Gradient Internal Standard Method for Absolute Quantification of Microbial Amplicon Sequencing Data

ABSTRACT High-throughput amplicon sequencing is a critical tool for studying microbiota; however, it results only in relative abundance data. Thus, changes in absolute abundance of microbiota cannot be determined, which hinders further microbiology research. We have therefore established a gradient...

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Autores principales:	Shilei Wang, Qun Wu, Ying Han, Rubing Du, Xiaoyong Wang, Yao Nie, Xiaowei Du, Yan Xu
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2021
Materias:	internal standards absolute quantification microbiota solid-state fermentation Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/06fcc614b6ba473390b51fe9e0d65773
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spelling	oai:doaj.org-article:06fcc614b6ba473390b51fe9e0d657732021-12-02T19:36:37ZGradient Internal Standard Method for Absolute Quantification of Microbial Amplicon Sequencing Data10.1128/mSystems.00964-202379-5077https://doaj.org/article/06fcc614b6ba473390b51fe9e0d657732021-02-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mSystems.00964-20https://doaj.org/toc/2379-5077ABSTRACT High-throughput amplicon sequencing is a critical tool for studying microbiota; however, it results only in relative abundance data. Thus, changes in absolute abundance of microbiota cannot be determined, which hinders further microbiology research. We have therefore established a gradient internal standard absolute quantification (GIS-AQ) method to overcome this issue, which can simultaneously obtain the absolute abundances of bacteria and fungi. Deviations from the quantitative equations of microbes and internal standards were eliminated through calibration. Compared with traditional quantitative real-time PCR and microscopy quantifications, this method is reliable (R2average = 0.998; P < 0.001) and accurate (Pinternals versus microscopy > 0.05). The GIS-AQ method can be adapted to any amplicon primer choice (e.g., 336F/806R and ITS3/ITS4), rendering it applicable to ecosystem studies including food, soil, and water samples. Crucially, when using solid-state fermentation samples from various temporal dimensions, the results obtained from the relative and absolute abundance are different. The absolute abundance can be used to study the difference in communities between different samples, and the GIS-AQ method allows this to be done rapidly. Therefore, combining the absolute abundance with relative abundance can accurately reflect the microbiota composition. IMPORTANCE To solve the problem of amplicon sequencing cannot discern the microbiota absolute abundance, we proposed a gradient internal standard absolute quantification method. We used Chinese liquor fermentation as a model system to demonstrate the reliability and accuracy of the method. By comparing the relative and absolute abundances of microbiota in various temporal dimensions, we found dynamic changes in the absolute abundance of communities under various temporal dimensions from the relative abundance. Based on its design principle, this method can be widely applied to different ecosystems. Therefore, we believe that the GIS-AQ method can play an immeasurably useful role in microbiological research.Shilei WangQun WuYing HanRubing DuXiaoyong WangYao NieXiaowei DuYan XuAmerican Society for Microbiologyarticleinternal standardsabsolute quantificationmicrobiotasolid-state fermentationMicrobiologyQR1-502ENmSystems, Vol 6, Iss 1 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	internal standards absolute quantification microbiota solid-state fermentation Microbiology QR1-502
spellingShingle	internal standards absolute quantification microbiota solid-state fermentation Microbiology QR1-502 Shilei Wang Qun Wu Ying Han Rubing Du Xiaoyong Wang Yao Nie Xiaowei Du Yan Xu Gradient Internal Standard Method for Absolute Quantification of Microbial Amplicon Sequencing Data
description	ABSTRACT High-throughput amplicon sequencing is a critical tool for studying microbiota; however, it results only in relative abundance data. Thus, changes in absolute abundance of microbiota cannot be determined, which hinders further microbiology research. We have therefore established a gradient internal standard absolute quantification (GIS-AQ) method to overcome this issue, which can simultaneously obtain the absolute abundances of bacteria and fungi. Deviations from the quantitative equations of microbes and internal standards were eliminated through calibration. Compared with traditional quantitative real-time PCR and microscopy quantifications, this method is reliable (R2average = 0.998; P < 0.001) and accurate (Pinternals versus microscopy > 0.05). The GIS-AQ method can be adapted to any amplicon primer choice (e.g., 336F/806R and ITS3/ITS4), rendering it applicable to ecosystem studies including food, soil, and water samples. Crucially, when using solid-state fermentation samples from various temporal dimensions, the results obtained from the relative and absolute abundance are different. The absolute abundance can be used to study the difference in communities between different samples, and the GIS-AQ method allows this to be done rapidly. Therefore, combining the absolute abundance with relative abundance can accurately reflect the microbiota composition. IMPORTANCE To solve the problem of amplicon sequencing cannot discern the microbiota absolute abundance, we proposed a gradient internal standard absolute quantification method. We used Chinese liquor fermentation as a model system to demonstrate the reliability and accuracy of the method. By comparing the relative and absolute abundances of microbiota in various temporal dimensions, we found dynamic changes in the absolute abundance of communities under various temporal dimensions from the relative abundance. Based on its design principle, this method can be widely applied to different ecosystems. Therefore, we believe that the GIS-AQ method can play an immeasurably useful role in microbiological research.
format	article
author	Shilei Wang Qun Wu Ying Han Rubing Du Xiaoyong Wang Yao Nie Xiaowei Du Yan Xu
author_facet	Shilei Wang Qun Wu Ying Han Rubing Du Xiaoyong Wang Yao Nie Xiaowei Du Yan Xu
author_sort	Shilei Wang
title	Gradient Internal Standard Method for Absolute Quantification of Microbial Amplicon Sequencing Data
title_short	Gradient Internal Standard Method for Absolute Quantification of Microbial Amplicon Sequencing Data
title_full	Gradient Internal Standard Method for Absolute Quantification of Microbial Amplicon Sequencing Data
title_fullStr	Gradient Internal Standard Method for Absolute Quantification of Microbial Amplicon Sequencing Data
title_full_unstemmed	Gradient Internal Standard Method for Absolute Quantification of Microbial Amplicon Sequencing Data
title_sort	gradient internal standard method for absolute quantification of microbial amplicon sequencing data
publisher	American Society for Microbiology
publishDate	2021
url	https://doaj.org/article/06fcc614b6ba473390b51fe9e0d65773
work_keys_str_mv	AT shileiwang gradientinternalstandardmethodforabsolutequantificationofmicrobialampliconsequencingdata AT qunwu gradientinternalstandardmethodforabsolutequantificationofmicrobialampliconsequencingdata AT yinghan gradientinternalstandardmethodforabsolutequantificationofmicrobialampliconsequencingdata AT rubingdu gradientinternalstandardmethodforabsolutequantificationofmicrobialampliconsequencingdata AT xiaoyongwang gradientinternalstandardmethodforabsolutequantificationofmicrobialampliconsequencingdata AT yaonie gradientinternalstandardmethodforabsolutequantificationofmicrobialampliconsequencingdata AT xiaoweidu gradientinternalstandardmethodforabsolutequantificationofmicrobialampliconsequencingdata AT yanxu gradientinternalstandardmethodforabsolutequantificationofmicrobialampliconsequencingdata
_version_	1718376361017475072

Gradient Internal Standard Method for Absolute Quantification of Microbial Amplicon Sequencing Data

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