Abstract：
:The objective of this study was to evaluate the effects of increased PCR cycle number on sequencing results from samples with low microbial biomass, including bovine milk, and murine pelage and blood. We hypothesized that subjecting DNA from such samples to higher PCR cycle numbers would increase 16S rRNA sequencing coverage. DNA was extracted from matched samples of each type and multiple PCR cycle numbers were evaluated to generate a total of 96 libraries from 24 milk samples, 46 libraries from 23 pelage samples, and 170 libraries from 85 blood samples. 16S rRNA sequencing was performed on the Illumina MiSeq platform, and the coverage per sample, detected richness, and beta-diversity were evaluated. Across all sample types, higher PCR cycle numbers were associated with increased coverage. Surprisingly however, while higher PCR cycle numbers resulted in greater number of useable datapoints, no differences were detected in metrics of richness or beta-diversity. While reagent controls amplified for 40 cycles yielded similarly increased coverage, control and experimental samples were clearly differentiated based on beta-diversity. The results from this study support the use of higher PCR cycle numbers to evaluate samples with low microbial biomass.
journal_name
J Microbiol Methodsjournal_title
Journal of microbiological methodsauthors
Witzke MC,Gullic A,Yang P,Bivens NJ,Adkins PRF,Ericsson ACdoi
10.1016/j.mimet.2020.106033subject
Has Abstractpub_date
2020-09-01 00:00:00pages
106033eissn
0167-7012issn
1872-8359pii
S0167-7012(20)30749-1journal_volume
176pub_type
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journal_title：Journal of microbiological methods
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pub_type： 杂志文章,评审
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journal_title：Journal of microbiological methods
pub_type： 杂志文章
doi：10.1016/j.mimet.2010.12.018
更新日期：2011-02-01 00:00:00
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journal_title：Journal of microbiological methods
pub_type： 杂志文章
doi：10.1016/j.mimet.2013.12.021
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journal_title：Journal of microbiological methods
pub_type： 杂志文章
doi：10.1016/j.mimet.2011.02.010
更新日期：2011-05-01 00:00:00
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journal_title：Journal of microbiological methods
pub_type： 杂志文章
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journal_title：Journal of microbiological methods
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abstract：BACKGROUND:The development of oral vaccines using yeast surface display technology is an area of intensive study in vaccine development, but the protein level displayed on yeast surfaces is not currently high enough to obtain a robust immune response. METHODS:To address this issue, we established an efficient and simp...
journal_title：Journal of microbiological methods
pub_type： 杂志文章
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