) the amount of DNA input. Initially, 1295 samples were sequenced using Q5 High-Fidelity DNA Polymerase. 112 samples (8.6%) had an exonic variant and an additional 104 samples (8.0%) had an exonic variant that did not pass the variant frequency calling threshold of 30%. After changing the polymerase to TaKaRa LA Taq DNA Polymerase Hot-Start Version: RR042B, all samples had an allele frequency passing the calling threshold. Allele frequency was unaffected by a change in primer, annealing temperature or amount of DNA input. Sequencing of the GBA1 gene using next generation sequencing might be susceptible to a polymerase specific allelic imbalance, which can result in a large amount of flase-negative results. This was resolved in our case by changing the polymerase. Regions displaying low variant calling frequencies in GBA1 sequencing output in previous and future studies might warrant additional scrutiny.
format	article
author	Jonas M. den Heijer Arnoud Schmitz Peter Lansbury Valerie C. Cullen Dana C. Hilt Vincenzo Bonifati Geert Jan Groeneveld
author_facet	Jonas M. den Heijer Arnoud Schmitz Peter Lansbury Valerie C. Cullen Dana C. Hilt Vincenzo Bonifati Geert Jan Groeneveld
author_sort	Jonas M. den Heijer
title	False negatives in GBA1 sequencing due to polymerase dependent allelic imbalance
title_short	False negatives in GBA1 sequencing due to polymerase dependent allelic imbalance
title_full	False negatives in GBA1 sequencing due to polymerase dependent allelic imbalance
title_fullStr	False negatives in GBA1 sequencing due to polymerase dependent allelic imbalance
title_full_unstemmed	False negatives in GBA1 sequencing due to polymerase dependent allelic imbalance
title_sort	false negatives in gba1 sequencing due to polymerase dependent allelic imbalance
publisher	Nature Portfolio
publishDate	2021
url	https://doaj.org/article/db9c836049f440f78b8e0d48151c2188
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False negatives in GBA1 sequencing due to polymerase dependent allelic imbalance

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