Metagenomic Sequencing (ONT)

Third-generation Sequencing

Service Overview

Nanopore sequencing distinguishes itself from other sequencing platforms, in that the nucleotides are read directly without DNA synthesis process. Leading by enzyme motor, a DNA/RNA untwists and passes a nano-sized protein pore (nanopore) at a certain speed on an electrically resistant membrane. Due to different chemical properties of different nucleotides, a fluctuation in current will be generated when a single nucleotide passes through the nanopore, which can be captured and transferred into sequence of bases.
Figure 1. Principle of ONT sequencing
Base calling on Nanopore platform is based on the mean and variation of the electric current, which will be translated by ONT platform using Recurrent Neural Network.
Figure 2. Principle of ONT sequencing
Metagenome refers to a collection of total genetic material of a mixed community of organisms, such as environmental metagenome, human metagenome, etc. It contains genomes of both cultivatable and uncultivatable microorganisms. Metagenomics is a molecular tool used to analyse the mixed genomic materials extracted from environmental samples, which provides detailed information in species diversity and abundancy, population structure, phylogenetic relationship, functional genes and correlation network with environmental factors, etc.

ONT sequencing platforms has recently introduced to metagenomic studies. Its outstanding performance in read length largely enhanced down stream metagenomic analysis in terms of species identification and functional gene analysis.

Service Advantages

High resolution in speices annotation
Low-abundant species identification
More complete bacteria genomes (Uncultivatable bacteria)
Reconstructing genomes rom environmental samples (BINNING)


In metagenomic analysis, binning refers to the process of clustering reads or contigs into groups and assigning each group to an individual species or operational taxonomic unit (OTU).

Contigs binning, i.e. clustering assembled contigs, enables assembly of single bacteria genome and metabolome-wide association study (MWAS). Based on metagenome assembly, similar contigs with comparable abundancy can be clustered into a same species, which can be further processed for single bacteria genome assembly. Subsequently, advanced analysis including gene functional annotation, comparative genomic analysis and evolutionary analysis can be processed at single bacteria genome level.

Contigs assembled by short reads and long reads

Short reads-based assembly is not able to identify repetitive sequences. Long reads can achieve more continuous assembly.

Experiment workflow

Sample quality assessment, library construction, library quality control and sequencing are processed strictly following standard protocol provided by Oxford Nanopore Technologies (ONT).

1. High-quality DNA extraction. Purity, concentration and integrity of DNA are examined by Nanodrop, Qubit and 0.35% Agarose gel electrophoresis.

2. Large DNA fragment collection by Blue Pippin automatic DNA collection system

3. Library construction (SQK-LSK109 ligation kit)

a. DNA repairing and purification with magnetic beads
b. Adaptor ligation and purification with magnetic beads
c. Library quantification with Qubit

4. Sequencing
Nanopore library construction

Bioinformatic Analysis

Results Demo

Species annotation

Species distribution and relative abundancy is acquired by mapping clean data against Nr database with “diamond” software .

Phylogenetic analysis

To reveal the phylogenetic relationships of the species in samples, phylogenetic tree is generated basing on species components of samples by “ MetaPhlAn2” software.

Gene functional annotation

The predicted non-redundant genes are mapped against Nr, GO, COG and KEGG database to acquire functional information of the genes. These gene annotations provide crucial evidences in predicting biological functions of the microbial community in specific environmental conditions.

CAZy annotation

Carbohydrate-Active Enzymes (CAZy) database describes the families of structural-related catalytic and carbonhydrate-binding molecules of enzymes that degrade, modify or create glycosidic bonds.

Differential gene functional enrichment

To reveal the biological functional influence of different environmental conditions, differential genes are enriched to biological function terms.