Prokayotic metabarcoding

Metagenomics sequencing enables the study of genome diversity of an entire microbial flora (including non-cultivable bacteria) or micro-organisms present in a given sample.

Prokaryotic metabarcoding studies are mostly based on the prokaryotic 16S ribosomal RNA gene sequencing, which is a universal region of the prokaryotic ribosomal 30S subunit. The region is 1,500 bp long and contains 9 hypervariable regions interspersed between conserved regions. Sequencing this variable regions using universal primers allows the microbial taxonomic diversity identification, the structure of prokaryotic communities and their phylogenetic classifications within a sample.

Primer nameSequence (5' → 3')RegionTargetFeatures
27F_HMP
534R_HMP
AGA GTT TGA TCC TGG CTC AG
ATT ACC GCG GCT GCT GG
V1-V3507 bp- Better resolution for many human-associated microbes
341F_ILMN
805R_ILMN
CCT ACG GGN GGC WGC AG
GAC TAC HVG GGT ATC TAA TCC
V3-V4464 bp- Broader phylogenetic coverage than V1-V3 region
- Broad coverage for Archea
515F_EMP
926R_EMP
GTG CCA GCM GCC GCG GTA A
CCG YCA ATT YMT TTR AGT TT
V4-V5411 bp

Eukaryotic metabarcoding

Metagenomics sequencing enables the study of genome diversity of an entire microbial flora (including non-cultivable bacteria) or organisms present in a given sample.

The eukaryotic 18S ribosomal RNA gene is an universal region of the eukaryotic ribosomal 40S subunit. The region is 1,869 bp long and contains both hypervariable regions interspersed between conserved regions. Sequencing this variable regions using universal primers allows the eukaryotic taxonomic diversity identification, the structure of eukaryotic communities and their phylogenetic classifications within a sample.

Their associated internal transcribed spacer region ITS has a higher degree of sequence variation. ITS 1 is located between 18S and 5.8S rDNA and ITS 2 between 5.8S and 28S rDNA. They are used as a universal fungi barcode marker.

While the 18S rRNA is mainly used for high resolution taxonomic studies of eukaryotes, the ITS region is used to classify fungal diversity in environmental samples. ITS1 and ITS2 have been found to be the most suitable markers for fungal phylogenetic analysis due to their variable sequences, conserved primers and multicopy nature.

Primer nameSequence (5' → 3')RegionTarget
Euk_1391F_EMP
EukBr_R_EMP
GTA CAC ACC GCC CGT C
TGA TCC TTC TGC AGG TTC ACC TAC
18S V9Microbial eukaryotic metabarcoding
gITS7
ITS4
GTG AAT CAT CGA RTC TTT G
TCC TCC GCT TAT TGA TAT GC
ITS 2Fungal metabarcoding

Image credit : Jean-Pierre Flandrois, Claude Bernard University Lyon 1, UMR 5558 Biometrics and Evolutionary Biology Laboratory

Metabarcoding: other organisms and genes investigation

Metagenomics sequencing enables the study of genome diversity of an entire microbial flora (including non-cultivable bacteria) or organisms present in a given sample.

Even if the 16S ribosomal RNA gene sequencing is mostly used in prokaryotic metabarcoding studies, it is also possible to analyze other markers or functional genes (like rpoB, groEL, gyrB, recA, hsp, nirK, nosZ genes…) to identify specific bacterial genus from a specific pathway.

Depending on your sample type, it is also possible to identify others types of organisms with others markers: COI (mitochondrial cytochrome C oxidase subunit I gene) for arthropods, fish, birds and mammals, rcbl or matK plastid markers (in chloroplast genome) for plant or seaweed studies, etc.

List of the available primers for metabarcoding projects.

How to use our Metabarcoding Sequencing by NGS services ?

After discussion with our team, we offer a personalized solution to your project, and tailor-made according to your needs, depending on your targeted regions and your sample diversity.

gDNA or total RNA extraction, marker specific amplification, run sequencing and bioinformatics analysis for genus or species identification are the complete personalized workflow solution that Biofidal can propose, allowing to multiplex up to 864 PCR products simultaneously on a single run. With a paired-end 2×250 bp or 2×300 bp run on MiSeq system and the sequencing deep coverage, the communities investigation will be with high resolution.

You can trust on our reactivity, fast delivery, low prices and scientific support for the best adaptation of protocols and techniques to the goals of your projects.

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