Keywords

1 Methods

Two approaches are used to study environmental samples including soil, water, and air:

  1. 1.

    Targeted 16S or 18S rRNA analysis

  2. 2.

    Shotgun sequencing

For either of these approaches, methodology of metagenomic workflow remains more or less similar, and they can be applied for any environment from shallow river to deep lake, sea or bay, air, and sediment.

1.1 Methodology for 16S rRNA Environmental Sequence Analyses

1.1.1 Sample Collection

The foremost part of environmental metagenomic study is sampling. Before planning for sampling, proper permission should be taken from the city government office, mayor’s office, etc. Consent from fishery cooperatives may sometimes be necessary. In the case of sampling from deep waters, safety of the researchers is an important issue. Appropriate lifesaving gears should be taken. Care should be given to preserve endangered species or wildlife. Finally, proper cautions should be taken to ensure collection of representative samples as it may directly affect downstream analyses.

During collection of samples, proper care should be taken to minimize contamination (Fig. 1.1). It may be suggested to use sterilized glassware for collecting water samples from a river or sea. Alternatively, water collection vessels can be rinsed with water three times before being used for sample collection. In case of collection of sediments from the river or seabed, it is always better to use “Marine Biology Dredge,” e.g., Ekman Dredge, when the water is deep. Otherwise, sediments can be directly scrapped from the riverbed when the water depth is low.

Fig. 1.1
figure 1

Metagenomic sampling from a shallow river. (a) Monitoring environmental parameters, (b) recording environmental data, (c) sampling from surface layer of the river, (d) prefiltration using 100 μm filter, (e) pouring water sample into a sterile bottle, and (f) collection of sediments from riverbed of the Tama River, Tokyo, Japan

Full size image

1.1.2 Transport and Preservation of Sample

It is always important to quickly transport the samples to the laboratory for processing. Care should be taken not to allow rising of the temperature inside the samplers. It is suggested to use ice packs and coolers with an icebox for transportation, thereby minimizing multiplication of microorganisms inside the bottle. Sometimes, it may become necessary to preserve it as samples may be collected from places far from the laboratory. In case of lack of on-site sample preparation facilities, the samples can be preserved at 4 °C for 8–10 h.

1.1.3 Sample Treatment and Filtration

Size fractionation of water is an important part of metagenomic analyses as it separates planktonic microorganisms according to their size. It is a routine to use size fractionation, often equipped with a prefilter of larger pore size (typically 1.0–100 μm) and smaller pore sizes (0.1–0.2 μm) over a sample volume ranging from 0.05 to 20 l (Ganesh et al. 2014; Padilla et al. 2015; Reza et al. 2018a) (Fig. 1.2). It is generally accepted that free-living bacterioplankton that consist of more than 90% of the total bacterial population in the marine and freshwater ecosystems are trapped on the small-size pore filters, while their particle-associated counterparts get trapped on the larger-size pore filters as they remain attached to suspended particles particularly in the 5-μm filter fraction during filtration (Reza et al. 2018a). While the wide range of sample volumes used for filtration in different samplings is typical for coastal and open water environments (Padilla et al. 2015), the use of prefilter by larger pore size is generally practiced to remove debris that may be present in the sample (Reza et al. 2018b).

Fig. 1.2
figure 2

Size fractionation of water samples. (a) Filtration of water, (b) trapping microorganisms on a filter, (c) rolling a filter paper, and (d) insertion of a filter paper into a 5-ml PowerWater Bead Tube

Full size image

Different types of water filtration systems are available in the market such as:

  1. (i)

    Nalgene™ polysulfone reusable bottle top filter with receiver (Thermo Fisher Scientific, Waltham, MA, USA)

  2. (ii)

    Advantec purification system (Advantec Co. Ltd., Tokyo, Japan)

  3. (iii)

    Sterivex™ cartridge filters (Millipore, Merck KGaA, Darmstadt, Germany)

In the case of using Nalgene™ polysulfone reusable bottle top filter with a receiver, ~350 ml of water is poured onto the filter with receiver (Thermo Fisher Scientific) and filtered sequentially through 45-mm diameter each of 5-, 0.8-, and 0.2-μm pore-sized Isopore™ membrane filters (Merck Millipore Ltd., Tullagreen, Ireland) using a vacuum pump. The pressure of the vacuum pump should be monitored carefully as it may disrupt cells of many soft-bodied protists and eukaryotes.

