Comparing PCR-generated artifacts of different polymerases for improved accuracy of DNA metabarcoding

Accuracy of PCR amplification is vital for obtaining reliable amplicon-sequencing results by metabarcoding. Here, we performed a comparative analysis of error profiles in the PCR products by 14 different PCR kits using a mock eukaryotic community DNA sample mimicking metabarcoding analysis. To prepa...
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Gespeichert in:

Autor*in:	Satoshi Nagai [verfasserIn] Sirje Sildever [verfasserIn] Noriko Nishi [verfasserIn] Satoshi Tazawa [verfasserIn] Leila Basti [verfasserIn] Takanori Kobayashi [verfasserIn] Yoshizumi Ishino [verfasserIn]

Format:	E-Artikel
Sprache:	Englisch

Erschienen:	2022

Übergeordnetes Werk:	In: Metabarcoding and Metagenomics - Pensoft Publishers, 2018, 6(2022), Seite 27-39
Übergeordnetes Werk:	volume:6 ; year:2022 ; pages:27-39

Links:	Link aufrufen Link aufrufen Link aufrufen Link aufrufen Link aufrufen Journal toc

DOI / URN:	10.3897/mbmg.6.77704

Katalog-ID:	DOAJ076182843

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author	Satoshi Nagai
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topic_title	QH540-549.5 Comparing PCR-generated artifacts of different polymerases for improved accuracy of DNA metabarcoding
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title	Comparing PCR-generated artifacts of different polymerases for improved accuracy of DNA metabarcoding
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title_full	Comparing PCR-generated artifacts of different polymerases for improved accuracy of DNA metabarcoding
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author_browse	Satoshi Nagai Sirje Sildever Noriko Nishi Satoshi Tazawa Leila Basti Takanori Kobayashi Yoshizumi Ishino
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title_sort	comparing pcr-generated artifacts of different polymerases for improved accuracy of dna metabarcoding
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title_auth	Comparing PCR-generated artifacts of different polymerases for improved accuracy of DNA metabarcoding
abstract	Accuracy of PCR amplification is vital for obtaining reliable amplicon-sequencing results by metabarcoding. Here, we performed a comparative analysis of error profiles in the PCR products by 14 different PCR kits using a mock eukaryotic community DNA sample mimicking metabarcoding analysis. To prepare a mock eukaryotic community from the marine environment, equal amounts of plasmid DNA from 40 microalgal species were mixed and used for amplicon-sequencing by a high-throughput sequencing approach. To compare the differences in PCR kits used for this experiment, we focused on the following seven parameters: 1) Quality, 2) Chimera, 3) Blast top hit accuracy, 4) Deletion, 5) Insertion, 6) Base substitution and 7) Amplification bias amongst species. The results showed statistically significant differences (p < 0.05) for all of the seven parameters depending on the PCR kits used. These differences may result from the different DNA polymerases included in each kit, although the result can also be influenced by PCR reaction conditions. Simultaneous analysis of several parameters suggested that kits containing KOD plus Neo (TOYOBO) and HotStart Taq DNA polymerase (BiONEER, CA, US) at the annealing temperature of 65 °C displayed better results in terms of parameters associated with chimeras, top hit similarity and deletions.
abstractGer	Accuracy of PCR amplification is vital for obtaining reliable amplicon-sequencing results by metabarcoding. Here, we performed a comparative analysis of error profiles in the PCR products by 14 different PCR kits using a mock eukaryotic community DNA sample mimicking metabarcoding analysis. To prepare a mock eukaryotic community from the marine environment, equal amounts of plasmid DNA from 40 microalgal species were mixed and used for amplicon-sequencing by a high-throughput sequencing approach. To compare the differences in PCR kits used for this experiment, we focused on the following seven parameters: 1) Quality, 2) Chimera, 3) Blast top hit accuracy, 4) Deletion, 5) Insertion, 6) Base substitution and 7) Amplification bias amongst species. The results showed statistically significant differences (p < 0.05) for all of the seven parameters depending on the PCR kits used. These differences may result from the different DNA polymerases included in each kit, although the result can also be influenced by PCR reaction conditions. Simultaneous analysis of several parameters suggested that kits containing KOD plus Neo (TOYOBO) and HotStart Taq DNA polymerase (BiONEER, CA, US) at the annealing temperature of 65 °C displayed better results in terms of parameters associated with chimeras, top hit similarity and deletions.
abstract_unstemmed	Accuracy of PCR amplification is vital for obtaining reliable amplicon-sequencing results by metabarcoding. Here, we performed a comparative analysis of error profiles in the PCR products by 14 different PCR kits using a mock eukaryotic community DNA sample mimicking metabarcoding analysis. To prepare a mock eukaryotic community from the marine environment, equal amounts of plasmid DNA from 40 microalgal species were mixed and used for amplicon-sequencing by a high-throughput sequencing approach. To compare the differences in PCR kits used for this experiment, we focused on the following seven parameters: 1) Quality, 2) Chimera, 3) Blast top hit accuracy, 4) Deletion, 5) Insertion, 6) Base substitution and 7) Amplification bias amongst species. The results showed statistically significant differences (p < 0.05) for all of the seven parameters depending on the PCR kits used. These differences may result from the different DNA polymerases included in each kit, although the result can also be influenced by PCR reaction conditions. Simultaneous analysis of several parameters suggested that kits containing KOD plus Neo (TOYOBO) and HotStart Taq DNA polymerase (BiONEER, CA, US) at the annealing temperature of 65 °C displayed better results in terms of parameters associated with chimeras, top hit similarity and deletions.
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title_short	Comparing PCR-generated artifacts of different polymerases for improved accuracy of DNA metabarcoding
url	https://doi.org/10.3897/mbmg.6.77704 https://doaj.org/article/da63cc4383144e038f00ac0e93574229 https://mbmg.pensoft.net/article/77704/download/pdf/ https://mbmg.pensoft.net/article/77704/download/xml/ https://mbmg.pensoft.net/article/77704/ https://doaj.org/toc/2534-9708
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﻿Comparing PCR-generated artifacts of different polymerases for improved accuracy of DNA metabarcoding

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Comparing PCR-generated artifacts of different polymerases for improved accuracy of DNA metabarcoding