Optimized DNA extraction and metagenomic sequencing of airborne microbial communities

Journal name:
Nature Protocols
Volume:
10,
Pages:
768–779
Year published:
DOI:
doi:10.1038/nprot.2015.046
Published online

Abstract

Metagenomic sequencing has been widely used for the study of microbial communities from various environments such as soil, ocean, sediment and fresh water. Nonetheless, metagenomic sequencing of microbial communities in the air remains technically challenging, partly owing to the limited mass of collectable atmospheric particulate matter and the low biological content it contains. Here we present an optimized protocol for extracting up to tens of nanograms of airborne microbial genomic DNA from collected particulate matter. With an improved sequencing library preparation protocol, this quantity is sufficient for downstream applications, such as metagenomic sequencing for sampling various genes from the airborne microbial community. The described protocol takes ~12 h of bench time over 2–3 d, and it can be performed with standard molecular biology equipment in the laboratory. A modified version of this protocol may also be used for genomic DNA extraction from other environmental samples of limited mass or low biological content.

At a glance

Figures

  1. Workflow for particulate matter sample pretreatment.
    Figure 1: Workflow for particulate matter sample pretreatment.

    After sample collection (Steps 1–5), the sample pretreatment workflow includes cutting and rolling the quartz filter containing the collected particulate matter (Step 8), buffer wash (Step 9), PES filter recollection (Steps 10–11) and shredding the PES filter into small pieces (Step 12) before the subsequent cell lysis and DNA extraction steps (Steps 13–25).

  2. Experimental setup for particulate matter collection and sample pretreatment.
    Figure 2: Experimental setup for particulate matter collection and sample pretreatment.

    (a–c) Setup of a VFC high-volume air sampler (Step 4) (a), with a clean PM2.5 inlet gasket (b) and a gasket after several days of particulate matter collection (c). (d) A Tissuquartz filter after 23 h of particulate matter collection. (e,f) A Tissuquartz filter is cut into four equal-sized pieces (Step 8) (e), before being rolled and inserted into 50-ml Falcon tubes filled with sterilized 1× PBS buffer, and then pelleted at 4 °C by low-speed centrifugation at 200g for 3 h (Step 9) (f). (g) The setup for re-filtration of the resuspension using a magnetic filter funnel (Step 10). (h) The re-collected sample on a 0.2-μm Supor 200 PES membrane disc filter (Step 11).

  3. Quality assessment of extracted genomic DNA and validation of the quality of prepared sequencing libraries.
    Figure 3: Quality assessment of extracted genomic DNA and validation of the quality of prepared sequencing libraries.

    (a) qPCR results from genomic DNA samples extracted as described in Table 1 using 16S rRNA gene universal primers (Step 25). PM10-pretreated beads: DNA extracted from pretreated PM10 sample (using the MO-BIO PowerSoil kit) and purified using AMPure XP beads; PM10 without (w/o) pretreatment beads: DNA extracted from PM10 sample by shredding the quartz filter without pretreatment (using the MO-BIO PowerMax kit) and purified using AMPure XP beads; PM10-pretreated column: DNA extracted from pretreated PM10 sample (using the MO-BIO PowerSoil kit) and purified using traditional column purification; PM10 w/o pretreatment column: DNA extracted from PM10 sample by shredding the quartz filter without pretreatment (using the MO-BIO PowerMax kit) and purified using traditional column purification; PM2.5 pretreated beads: DNA extracted from pretreated PM2.5 sample (using the MO-BIO PowerSoil kit) and purified using AMPure XP beads. qPCR of the above five samples resulted in successful 16S rRNA gene amplification, whereas the negative control did not. Amplification curves with lower Ct values suggest the presence of more bacterial DNA in the extracted genomic DNA and thus better sample quality. The qPCR data (color-coded and ranked top-down from the lowest Ct to the highest), together with the DNA concentration data presented in Table 1, suggest that the method of using pretreatment with bead purification provides higher DNA yield than all the other methods tested above. The addition of the pretreatment step improves the yield of DNA extraction, and using beads for DNA purification is more effective than using column. (bd) Agilent 2100 Bioanalyzer analysis of six examples of prepared good-quality sequencing libraries (Step 34), including those of four PM2.5 samples and two PM10 samples (all DNA samples were extracted using the pretreated beads method). (b) Electrophoresis of the six libraries: a single, smeary band at 500–800 bp suggests good library quality (the green and purple bands correspond to Bioanalyzer-called lower and upper markers, respectively). (c,d) Electropherograms of two of the libraries (PM2.5-1 and PM10-1, as shown in b) with 15-bp and 1,500-bp markers; a single, wide peak at 500–800 bp suggests good library quality. a.u., arbitrary units; FU, fluorescence units.

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Affiliations

  1. School of Life Sciences, Center for Synthetic and Systems Biology, Ministry of Education Key Laboratory of Bioinformatics, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Tsinghua University, Beijing, China.

    • Wenjun Jiang,
    • Peng Liang &
    • Ting F Zhu
  2. State Key Joint Laboratory of Environment Simulation and Pollution Control, School of Environment, State Environmental Protection Key Laboratory of Sources and Control of Air Pollution Complex, Tsinghua University, Beijing, China.

    • Buying Wang &
    • Jingkun Jiang
  3. Center of Biomedical Analysis, School of Life Sciences, Tsinghua University, Beijing, China.

    • Jianhuo Fang,
    • Jidong Lang &
    • Geng Tian

Contributions

W.J., P.L., B.W., J.F. and J.L., with inputs from T.F.Z., J.J. and G.T., performed the experiments and analyzed the data. T.F.Z. conceived and organized the study. W.J. and T.F.Z., with inputs from the other coauthors, wrote the paper.

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The authors declare no competing financial interests.

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