1.1.4 DNA Extraction

Generally microorganisms adhere to the filters, and different methods are used to extract DNA from these filters. Here we will discuss Qiagen’s bead-beating technique (Qiagen GmbH, Hilden, Germany). After removing the filters from the filtration unit using sterile forceps, the following steps can be used:

  1. (i)

    First the filter membrane should be inserted into the 5-ml PowerWater® Bead Tube and subsequently be rolled in such a way that its top side faces inward.

  2. (ii)

    Then 1 ml of Solution PW1 preheated at 55 °C is added to the PowerWater® Bead Tube.

  3. (iii)

    The PowerWater® Bead Tube is secured horizontally to a Vortex adapter and vortexed at the maximum speed for 5 min.

  4. (iv)

    Then the tubes are centrifuged at ≤4000 × g for 1 min at room temperature. The speed will depend on the capability of the centrifuge in use.

  5. (v)

    The supernatant is transferred to a clean 2-ml Collection Tube by drawing up the supernatant using a 1-ml pipette tip by placing it down into the beads. Here, placing the pipette tip down into the beads and pipetting more than once are required to ensure the removal of the supernatant. Any carryover of beads will not affect subsequent steps. In this way, recovery between 600 and 650 μl of the supernatant is possible depending on the type of filter membranes used.

  6. (vi)

    Then the tubes are centrifuged at 13,000 × g for 1 min.

  7. (vii)

    To avoid the pellet, the supernatant is transferred to a clean 2-ml Collection Tube, and 200 μl of Solution PW2 are added, and the mixture is vortexed briefly to mix. They are subsequently incubated at 4 °C for 5 min and centrifuged at 13,000 × g for 1 min.

  8. (viii)

    Then the supernatant is transferred to a clean 2-ml Collection Tube by carefully avoiding the pellet.

  9. (ix)

    To the 2-ml Collection Tube, 650 μl of Solution PW3 is added, and the mixture is vortexed briefly to mix. Then 650 μl of supernatant is loaded onto a Spin Filter and centrifuged at 13,000 × g for 1 min.

  10. (x)

    The flow is discarded through, and the process is repeated until all the supernatant has been loaded onto the Spin Filter. In this way, a total of two loads for each sample processed are required.

  11. (xi)

    Then the Spin Filter basket is placed into a clean 2-ml Collection Tube, and 650 μl of Solution PW4 is added and centrifuged at 13,000 × g for 1 min.

  12. (xii)

    The flow through is discarded, and 650μl of Solution PW5 is again added, and the mixture is centrifuged at 13,000 × g for 1 min. The flow through is again discarded and centrifuged again at 13,000 × g for 2 min to remove residual wash.

  13. (xiii)

    Finally the Spin Filter basket is placed into a clean 2-ml Collection Tube and centrifuged at 13,000 × g for 1 min after adding 100 μl of Solution PW6 to the center of the white filter membrane.

  14. (xiv)

    The Spin Filter basket is discarded, and the obtained DNA is now ready for any downstream application.

After extraction, DNA concentration needs to be measured as most of the library preparation kits require a specified amount of DNA for downstream analyses. For example, Nextera XT DNA Sample Preparation Kit (Illumina Inc., San Diego, CA, USA) requires only 1 ng of input DNA. In such cases, DNA quantitation can be done with a Qubit 2.0 Fluorometer (Invitrogen, Carlsbad, CA, USA) using Qubit dsDNA HS Assay Kit (Invitrogen) that uses highly sensitive and accurate fluorescence-based Qubit quantitation assays for quantitation of DNA, RNA, and protein. For each assay, it may be necessary to run new standards for calibrating the Qubit 2.0 Fluorometer. For later uses, the values from the previously done calibration can be used once calibration is completed for the first time. The process is briefly described below:

  1. (i)

    Calibration of the Qubit 2.0 Fluorometer requires the preparation of the appropriate standard solutions. We need to set up two assay tubes: one for standards and the other for our sample. To prepare the Qubit Working Solution by diluting the Qubit reagent 1:200 in Qubit buffer, we need 200 μl of working solution each for standard and sample. We need to add 190 μl of working solution and 10 μl of standard solution (supplied with the kit) to make total volume in each assay tube to 200 μl. As for assay of our sample, we need to add 180–199 μl working solution and 1–20 μl of our sample to make the total volume to 200 μl. Generally, we use 1–2 μl of our sample and 198–199 μl of working solution for each sample assay.

