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16S/18S/ITS Amplicon Sequencing-NGS

Amplicon sequencing with Illumina technology, specifically targeting the 16S, 18S, and ITS genetic markers, is a powerful method for unraveling the phylogeny, taxonomy, and species abundance within microbial communities. This approach involves sequencing the hypervariable regions of housekeeping genetic markers. Originally introduced as a molecular fingerprint by Woeses et al in 1977, this technique has revolutionized microbiome profiling by enabling isolation-free analyses. Through the sequencing of 16S (bacteria), 18S (fungi), and Internal Transcribed Spacer (ITS, fungi), researchers can identify not only abundant species but also rare and unidentified ones. Widely adopted as a pivotal tool, amplicon sequencing has become instrumental in discerning differential microbial compositions across diverse environments, including the human mouth, intestines, stool, and beyond.


Service Details

Demo Results

Case Study

Service Features

●  Sequencing platform: Illumina NovaSeq.

●  Amplification of short regions of 16S, 18S and ITS, among other amplification targets.

●  Flexible choices of amplicon.

●  Previous project experience with multiple amplification targets.

Service Advantages

●  Isolation-free: and rapid identification of microbial composition in environmental samples.

●  High resolution: in low-abundant components in environmental samples.

●  Widely applicable: to diverse microbial community studies.

●  Comprehensive bioinformatic analysis: including the with latest QIIME2 package (quantitative insight into microbial ecology) with diverse analyses in terms of database, annotation, OTU/ASV.

●  Extensive Expertise: with thousands of amplicon sequencing projects conducted annually, BMKGENE brings over a decade of experience, a highly skilled analysis team, comprehensive content, and excellent post-sales support.

Service Specifications

Library

Sequencing Strategy

Data recommended

Quality control

Amplicon

Illumina PE250

50/100K tags (read pairs)

Q30≥85%

Service Requirements

Concentration (ng/µL)

Total amount (ng)

Volume (µL)

OD260/280

≥1

≥300

≥20

1.6-2.5

● Soil/sludge: 1-2g
● Intestinal content-animal: 0.5-2g
● Intestinal contents-insect: 0.1-0.25g
● Plant surface (enriched sediment): 0.1-0.5g
● Fermentation broth enriched sediment): 0.1-0.5g
● Faeces (large animals): 0.5-2g
● Faeces (mouse): 3-5grains
● Pulmonary alveolar lavage fluid: filter paper
● Vaginal swab: 5-6 swabs
● Skin/genital swab/saliva/oral soft tissue/pharyngeal swab/rectal swab: 2-3 swabs
● Surface microorganisms: filter paper
● Waterbody/air/biofilm: filter paper
● Endophytes: 1-2g
● Dental Plaque: 0.5-1g

Service Work Flow

sample delivery

Sample delivery

Library Preparation

Library construction

Sequencing

Sequencing

Data analysis

Data analysis

After sale Services

After-sale services


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  • 1.Species distribution 

    3

    2.Heat map: Species richness clustering

    4

    3.Rare faction curve

    5

    4.NMDS analysis

    6

    5.Lefse analysis

    7

     

     

     

    BMK Case

    Obese Individuals with and without Type 2 Diabetes show different gut microbial  functional capacity and composition

    Published: Cell Host & Microbe, 2019

    Sequencing strategy:

    Lean non-diabetes (n=633); Obese non-diabetes (n=494); Obese-Type 2 diabetes (n=153);
    Target region: 16S rDNA V1-V2
    Platform: Illumina Miseq (NGS-based amplicon sequencing)
    Subset of DNA extracts were subjected to metagenomic sequencing on Illumina Hiseq

    Key results

    Microbial profilings of these metabolic diseases were successfully differentiated.
    By comparing microbial features generated by 16S sequencing, obesity was found to associated with changes in microbial composition, individual features, especially significant decrease in Akkermansia, Faecalibacterium, Oscillibacter, Alistipes, etc. In addtion, T2D was found associated with increase in Escherichia/shigella.

    Reference

    Thingholm, L. B. , et al. “Obese Individuals with and without Type 2 Diabetes Show Different Gut Microbial Functional Capacity and Composition.” Cell Host & Microbe 26.2(2019).

     

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