Abstract
Metagenomics is about the sequencing and characterization of genomic DNA of uncultured microbes sampled directly from their habitats. Next-generation sequencing (NGS) technologies and the ability of sequencing uncultured microbes have dramatically expanded and transformed our knowledge of the microbial world. In this chapter, we provide an introduction and flavor to metagenomic studies from sampling to data analysis. Also, workflow and several common methodologies are summarized for the sequence-driven metagenomic analysis to identify the composition of microbes, compare different microbial communities, characterize the functional potential of microbial communities and infer the microbes, which are involved in the metabolic pathways. Additionally, we describe some well-established platforms and software, and briefly review their utilities. Finally, an explication of interactions between genomics and meta-genomics gives a new view for host phenotype-genotype analysis.
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Abbreviations
- Egg:
-
NOG Evolutionary genealogy of genes: non-supervised ortholog groups
- EVA:
-
Entity attribute value
- FDR:
-
False discovery rate
- FP:
-
Fecal protease
- GWAS:
-
Genome-wide association study
- HMP:
-
Human Microbiome Project
- IBD:
-
Inflammatory bowel disease
- IBS:
-
Irritable bowel syndrome
- KEGG:
-
Kyoto encyclopedia of genes and genomes
- KR:
-
Kantorovich-Rubinstein
- MDA:
-
Multiple displacement amplification
- MGWAS:
-
Metagenome-wide association study
- MiRKAT:
-
Microbiome regression based on kernel association test
- MSA:
-
Multiple sequence alignment
- NGS:
-
Next-generation sequencing
- OTU:
-
Operational taxonomic unit
- PCR:
-
Polymerase chain reaction
- PCoA:
-
Principal Coordinate Analysis
- PERMANOVA:
-
Permutational multivariate analysis of variance
- QIIME:
-
Quantitative insights into microbial ecology
- RDP:
-
Ribosomal Database Project
- T2D:
-
Type 2 diabetes
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Appendix
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DNA isolation of fecal sample
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Collection and preparation of fecal samples
Fecal samples should be collected and processed timely, ideally within 4 h. After adding equal volume of sterile milli-q water (1:1 feces/water), fecal samples need to be homogenized thoroughly. Then about 1–10 ml slurries will be transferred to cryogenic tubes and frozen at −80 °C until DNA extraction.
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DNA extraction and purification
There are two common methods for fecal DNA purification. One is from the Metagenomics of the Human Intestinal Tract (MetaHIT) project in Europe and the other one is from the National Institutes of Health's Human Microbiome Project (HMP). The MetaHIT protocol mainly uses laboratory-made buffers and solutions, while HMP protocol is based on Mobio PowerLyzer™ PowerSoil® DNA isolation Kit (MO BIO Laboratories) (Wesolowska-Andersen et al. 2014). The MetaHIT method yields significantly higher DNA amount than HMP approach; however, yield and purity of DNA extracted with both protocols were sufficient for Illumina-based deep metagenome sequencing (Wesolowska-Andersen et al. 2014).
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Wang, R., Zhou, Y., Cao, S., Wang, Y., Zhang, J., Deng, HW. (2016). Metagenomic Profiling, Interaction of Genomics with Meta-genomics. In: Wang, X., Baumgartner, C., Shields, D., Deng, HW., Beckmann, J. (eds) Application of Clinical Bioinformatics. Translational Bioinformatics, vol 11. Springer, Dordrecht. https://doi.org/10.1007/978-94-017-7543-4_9
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