  2. (ii)

    Then all tubes are to be vortexed for 2–3 s and incubated at room temperature for 2 min.

  3. (iii)

    After completion, they should be inserted into the Qubit 2.0 Fluorometer to take readings.

The extracted DNA can be stored at −20 °C, and aliquots of the extracted DNA can be used for metagenomic 16S rRNA sequencing.

1.1.5 PCR Amplification of 16S rRNA Gene and Next-Generation Sequencing

To determine the diversity and composition of the bacterial communities in the size-fractionated microorganism samples, different variable regions such as V1, V2, or V3 of the bacterial 16S rRNA gene can be targeted in metagenomic studies. The selection of appropriate primer is very important, as success of such study depends on the selection and design of primers. In this section, we discuss about sequencing methodology using Ion PGM (Thermo Fisher Scientific) and MiSeq (Illumina Inc., San Diego, CA) platforms.

  1. (i)

    For Ion PGM:

Polymerase chain reaction (PCR) is performed as follows: 20 μl each of PCR mixture comprises 0.1 μl Ex Taq HS DNA polymerase (Takara, Otsu, Japan), 2 μl of 10× Ex Taq DNA polymerase buffer (Takara), 1.6 μl of 2.5 mM dNTP, 12.3 μl of nuclease-free water, 1 μl of 2 mM adaptor-labeled forward primer 1 μl of 2 mM adaptor-labeled and barcoded reverse primer, and 2 μl of undiluted template DNA. PCR conditions can consist of 5 min incubation at 95 °C followed by 30 cycles of 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 30 s with the last cycle employing an extension for 5 min at 72 °C. Required number of independent PCRs can be carried out for the same amount of sample with a combination of forward primers and the common reverse primer.

Following amplification, all PCR products are examined for their size and specificity by electrophoresis on 2.5% w/v agarose and gel purified using FastGene Gel/PCR Extraction Kit (Nippon Genetics Co. Ltd., Tokyo, Japan). Prior to sequencing, all amplicon types are assessed for fragment size distribution and DNA concentration using a Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA). Ion Torrent sequencing is then performed according to the Ion Torrent standard workflow (Thermo Fisher Scientific). All barcoded amplicons are diluted to 13 pM and equimolarly pooled. Then 25 μl of amplicon library is used for downstream application using the Ion PGM Template OT2 400 Kit for use with the Ion OneTouch 2 System. This procedure results in the generation of template-positive Ion PGM Template OT2 400 Ion Sphere Particles. After enrichment of the particles, a sequencing run is performed with the Ion 318 Chip Kit v2 based on the Ion PGM Sequencing 400 Kit User Guide, and FASTQ files are generated with Torrent Suite software version 4.2.1.

  1. (ii)

    For MiSeq:

For targeted 16S rRNA analyses using MiSeq, PCR libraries need to be constructed using forward (F) and reverse (R) indexed primes targeting variable regions between V1 and V3. For making barcoded libraries using Illumina’s TruSeq or Nextera kits, 12 barcodes come with the library preparation kit that Illumina provides. Therefore, indexing primers included in the sequencing reagent kits are used for running MiSeq (Reza et al. 2018c). After PCR amplification, the amplicons are quantified using a Bioanalyzer and pooled in equimolar amounts as described in Illumina’s “16S Metagenomic Sequencing Library Preparation” Guide (Part # 15044223 Rev. B). Finally, sequencing is performed as paired-end reads using an Illumina MiSeq platform and a MiSeq Reagent Kit v3 (600 cycles).

