Last time, the editor mainly introduced common microorganisms and pollutants in samples with low biomass such as oral cavity and vagina, but the high sensitivity of sequencing technology also amplifies the impact of DNA contamination in samples, so how to carry out pollution control for samples with low biomass It's very important~
In 2019, in the document "Contamination in Low Microbial Biomass Microbiome Studies: Issues and Recommendations" [1], the pollution control theory for low biomass samples was first proposed, mainly the detailed process of low biomass sample pollution treatment, which simplifies the process of contamination. The processing method is described in detail as follows:
01
Experimental Design for Pollution Reduction:
DNA from samples is extracted in a clean room environment using ultra-clean kits and reagents, and pre- and post-PCR experiments are performed in separate designated areas.
02
Control Contaminating DNA:
Record negative control samples, such as: different countries, different hospitals, different types of samples, batch sources (address source, DNA extraction batch, PCR amplification batch and sequencing library number). Incorporating negative controls into each batch type enables detection of different types of batch effects.
At the same time, the RIDE checklist is proposed to provide researchers with a minimum standard checklist for microbiome research in low microbial biomass samples.
This checklist consists of four parts:
01
Report experimental designs and methods designed to reduce pollution and assess the effects of pollution;
02
Include includes assessing controls for contaminating DNA. Each batch of sampling, nucleic acid extraction, and amplification must include one type of negative control (sampling blank control, DNA extraction blank control, and no-template amplification control);
03
Determine determines contamination levels by comparing biological samples to controls;
04
Explore explores the contaminating populations in each study and reports their impact on biological sample interpretation.
Diagram Flow chart of method to minimize contaminating DNA in low microbial biomass
In fact, after knowing so much, the purpose is to put forward some suggestions for the study of low-biomass microbial communities to avoid pollution as much as possible [2-3]:
1. Wherever possible, maximize initial sample biomass through sample type selection, filtration, or concentration. If the microbial load is less than approximately 103 to 104 cells, reliable results may not be obtained due to contamination predominating.
Results can be optimized using Gram staining, fluorescence in situ hybridization (FISH), qPCR, or other methods for DNA quantification prior to sequencing.
2. Minimize the risk of contamination during sample collection.
3. For each batch of samples, each extraction kit and each PCR kit, a negative control and target environmental samples are collected, processed and sequenced at the same time.
4. Samples should be processed in a random order to avoid erroneous patterns, ideally in duplicate, and should be processed with different kits/reagent batches.
5. Which kit was used to process which sample should be documented so that contamination of a specific kit lot number can be traced back to the final data set.
6. Quantification of negative control samples should be carried out during each process to monitor the occurrence of contamination.
7. After sequencing, note taxa in negative controls, taxa that are statistically associated with a particular batch of reagents, and taxa that are biologically insignificant, taxa consistent with previously reported contaminants.
8. If the presence of suspected contaminating flora is considered to be meaningful, repeat sequencing should be performed using different lots of DNA extraction kits/reagents, and ideally, non-sequencing methods such as traditional culture or using appropriate probes should be used. in situ hybridization) to further confirm their true existence.
references
[1] Eisenhofer R,Minich JJ,Marotz C, et al. Contamination in Low Microbial Biomass Microbiome Studies: Issues and Recommendations. Trends Microbiol. 2019
[2] Salter S J, Cox M J, Turek E M, et al. Reagent and laboratory contamination can critically impact sequence-based microbiome analyses.BMC biology, 2014
[3] Nejman D,Livyatan I,Fuks G, et al. The human tumor microbiome is composed of tumor type-specific intracellular bacteria. Science. 2020