TY - JOUR
T1 - Impact of contaminating DNA in whole-genome amplification kits used for metagenomic shotgun sequencing for infection diagnosis
AU - Thoendel, Matthew
AU - Jeraldo, Patricio
AU - Greenwood-Quaintance, Kerryl E.
AU - Yao, Janet
AU - Chia, Nicholas
AU - Hanssen, Arlen D.
AU - Abdel, Matthew P.
AU - Patela, Robin
N1 - Publisher Copyright:
© 2017 American Society for Microbiology. All Rights Reserved.
PY - 2017/6
Y1 - 2017/6
N2 - Whole-genome amplification (WGA) is a useful tool for amplification of very small quantities of DNA for many uses, including metagenomic shotgun sequencing for infection diagnosis. Depending on the application, background DNA from WGA kits can be problematic. Three WGA kits were tested for their utility in a metagenomics approach to identify the pathogens in sonicate fluid comprised of biofilms and other materials dislodged from the surfaces of explanted prosthetic joints using sonication. The Illustra V2 Genomiphi, Illustra single cell Genomiphi, and Qiagen REPLI-g single cell kits were used to test identical sonicate fluid samples. Variations in the number of background reads, the genera identified in the background, and the number of reads from known pathogens known to be present in the samples were observed between kits. These results were then compared to those obtained with a library preparation without prior WGA using an NEBNext Ultra II paired-end kit, which requires a very small amount of input DNA. This approach also resulted in the presence of contaminant bacterial DNA and yielded fewer reads from the known pathogens. These findings highlight the impact that WGA kit selection can have on metagenomic analysis of low-biomass samples and the importance of the careful selection and consideration of the implications of using these tools.
AB - Whole-genome amplification (WGA) is a useful tool for amplification of very small quantities of DNA for many uses, including metagenomic shotgun sequencing for infection diagnosis. Depending on the application, background DNA from WGA kits can be problematic. Three WGA kits were tested for their utility in a metagenomics approach to identify the pathogens in sonicate fluid comprised of biofilms and other materials dislodged from the surfaces of explanted prosthetic joints using sonication. The Illustra V2 Genomiphi, Illustra single cell Genomiphi, and Qiagen REPLI-g single cell kits were used to test identical sonicate fluid samples. Variations in the number of background reads, the genera identified in the background, and the number of reads from known pathogens known to be present in the samples were observed between kits. These results were then compared to those obtained with a library preparation without prior WGA using an NEBNext Ultra II paired-end kit, which requires a very small amount of input DNA. This approach also resulted in the presence of contaminant bacterial DNA and yielded fewer reads from the known pathogens. These findings highlight the impact that WGA kit selection can have on metagenomic analysis of low-biomass samples and the importance of the careful selection and consideration of the implications of using these tools.
KW - Metagenomics
KW - Prosthetic joint infection
KW - Whole-genome amplification
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U2 - 10.1128/JCM.02402-16
DO - 10.1128/JCM.02402-16
M3 - Article
C2 - 28356418
AN - SCOPUS:85019556084
SN - 0095-1137
VL - 55
SP - 1789
EP - 1801
JO - Journal of clinical microbiology
JF - Journal of clinical microbiology
IS - 6
ER -