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Shotgun Metagenomic Sequencing

Overview

In shotgun metagenomic sequencing, genomes from environmental samples are analyzed without the prior isolation and cultivation of individual species; therefore, it is a powerful technique for studying microbial communities in their natural habitat, with a broad range of applications. At Novogene, our customers can rely on our expertise in hyphenate Sequencing (NGS) to help them explore the rich genetic repertoire of microbial communities, while also benefiting from our bioinformatics expertise to help identify the species, genes, and pathways represented in their samples. Novogene provides metagenomic sequencing service with an Illumina HiSeq platform and an assembly-first strategy. Additionally, our bioinformatic analysis provide gene predictions, functional annotations, and taxonomic annotations. Our standard analysis package includes, mPATH, heatmaps, PCA, cluster analysis and other programs, generating high-quality, publication-ready data.

Service Specifications

Applications

  • Microbial diversity research
  • Community structure research
  • Functional genes research
  • Metabolic pathways research

Advantages

  • Highly experienced: We have completed numerous metagenomic sequencing projects for our customers, and have published multiple metagenomic studies.
  • Outstanding service: We provide high-quality sequencing (with Q30 score ≥ 80%), an efficient standard workflow, and cost-effective bioinformatics analysis.
  • Effective methodology: Our techniques enhance the generation of data from low-abundance species.
  • Comprehensive analysis: Expert bioinformatics analysis with three databases (KEGG, eggNOG, CAZy) provides comprehensive data on annotated genes and metabolic pathways.

Sample Requirements

 

 

Sample Type Amount Concentration Purity
Genomic DNA ≥ 200 ng ≥ 10 ng/μL OD 260/280=1.8-2.0,

Sequencing Parameter and Analysis

Platform Illumina Novaseq 6000
Read length Pair-end 150
Recommended Sequencing Depth ≥ 40 million read pair per sample for the species with reference genome
Standard Data Analysis
  • Data Quality Control
  • De novo Assembly
  • Gene Functional Annotation
  • Comparative Analysis between Various Samples

Note: For detailed information, please refer to the Service Specifications and contact us for customized requests.

Project Workflow

Hypoxia induces senescence of bone marrow mesenchymal stem cells via altered gut microbiota

Background

Systemic chronic hypoxia is a feature of many diseases and may influence the communication between bone marrow (BM) and gut microbiota. We observe premature senescence of BMSCs and abnormal D-galactose accumulation in patients with CCHD. Together, our findings show that chronic hypoxia predisposes BMSCs to premature senescence, which may be due to gut dysbiosis and thus induced D-galactose accumulation.

Sampling

Genomic DNA extracted from fresh fecal samples that were collected from 40 randomly chosen children with congenital heart disease in two groups (NCy and Cy)

Sequencing Strategy

Illumina platform, paired-end 150 bp

Figure. 1 Relative abundance of NCy and Cy

Figure. 2 LEfSe analysis
Conclusion:

BMSCs from patients with CCHD were predisposed to premature senescence, which might have resulted from gut dysbiosis and gut microbiota-derived D-galactose accumulation. Lactobacillus supplementation restored the deficient BMSCs. Our findings suggested that oral Lactobacillus supplementation might enhance the efficiency of autologous BMSC-based regenerative therapy and open possibilities to improve stem cell-based cures for patients with CCHD.

Temporal Stability of the Human Skin Microbiome

Background:

Biogeography and individuality shape the structural and functional composition of the human skin microbiome. To explore these factors’ contribution to skin microbial community stability, we generated metagenomic sequence data from longitudinal samples collected over months and years. Analyzing these samples using a multi-kingdom, reference-based approach, we found that despite the skin’s exposure to the external environment, its bacterial, fungal, and viral communities were largely stable over time.

Sampling:

DNA samples extracted from skin of 12 healthy people

Sequencing Strategy:

Illumina platform, paired-end 150 bp

Figure. 3 Mean theta heatmap
Conclusion:

Site, individuality, and phylogeny were all determinants of stability. Foot sites exhibited the most variability; individuals differed in stability; and transience was a particular characteristic of eukaryotic viruses, which showed little site-specificity in colonization. Strain and single-nucleotide variant-level analysis showed that individuals maintain, rather than reacquire, prevalent microbes from the environment. Longitudinal stability of skin microbial communities generates hypotheses about colonization resistance and empowers clinical studies exploring alterations observed in disease states.

Dynamics and Stabilization of the Human Gut Microbiome during the First Year of Life

Background:

The gut microbiota is central to human health, but its establishment in early life has not been quantitatively and functionally examined. Applying metagenomic analysis on fecal samples from a large cohort of Swedish infants and their mothers, we characterized the gut microbiome during the first year of life and assessed the impact of mode of delivery and feeding on its establishment.

Sampling:

DNA samples extracted Fecal sample series including mother at birth and infant as newborn, aged 4 (time for introduction of solid foods) and 12 months (when children are generally fed full meals)

Sequencing Strategy:

Illumina platform, paired-end 150 bp

Figure 4. Compromised Resemblance to the Mother’s Gut Microbiome in C-Section Infants
Conclusion:

We observed nonrandom transitions in the infants’ gut, probably induced by the establishment of an anaerobic environment, nutrient availability, and microbial interactions during community succession. We developed a MetaOTU method that allowed the identification of more than 4,000 new microbial genomes, thus making it highly useful for comprehensive investigations on microbial genomes in various environments.


Core-pan gene rarefaction curve


Heatmap for the relation among samples


Venn figures for gene number among sample


Gene number and abundance clustering heatmap in genus level


Anosim analysis at phylum level


linear discriminant analysis (LDA) and cladogram


Kyoto Encyclopedia of Genes and Genomes (KEGG) annotation