1.1.6 Sequence Analyses

For Ion PGM datasets, individual sequence reads are first filtered within the PGM software to remove low-quality and polyclonal sequences. To avoid possible bias and to minimize taxonomic ambiguity, a stringent criterion is generally set to use only longer reads where sequences are again quality trimmed to remove <270 bp using Genomics Workbench 8.0 (CLC bio, Cambridge, MA, USA). They are then processed by the NGS analysis pipeline of the SILVA rRNA gene database project (SILVAngs 1.2) (Quast et al. 2013). Downstream analyses are performed as described by Klindworth et al. (2013), where different tools in the pipeline including SINA for the alignment of sequences (Pruesse et al. 2012), CD-HIT for the clustering of sequences (Li and Godzik 2006), and BLAST for the classification of sequences (Camacho et al. 2009) are included.

As for 16S targeted libraries generated by MiSeq, all reads can be uploaded to BaseSpace server, and sequences of at least 100 bp are analyzed using the Illumina 16S Metagenomic App (v1.0.1) for taxonomic classification using an Illumina-curated version of Greengenes May 2013 reference taxonomy database.

1.1.7 Results Obtained for 16S rRNA Analyses for the Tama River, Tokyo, Japan

In this section, we present results of one of our studies conducted on Tama River, Tokyo, Japan, that employed next-generation sequencing technology targeting a 16S ribosomal RNA (rRNA) gene amplicon. Both the particle-associated and free-living portions collected from the river were studied separately after size fractionation of water samples trapped by 5.0-, 0.8-, and 0.2-μml filters. It was revealed that this urban river was most dominated by Proteobacteria, followed by Bacteroidetes, Actinobacteria, and Cyanobacteria (Fig. 1.3). Through this approach, we did not detect any archaeal and eukaryotic sequences as we used bacteria-specific primers (Reza et al. 2018c).

Fig. 1.3
figure 3

Relative abundances of the major bacterial and archaeal phyla and classes in 5.0-, 0.8-, and 0.2-μm filter fractions from the Tama River based on the taxonomic identification of 16S rRNA gene fragments. The artificial group “Others” contains unidentified sequences, as well as sequences with a relative abundance lower than 1%. (From Reza et al. 2018c)

Full size image

Our study was the first river metagenomic report from Japan. Through the brief analysis, we were able to identify genus-level taxonomic profile of the Tama River that can work as a baseline for the river system concerned and can be used to enhance the existing water quality monitoring efforts to improve well-being of the residents in the area.

1.2 Methodology for Shotgun Metagenomic Sequence Analyses

1.2.1 Sample Collection

Generally for deep water expedition in the marine water, no specific permits are required for sampling. This gives freedom for researchers who conduct sampling. However, proper permissions should be obtained for protecting endangered or protected species at sea. Usually vertical water sampler is used to collect water sample from a specific water depth. The volume of water sample collected varies depending on biomass present in the water. Generally 5–10 l of seawater is collected and prefiltered through a 100-μm filter to remove debris (Fig. 1.4). Corresponding seawater quality parameters, including temperature, salinity, and dissolved oxygen, are also measured on site using a water quality profiler. Simultaneously representative water samples are collected in separate sunlight-protected bottles for measurement of other water quality parameters including the abundance of chlorophyll a (chl-a) and heterotrophic bacteria (Yamada et al. 2017).

Fig. 1.4
figure 4

Sampling of water at sea. (a) Travelling to sampling spot, (b) prefiltration using a 100-μm filter, and (c) and (d) sample collection in the Ofunato Bay, Iwate Prefecture, Japan

Full size image

1.2.2 Sample Treatment and Filtration

In this section, we will discuss about filtration using Advantec purification system (Advantec Co. Ltd.). Generally the prefiltered water is filtered sequentially through 20-μm pore-sized Nylon Net Filter NY20 with a 47-mm diameter (Merck Millipore Ltd.) and through 5-, 0.8-, and 0.2-μm pore-sized Isopore Membrane Filters, 142 mm in diameter (Merck Millipore Ltd.), using a peristaltic pump (Masterflex L/S, Cole-Parmer International, Vernon Hills, IL, USA) (Fig. 1.5).

Fig. 1.5
figure 5

Filtration of water. (a) Suction of water from tank, (b) and (c) sequential filtration using filtration system, and (d) peristaltic pump in use

Full size image

To minimize disruption of soft-bodied protists and other picoplankton, we maintained a low pump speed of 30–40 rpm. After filtration, all filters were stored at −80 °C until DNA extraction.

