Abstract
High-throughput amplicon sequencing technology has been widely used in soil microbiome studies. Here, we estimated the bias of amplicon sequencing data affected by DNA extraction methods in a saline soil, and a non-saline normal soil was used as a control. Compared with the normal soil, several unique points were observed in the saline soil. The soil washing pretreatment can improve not only DNA quantity and quality but also microbial diversities in the saline soil; therefore, we recommend the soil washing pretreatment for saline soils especially hypersaline soils that cannot be achieved with detectable DNA amounts without the pretreatment. Also, evenness indices were more easily affected by DNA extraction methods than richness indices in the saline soil. Moreover, proportions of Gram-positive bacteria had significant positive correlations with the achieved microbial diversities within replicates of the saline soil. Though DNA extraction methods can bias the microbial diversity or community and relative abundances of some phyla/classes can vary by a factor of more than five, soil types were still the most important factor of the whole community. We confirmed good comparability in the whole community, but more attention should be paid when concentrating on an exact diversity value or the exact relative abundance of a certain taxon. Our study can provide references for the DNA extraction from saline and non-saline soils and comparing sequencing data across studies who may employ different DNA extraction methods.
Highlights
The fast-growing high-throughput sequencing technology has considerably changed our understanding of microbial communities in all kinds of environments on Earth (Bates et al, 2011; Caporaso et al, 2011a,b)
The saline soil SS1 used for further sequencing had similar pH with the normal soil NS, but Total organic carbon (TOC) and WC were much lower in the saline soil than the normal soil (Table 1)
We employed several direct DNA extraction methods to optimize extraction methods for saline soils and estimated soil microbiome biases associated with different DNA extraction methods
Summary
The fast-growing high-throughput sequencing technology has considerably changed our understanding of microbial communities in all kinds of environments on Earth (Bates et al, 2011; Caporaso et al, 2011a,b). The amplicon sequencing was much more cost-effective and more widely used (Caporaso et al, 2011b), but additional PCR amplification may bias microbiome studies (Gohl et al, 2016). Other procedures, such as DNA extraction, library preparation, and downstream bioinformatic analysis, may cause biases (Wust et al, 2016; Sinha et al, 2017; Zielinska et al, 2017). DNA purification steps were needed for crude DNA from manual methods
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