Cargando…

Getting rid of ‘rain’ and ‘stars’: Mitigating inhibition effects on ddPCR data analysis, the case study of the invasive crayfish Pacifastacus leniusculus in the streams of Luxembourg

ddPCR is becoming one of the most widely used tool in the field of eDNA-based aquatic monitoring. Although emulsion PCR used in ddPCR confers a partial mitigation to inhibition due to the high number of reactions for a single sample (between 10K and 20K), it is not impervious to it. Our results show...

Descripción completa

Detalles Bibliográficos
Autores principales: Porco, David, Hermant, Sylvie, Purnomo, Chanistya Ayu, Horn, Mario, Marson, Guy, Colling, Guy
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9668142/
https://www.ncbi.nlm.nih.gov/pubmed/36383571
http://dx.doi.org/10.1371/journal.pone.0275363
_version_ 1784831850418536448
author Porco, David
Hermant, Sylvie
Purnomo, Chanistya Ayu
Horn, Mario
Marson, Guy
Colling, Guy
author_facet Porco, David
Hermant, Sylvie
Purnomo, Chanistya Ayu
Horn, Mario
Marson, Guy
Colling, Guy
author_sort Porco, David
collection PubMed
description ddPCR is becoming one of the most widely used tool in the field of eDNA-based aquatic monitoring. Although emulsion PCR used in ddPCR confers a partial mitigation to inhibition due to the high number of reactions for a single sample (between 10K and 20K), it is not impervious to it. Our results showed that inhibition impacts the amplitude of fluorescence in positive droplets with a different intensity among rivers. This signal fluctuation could jeopardize the use of a shared threshold among samples from different origin, and thus the accurate assignment of the positive droplets which is particularly important for low concentration samples such as eDNA ones: amplification events are scarce, thus their objective discrimination as positive is crucial. Another issue, related to target low concentration, is the artifactual generation of high fluorescence droplets (‘stars’). Indeed, these could be counted as positive with a single threshold solution, which in turn could produce false positive and incorrect target concentration assessments. Approximating the positive and negative droplets distribution as normal, we proposed here a double threshold method accounting for both high fluorescence droplets (‘stars’) and PCR inhibition impact in delineating positive droplets clouds. In the context of low concentration template recovered from environmental samples, the application of this method of double threshold establishment could allow for a consistent sorting of the positive and negative droplets throughout ddPCR data generated from samples with varying levels of inhibitor contents. Due to low concentrations template and inhibition effects, Quantasoft software produced an important number of false negatives and positive comparatively to the double threshold method developed here. This case study allowed the detection of the invasive crayfish P. leniusculus in 32 out of 34 sampled sites from two main rivers (Alzette and Sûre) and five of their tributaries (Eisch, Attert, Mamer, Wiltz and Clerve).
format Online
Article
Text
id pubmed-9668142
institution National Center for Biotechnology Information
language English
publishDate 2022
publisher Public Library of Science
record_format MEDLINE/PubMed
spelling pubmed-96681422022-11-17 Getting rid of ‘rain’ and ‘stars’: Mitigating inhibition effects on ddPCR data analysis, the case study of the invasive crayfish Pacifastacus leniusculus in the streams of Luxembourg Porco, David Hermant, Sylvie Purnomo, Chanistya Ayu Horn, Mario Marson, Guy Colling, Guy PLoS One Research Article ddPCR is becoming one of the most widely used tool in the field of eDNA-based aquatic monitoring. Although emulsion PCR used in ddPCR confers a partial mitigation to inhibition due to the high number of reactions for a single sample (between 10K and 20K), it is not impervious to it. Our results showed that inhibition impacts the amplitude of fluorescence in positive droplets with a different intensity among rivers. This signal fluctuation could jeopardize the use of a shared threshold among samples from different origin, and thus the accurate assignment of the positive droplets which is particularly important for low concentration samples