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Title: A Simple, Cost-Effective, and Automation-Friendly Direct PCR Approach for Bacterial Community Analysis

Journal Article · · mSystems
 [1];  [1];  [2];  [3]
  1. Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
  2. Univ. of California, Berkeley, CA (United States)
  3. Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Univ. of California, Berkeley, CA (United States)

Bacterial communities in water, soil, and humans play an essential role in environmental ecology and human health. PCR-based amplicon analysis, such as 16S rRNA sequencing, is a fundamental tool for quantifying and studying microbial composition, dynamics, and interactions. However, given the complexity of microbial communities, a substantial number of samples becomes necessary for analyses that parse the factors that determine microbial composition. A common bottleneck in performing these kinds of experiments is genomic DNA (gDNA) extraction, which is time-consuming, expensive, and often biased based on the types of species present. Direct PCR method is a potentially simpler and more accurate alternative to gDNA extraction methods that do not require the intervening purification step. In this study, we evaluated three variations of direct PCR methods using diverse heterogeneous bacterial cultures, including both Gram-positive and Gram-negative species, ZymoBIOMICS microbial community standards, and groundwater. By comparing direct PCR methods with DNeasy Blood and Tissue Kits for microbial isolates and DNeasy PowerSoil Kits for microbial communities, we found that a specific variant of the direct PCR method exhibits an overall efficiency comparable to that of the conventional DNeasy PowerSoil protocol in the circumstances we tested. We also found that the method showed higher efficiency for extracting gDNA from the Gram-negative strains compared to DNeasy Blood and Tissue protocol. This direct PCR method is 1,600 times less expensive ($0.34 for 96 samples) and 10 times simpler (15 min hands-on time for 96 samples) than the DNeasy PowerSoil protocol. The direct PCR method can also be fully automated and is compatible with small-volume samples, thereby permitting scaling of samples and replicates needed to support high-throughput large-scale bacterial community analysis.

Research Organization:
Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)
Sponsoring Organization:
USDOE
Grant/Contract Number:
AC02-05CH11231; AC02-05CH1123
OSTI ID:
1822977
Alternate ID(s):
OSTI ID: 1855188
Journal Information:
mSystems, Vol. 6, Issue 5; ISSN 2379-5077
Publisher:
American Society for MicrobiologyCopyright Statement
Country of Publication:
United States
Language:
English

References (43)

A simple method for the isolation of chromosomal DNA from Gram positive or acid-fast bacteria journal January 1991
The truth about metagenomics: quantifying and counteracting bias in 16S rRNA studies journal March 2015
A method for simultaneous measurement of soil bacterial abundances and community composition via 16S rRNA gene sequencing journal May 2016
Low concentration DNA extraction and recovery using a silica solid phase journal May 2017
The Impact of Different DNA Extraction Kits and Laboratories upon the Assessment of Human Gut Microbiota Composition by 16S rRNA Gene Sequencing journal February 2014
A novel programmable lysozyme-based lysis system in Pseudomonas putida for biopolymer production journal June 2017
A simple, inexpensive method for preparing cell lysates suitable for downstream reverse transcription quantitative PCR journal April 2014
Comparative study of the interaction of CHAPS and Triton X-100 with the erythrocyte membrane journal March 2014
A single protocol for extraction of gDNA from bacteria and yeast journal March 2015
Evaluation of the impact of six different DNA extraction methods for the representation of the microbial community associated with human chronic wound infections using a gel-based DNA profiling method journal September 2017
Resistance of cell membranes to different detergents journal April 2003
Effects of sampling strategy and DNA extraction on human skin microbiome investigations journal November 2019
The Interplay between Cell Wall Mechanical Properties and the Cell Cycle in Staphylococcus aureus journal December 2014
Absolute quantitation of microbiota abundance in environmental samples journal June 2018
DNA extraction approaches substantially influence the assessment of the human breast milk microbiome journal January 2020
A simple "universal" DNA extraction procedure using SDS and proteinase K is compatible with direct PCR amplification. journal June 1995
A Direct PCR Approach to Accelerate Analyses of Human-Associated Microbial Communities journal September 2012
An optimised direct lysis method for gene expression studies on low cell numbers journal August 2015
Rapid, Simple Alkaline Extraction of Human Genomic DNA from Whole Blood, Buccal Epithelial Cells, Semen and Forensic Stains for PCR journal October 1998
The Human Microbiome Project journal October 2007
Naive Bayesian Classifier for Rapid Assignment of rRNA Sequences into the New Bacterial Taxonomy journal June 2007
Consistent and correctable bias in metagenomic sequencing experiments journal September 2019
The Earth Microbiome project: successes and aspirations journal August 2014
Bacterial membrane lipids: diversity in structures and pathways journal April 2015
scFTD-seq: freeze-thaw lysis based, portable approach toward highly distributed single-cell 3′ mRNA profiling journal November 2018
Quantifying and Understanding Well-to-Well Contamination in Microbiome Research journal June 2019
Quantifying spatiotemporal variability and noise in absolute microbiota abundances using replicate sampling journal July 2019
Environmental Selection, Dispersal, and Organism Interactions Shape Community Assembly in High-Throughput Enrichment Culturing journal August 2017
Structural bases of peptidoglycan recognition by lysostaphin SH3b domain journal April 2019
Establishing microbial composition measurement standards with reference frames journal June 2019
A modular method for the extraction of DNA and RNA, and the separation of DNA pools from diverse environmental sample types journal May 2015
High-Throughput Miniaturized 16S rRNA Amplicon Library Preparation Reduces Costs while Preserving Microbiome Integrity journal November 2018
Comparison of six commercial kits to extract bacterial chromosome and plasmid DNA for MiSeq sequencing journal June 2016
Improved yield and accuracy for DNA extraction in microbiome studies with variation in microbial biomass journal June 2019
Mechanical Disruption of Lysis-Resistant Bacterial Cells by Use of a Miniature, Low-Power, Disposable Device journal July 2011
A Modified SDS-Based DNA Extraction Method for High Quality Environmental DNA from Seafloor Environments journal June 2016
The use of sarkosyl in generating soluble protein after bacterial expression. journal February 1991
Damage to Escherichia coli on Exposure to Moist Heat journal September 1968
Influence of the level of ceramides on the permeability of stratum corneum lipid liposomes caused by a C12-betaine/sodium dodecyl sulfate mixture journal June 1999
Cell disruption by cationic surfactants affects bioproduct recovery from Synechocystis sp. PCC 6803 journal September 2018
A rapid DNA isolation procedure for the identification of Campylobacter jejuni by the polymerase chain reaction journal December 2000
Deciphering microbial interactions in synthetic human gut microbiome communities journal June 2018
Improved extraction of PCR-quality community DNA from digesta and fecal samples journal May 2004