**4. Comparison of metagenome analysis techniques**

The metagenome of polar microorganisms has been widely studied in recent years. Their results can provide a great amount of information about the biodiversity, survival capacity, and functioning of microbial communities in these extreme environments. In addition, information about ancient communities preserved within glacial ice through time can be obtained [33].

#### **4.1 Sanger**

Between 1975 and 2005, most of the DNA sequences were obtained through the application of the Sanger techniques [34], which led to the first generation of automated DNA sequencers [35] (**Figure 4**). For 16S or 18S rRNA sequencing, PCR amplification was carried out with specific primers (**Table 1**) and sequencing instruments based on capillary electrophoresis. Nowadays, Sanger sequencing achieves high read lengths of up to 1000 bp and per base and accuracies of 99.999%

**Figure 4.** *Typical workflow in polar glacier metagenomics researches.*


*Microbial Community Structure and Metabolic Networks in Polar Glaciers DOI: http://dx.doi.org/10.5772/intechopen.84945*

> **Table 1.**

*Primer sequences for 16S or 18S rRNA sequencing.*

[36]. In the de novo metagenomics, randomly fragmented DNA was cloned into a high-copy-number plasmid and then transformed in *Escherichia coli*. However, whole-genome sequencing by this technology was extremely expensive and time consuming.

Some examples of microorganisms from polar glaciers analyzed with these technologies were Antarctic bacteria from the Dry Valleys [37] and the Arctic ice pack [38]. In general, the number of sequences identified by this technique was scarce. However, this method has the advantage of generating long reference sequences, which are very useful for studies of taxonomy and biodiversity.

#### **4.2 NGS**

Next-generation sequencing (NGS) technology is similar to capillary sequencing (**Figure 4**). The main difference is that, instead of sequencing a single DNA fragment, NGS develops this process with millions of DNA fragments.

The introduction of pyrosequencing technology by 454 life sciences in 2005 began the NGS innovation. This allowed the identification of thousands of shortsequencing reads without the need for cloning. This technique was used to research the microbial life in the Dry Valleys, Antarctica [39], and Ace Lake, Antarctica [40], and in Arctic glaciers from Svalbard [10].

Since then, many other NGS technologies have been developed. The Illumina platform (MiniSeq, MiSeq, NextSeq, HiSeq, and NovaSeq instruments) is based on sequencing by synthesis of the complementary strand and fluorescence-based detection of reversibly blocked terminator nucleotides. The platform includes multiple instruments with varying read length. For example, Illumina sequencing has been employed in a metagenomic research into diazotrophic communities across Arctic glacier forefields [41] and in the metagenomic analysis of basal ice from an Alaskan glacier [42]. Sequencing of 16S and 18S rDNA PCR amplicons is the most common approach to investigating environmental prokaryotic diversity, despite the known biases introduced during PCR. Recently this method has been improved with the use of 16S rDNA fragments derived from Illumina-sequenced environmental metagenomes [43]. Furthermore, newer Illumina sequencers produce longer reads (e.g., the HiSeq2500 and MiSeq produce 2 × 150bp and 2 × 250bp reads, respectively, which after merging can generate reads up to, e.g., 290 and 490 bp).

Other metagenomic studies based on the Ion Torrent platform were also based on sequencing by synthesis, but the detection was performed using semiconductor technology. Ion Torrent technology was applied to analyze red snow microbiomes and their role in melting Arctic glaciers [12].

The main drawback of the aforementioned second-generation sequencing platforms is that they generate relatively fragmented genome assemblies. In order to produce closed reference genomes, longer reads are required [36]. To meet this demand, third-generation sequencing platforms have been developed. These technologies directly target single DNA molecules without the need for PCR amplification. The PacBio RSII platform uses single-molecule real-time (SMRT) sequencing technology which allows to obtain extremely long DNA fragments of 20 kb and even longer [43].

#### **4.3 Genome analysis tools**

Environmental microbiome sequencing analysis consists of binning sequencing reads into taxonomic units to compare the microbial composition of samples. This information will allow the knowledge of the microbial population taxonomy, diversity, and functioning. When these data are correlated to certain environmental parameters,


*Microbial Community Structure and Metabolic Networks in Polar Glaciers DOI: http://dx.doi.org/10.5772/intechopen.84945*

*2 B, bacterial taxa; E, eukaryotic taxa.*

#### **Table 2.**

*Metagenome analysis tools.*

both ecological and biogeochemical analysis can be performed. Taxonomic binning of 16S and 18S rRNA reads is usually based on one of these four databases: SILVA, Ribosomal Database Project, Greengenes, and NCBI [44]. For instance, the Ribosomal Database Project was used to perform a metagenomic analysis if Illumina sequences to identify bacterial communities in Antarctic surface snow [45].

Several tools have been developed to investigate the taxonomic composition of metagenomes and, in some cases, the functional composition of the community. These tools can be classified into two groups: those that use all the available sequences (MEGAN/MEGAN4, MG-RAST, Genometa, Kraken, LMAT, Taxator-tk, CLARK, GOTTCHA, EBI) and those that use a set of genes (MetaPhyler, QIIME6, mOTU, MetaPhlAn, One Codex) [33]. These genome analysis tools are summarized in **Table 2**.

An example of the use of these tools is the metagenomics analysis with MG-RAST performed to study Arctic microbial communities [41]. Sequence analysis with QIIME was performed with cryoconite samples from Arctic glaciers [10] and with permafrost samples from the Antarctic Dry Valleys [39].

Although metagenomics is changing rapidly, still new improvements in the development of analytical tools and databases are required to answer important questions in polar glacier microbiology.

## **5. Conclusions**

• Extraordinary advances in metagenomics have allowed a great understanding of microbial ecology and function of polar glacier microbial communities.