1.2.3 DNA Extraction

Genomic DNA is extracted from 0.2-μm filters using the PowerWater® DNA Isolation Kit (Mo Bio Laboratories, Inc., Carlsbad, CA, USA), following the manufacturer’s recommendations as described earlier.

DNA concentrations also should be measured with Qubit dsDNA HS Assay Kit (Invitrogen), using a Qubit Fluorometer 2.0 (Invitrogen). The extracted DNA samples can be stored at −20 °C until use.

1.2.4 Sequence processing and analyses

Extracted DNA aliquots are used for metagenomic shotgun sequencing. For library preparation by Nextera XT DNA Sample Preparation Kit (Illumina Inc.), 1 ng of gDNA is required. So sufficient amount of DNA should be diluted with sterile distilled water to adjust the concentration to 0.2 ng/μl.

Five microliters of gDNA at a concentration of 0.2 ng/μl are then taken, whereas DNA samples for metagenomics are prepared employing the Nextera XT DNA Sample Preparation Kit (Illumina Inc.) and sequenced as paired-end reads using an Illumina MiSeq platform and a MiSeq Reagent Kit v3 (600 cycles) (Illumina Inc.).

Illumina paired-end reads for each library are first joined by overlapping forward and reverse reads of the same DNA fragment (paired-end sequences) using the software FLASH (Magoc and Salzberg 2011) with default parameters (overlap minimum, 10 nt; maximum allowed ratio between the number of mismatched base pairs and the overlap length, 0.25). Quality filtering of these shotgun sequencing reads is then performed by removing reads <50 bp and quality trimmed to Phred 20 using the Genomics Workbench (CLC bio). BLASTn is performed using the quality- and size-filtered sequences against the NCBI-nt reference database. Taxonomic analysis at the genus level is then performed using MEGAN v5.10.3 (Huson et al. 2007), after parsing the BLAST output. Comparative analysis in MEGAN is also performed after normalizing counts per the recommendations of the authors. The least common ancestor (LCA) assignment algorithm is set at the parameters: minimum support = 1; minimum score = 50; top percent = 10; and maximum expected 1.0E – 10.

The obtained sequence matrices are generally used for correlating with various environmental factors including chl-a concentrations, heterotrophic bacterial abundances, and other metadata using statistical packages, e.g., R software in the Ofunato Bay (R Development Core Team 2008), based on experiment objectives (Rashid et al. 2018).

1.2.5 Results Obtained for a Shotgun Sequence Analyses for the Ofunato Bay, Iwate Prefecture, Japan

In this section, we are going to present the results of one of our shotgun metagenomic studies conducted on the Ofunato Bay, a deep enclosed bay in Iwate Prefecture, Japan (Reza et al. 2018b). Unlike our Tama River study, only free-living portions of the bay were reported as this portion has been considered as the most dominant among the total bacterial population. Phylogenetic differences among spring, summer, autumn, and winter bacterioplankton of the bay was studied that showed members of some group decreased at high water temperatures but increased at low temperatures, indicating seasonal change of the free-living bacterioplankton in the Ofunato Bay (Fig. 1.6).

Fig. 1.6
figure 6

Principal component analyses (PCAs) based on relative abundances of bacterial genera obtained from shotgun metagenomic data. Panel “a” shows that several datasets from KSt. 3 derived from 1-m water depth were clustered (dotted parabola), while Panel “b” shows that datasets derived from spring, summer, and winter were clustered together (parabola filled with green, red, and blue, respectively). Data employed was only from 0.2-μm-size filters targeting the free-living fractions from samples collected during January to December in the Ofunato Bay. (From Reza et al. 2018b)

Full size image

As the Ofunato Bay is housed by Japan’s famous buoy-and-rope type oyster (Crassostrea gigas) culture facilities, we were able to find strong locality signal for bacterial communities based on bacterial functional analyses (Kobiyama et al. 2018). The dominant bacterial species Candidatus Pelagibacter ubique and Planktomarina temperata were reportedly found to show change seasonally, playing important roles in producing dimethyl sulfide (DMS) and methanethiol (MeSH) from dimethylsulfoniopropionate (DMSP) as signaling molecules for possible formation of scent of the tidewater or as fish attractants (Kudo et al. 2018).