such as eDNA ones: amplification events are scarce, thus their objective discrimination as positive is crucial. Another issue, related to target low concentration, is the artifactual generation of high fluorescence droplets (‘stars’). Indeed, these could be counted as positive with a single threshold solution, which in turn could produce false positive and incorrect target concentration assessments. Approximating the positive and negative droplets distribution as normal, we proposed here a double threshold method accounting for both high fluorescence droplets (‘stars’) and PCR inhibition impact in delineating positive droplets clouds. In the context of low concentration template recovered from environmental samples, the application of this method of double threshold establishment could allow for a consistent sorting of the positive and negative droplets throughout ddPCR data generated from samples with varying levels of inhibitor contents. Due to low concentrations template and inhibition effects, Quantasoft software produced an important number of false negatives and positive comparatively to the double threshold method developed here. This case study allowed the detection of the invasive crayfish P. leniusculus in 32 out of 34 sampled sites from two main rivers (Alzette and Sûre) and five of their tributaries (Eisch, Attert, Mamer, Wiltz and Clerve). Public Library of Science 2022-11-16 /pmc/articles/PMC9668142/ /pubmed/36383571 http://dx.doi.org/10.1371/journal.pone.0275363 Text en © 2022 Porco et al https://creativecommons.org/licenses/by/4.0/This is an open access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
spellingShingle Research Article
Porco, David
Hermant, Sylvie
Purnomo, Chanistya Ayu
Horn, Mario
Marson, Guy
Colling, Guy
Getting rid of ‘rain’ and ‘stars’: Mitigating inhibition effects on ddPCR data analysis, the case study of the invasive crayfish Pacifastacus leniusculus in the streams of Luxembourg
title Getting rid of ‘rain’ and ‘stars’: Mitigating inhibition effects on ddPCR data analysis, the case study of the invasive crayfish Pacifastacus leniusculus in the streams of Luxembourg
title_full Getting rid of ‘rain’ and ‘stars’: Mitigating inhibition effects on ddPCR data analysis, the case study of the invasive crayfish Pacifastacus leniusculus in the streams of Luxembourg
title_fullStr Getting rid of ‘rain’ and ‘stars’: Mitigating inhibition effects on ddPCR data analysis, the case study of the invasive crayfish Pacifastacus leniusculus in the streams of Luxembourg
title_full_unstemmed Getting rid of ‘rain’ and ‘stars’: Mitigating inhibition effects on ddPCR data analysis, the case study of the invasive crayfish Pacifastacus leniusculus in the streams of Luxembourg
title_short Getting rid of ‘rain’ and ‘stars’: Mitigating inhibition effects on ddPCR data analysis, the case study of the invasive crayfish Pacifastacus leniusculus in the streams of Luxembourg
title_sort getting rid of ‘rain’ and ‘stars’: mitigating inhibition effects on ddpcr data analysis, the case study of the invasive crayfish pacifastacus leniusculus in the streams of luxembourg
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9668142/
https://www.ncbi.nlm.nih.gov/pubmed/36383571
http://dx.doi.org/10.1371/journal.pone.0275363
work_keys_str_mv AT porcodavid gettingridofrainandstarsmitigatinginhibitioneffectsonddpcrdataanalysisthecasestudyoftheinvasivecrayfishpacifastacusleniusculusinthestreamsofluxembourg
AT hermantsylvie gettingridofrainandstarsmitigatinginhibitioneffectsonddpcrdataanalysisthecasestudyoftheinvasivecrayfishpacifastacusleniusculusinthestreamsofluxembourg
AT purnomochanistyaayu gettingridofrainandstarsmitigatinginhibitioneffectsonddpcrdataanalysisthecasestudyoftheinvasivecrayfishpacifastacusleniusculusinthestreamsofluxembourg
AT hornmario gettingridofrainandstarsmitigatinginhibitioneffectsonddpcrdataanalysisthecasestudyoftheinvasivecrayfishpacifastacusleniusculusinthestreamsofluxembourg
AT marsonguy gettingridofrainandstarsmitigatinginhibitioneffectsonddpcrdataanalysisthecasestudyoftheinvasivecrayfishpacifastacusleniusculusinthestreamsofluxembourg
AT collingguy gettingridofrainandstarsmitigatinginhibitioneffectsonddpcrdataanalysisthecasestudyoftheinvasivecrayfishpacifastacusleniusculusinthestreamsofluxembourg