Comparative Analysis of Sample Extraction and Library Construction for Shotgun Metagenomics
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Comparative Analysis of Sample Extraction and Library Construction for Shotgun Metagenomics. / Peng, Zonghui; Zhu, Xiaolong; Wang, Zhijiao; Yan, Xianting; Wang, Guangbiao; Tang, Meifang; Jiang, Awei; Kristiansen, Karsten.
In: Bioinformatics and Biology Insights, Vol. 14, 2020.Research output: Contribution to journal › Journal article › Research › peer-review
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TY - JOUR
T1 - Comparative Analysis of Sample Extraction and Library Construction for Shotgun Metagenomics
AU - Peng, Zonghui
AU - Zhu, Xiaolong
AU - Wang, Zhijiao
AU - Yan, Xianting
AU - Wang, Guangbiao
AU - Tang, Meifang
AU - Jiang, Awei
AU - Kristiansen, Karsten
PY - 2020
Y1 - 2020
N2 - Human fecal specimens, serve as important materials, are widely used in the field of microbiome research, in which inconsistent results have been a pressing issue. The possible attribute factors have been proposed including the specimen status after preservation, extracted DNA quality, library preparation protocol, and sample DNA input. In this study, quality comparisons for shotgun metagenomics sequencing were performed between 2 DNA extraction methods for fresh and freeze-thaw samples, 2 library preparation protocols, and various sample inputs. The results indicate that Mag-Bind® Universal Metagenomics Kit (OM) outperformed DNeasy PowerSoil Kit (QP) with a higher DNA quantity. Controlling on library preparation protocol, OM detected on-average more genes than QP. For library construction comparison by controlling on the same DNA sample, KAPA Hyper Prep Kit (KH) outperformed the TruePrep DNA Library Prep Kit V2 (TP) with the higher number of detected genes number and Shannon index. No significant differences were found in taxonomy between 2 library preparation protocols using the fresh, freeze-thaw and mock community samples. No significant difference was observed between 250 and 50 ng DNA inputs for library preparation on both fresh and freeze-thaw samples. Through the preliminary study, a combined protocol is recommended for performing metagenomics studies, by using OM method plus KH protocol as well as suitable DNA quantity on either fresh or freeze-thaw samples. Our findings provide clues for potential variations from various DNA extraction methods, library protocols, and sample DNA inputs, which are critical for consistent and comprehensive profiling of the human gut microbiome.
AB - Human fecal specimens, serve as important materials, are widely used in the field of microbiome research, in which inconsistent results have been a pressing issue. The possible attribute factors have been proposed including the specimen status after preservation, extracted DNA quality, library preparation protocol, and sample DNA input. In this study, quality comparisons for shotgun metagenomics sequencing were performed between 2 DNA extraction methods for fresh and freeze-thaw samples, 2 library preparation protocols, and various sample inputs. The results indicate that Mag-Bind® Universal Metagenomics Kit (OM) outperformed DNeasy PowerSoil Kit (QP) with a higher DNA quantity. Controlling on library preparation protocol, OM detected on-average more genes than QP. For library construction comparison by controlling on the same DNA sample, KAPA Hyper Prep Kit (KH) outperformed the TruePrep DNA Library Prep Kit V2 (TP) with the higher number of detected genes number and Shannon index. No significant differences were found in taxonomy between 2 library preparation protocols using the fresh, freeze-thaw and mock community samples. No significant difference was observed between 250 and 50 ng DNA inputs for library preparation on both fresh and freeze-thaw samples. Through the preliminary study, a combined protocol is recommended for performing metagenomics studies, by using OM method plus KH protocol as well as suitable DNA quantity on either fresh or freeze-thaw samples. Our findings provide clues for potential variations from various DNA extraction methods, library protocols, and sample DNA inputs, which are critical for consistent and comprehensive profiling of the human gut microbiome.
KW - DNA isolation method
KW - library preparation protocol
KW - Metagenomics
KW - sample input
KW - sample preservation
U2 - 10.1177/1177932220915459
DO - 10.1177/1177932220915459
M3 - Journal article
C2 - 32546984
AN - SCOPUS:85085987153
VL - 14
JO - Bioinformatics and Biology Insights
JF - Bioinformatics and Biology Insights
SN - 1177-9322
ER -
ID: 243191407