High-quality metagenomic DNA from marine sediment samples for genomic studies through a preprocessing approach

Abstract

Recent advances in culture-independent studies of microbes had proved to be more reliable and efficient than the conventional ones. The isolation of good quality and quantity of total community DNA are one of the major hurdles in this endeavour. Shearing of DNA during the extraction process and the co-extraction of inhibitory compounds reduce the quality of the isolated nucleic acids making it unsuitable for the construction of large insert metagenomic libraries. In the present study, a multi-level filtration step was brought in which efficiently isolated total bacterial DNA from three different environment samples. The preprocessing method could efficiently improve the 260/230 ratio of the isolated DNA by 2.3–45 % and decreased the protein contamination by 22.5–34.5 % on saltpan and arctic sediment samples, respectively. The more significant part of the experiment was that the DNA obtained was of high quality with minimal shearing making it most suitable for the construction of large insert genomic libraries. PCR amplification of 16S rRNA gene confirmed that the filtration method was effective in the isolation of high-quality DNA.

Amplicon sequencing based profiling of bacterial diversity from Krossfjorden, Arctic

“…Sediment samples were suspended in sterilized virus free seawater and subjected to low speed (320 × g ) centrifugation for the removal of coarse sediment particles followed by high speed (9000 × g ) centrifugation for collecting the fine sediment fraction with bacteria. The supernatant was subjected to sequential filtration using 0.8 μ, 0.45 μ and 0.22 μ filter membranes for the separation of bacterial fraction [5] . Fine sediment obtained after high speed centrifugation and the residue on filter membranes were used for metagenomic DNA extraction as per modified Zhou et al [6] protocol.…”

Sediment-associated microbial community profiling: sample pre-processing through sequential membrane filtration for 16s rDNA amplicon sequencing

“…The samples were then suspended in a buffer consisting of 10 mM EDTA, 50 mM Tris-HCl, 50 mM Na2HPO4•7H2O at pH 8.0 to remove PCR inhibitors (Zhou et al 1996;Poulain et al, 2015). Genomic DNA was extracted from both the non-processed and filtered subsamples following the protocol of Zhou et al (1996) (as employed in Solomon et al, 2016). The DNA extracted from non-processed sediment subsamples were combined accordingly before amplification.…”

“…Most of the studies on applying pre-processing sediment samples by sequential membrane filtration focused on the quality assessment and efficiency of the extracted metagenomic DNA. Solomon et al (2016) demonstrated that community DNA with minimal shearing was obtained from pre-processing marine sediment samples and performed PCR amplification of the 16S rDNA gene to confirm that the filtration method isolated high-quality DNA. A similar protocol was employed to process arctic sediment samples to characterize the bacterial community structure by 16S rDNA amplicon sequencing (Kachiprath et al, 2017).…”

“…Most studies would directly extract microbial DNA from sediment samples, amplify a target hypervariable region of the 16s rDNA gene through polymerase chain reaction (PCR), process for amplicon library construction, and sequence on a high-throughput platform (e.g., Illumina-based technologies). One major challenge with such an approach would be the isolation and capture of good quality and quantity DNA from sediment samples (Harnpicharnchai et al, 2007;Solomon et al, 2016), which mostly contain impurities that inhibit amplification through PCR (Albers et al, 2013). Various commercial extraction kits are available for the rapid processing of environmental samples tailored to yield abundant and high-quality DNA minimizing the effects of enzyme inhibitors, e.g., humic acid, polysaccharides, metals, etc.…”

“…Pre-processing sediment samples by multi-level or sequential membrane filtration have been reported to efficiently isolate high-quality DNA while reducing inhibitory enzyme Pre-processing for Sediment Microbial Community Profiling 4 compounds (Solomon et al, 2016;Kachiprath et al, 2017;Mathai et al, 2019;Sakami, 2019). Sequential filtration has been used to concentrate microbial biomass and assess communities based on size fractions using filter membranes with different pore sizes (Padilla et al, 2015;Bae, Lyons, and Onstad, 2019).…”