Microbiome/Amplicon Service


Advances in next-generation sequencing technologies have greatly expanded the field of metagenomics (also referred to as community genomics, or environmental genomics), giving researchers better tools to study genetic material from environmental or microbiome samples without cultivating cultures. There are several approaches, applications, and goals within the field of Metagenomics; an important and rapidly growing one is the use of targeted amplicon sequencing to understand the diversity of community samples.

The targeted amplicon approach involves sequencing a phylogenetically informative marker to identify organisms in community samples. The marker used should be present in all of the expected organisms, and, conserved such that the primers can amplify genes from a wide range of individuals but variable enough to offer resolution that is taxonomically useful. A number of different markers are commonly used and of course vary by taxa of interest, but the most commonly used is the 16S rRNA gene for bacterial samples, the 18S for eukaryotes, and ITS for fungal samples.

GGBC uses a targeted amplicon sequencing protocol for Illumina platforms that is simple, cost-effective, and produces good data. In short, the library preparation process involves two PCR steps. The first round of PCR uses target specific primers with overhang sequences to amplify the selected marker and allow for barcoding. In the second round of PCR, Illumina compatible barcodes are added to each amplicon.

GGBC has successfully worked on projects with several different target markers, primers, and types/qualities of samples.

There are several references listed below for further reading and information both on our specific protocol and metagenomics as a whole.

Consultation and Assistance

Contact for Genomics and Bioinformatics Consultation

Email Dr. Walt Lorenz at wlorenz@uga.edu for a consultation on new or existing Genomics and Bioinformatics projects. Also, you can contact Dr. Lorenz for assistance with grant proposals and for obtaining a letter of support from GGBC.

Contact for Microbiome Technical Assistance

For technical questions about existing or new Illumina projects, please contact:

More contacts

Please visit our “All Inquiries” page for detailed information about who you should contact at GGBC to receive a quick and accurate response.

Sample Preparation

As of April 1st, 2022, GGBC implemented the following changes to the Microbiome Amplicon Sequencing Workflow in order to make the greatest use of our time and resources at GGBC to assist the larger genomic community at UGA and beyond.

1. Researchers must amplify the target regions (16S/18S/ITS/etc.) in their laboratories utilizing their specific primers. The specific primers must have overhangs (universal sequence added to the 5’ end of the specific primers to create binding sites for the barcoded primers in the 2nd PCR step) as follows:



2. Researchers must give evidence of an acceptable target region amplification and the lack of secondary PCR products. This can be a good-resolution gel image or bioanalyzer DNA or fragment analyzer NGS traces. The latter two can be done at GGBC for a fee.

3. Researchers are encouraged to use beads or columns to separate the specific target amplificons from free primers and nucleotides. If they can’t do this step in their laboratories, it can be done at GGBC.

4. Researchers must submit at least 15 µl of the amplicon products in a 96-well v-bottom plates.

With these modifications, the New Microbiome Amplicon Sequencing Workflow will include the following steps:

  1. Clean the initial PCR product provided by the researcher, if necessary.

  2. Check the concentration with the Synergy HT plate reader.

  3. Performing the second PCR to add the barcoded Illumina adapters to all samples.

  4. Clean the second PCR products using Ampure Beads.

  5. Determine the concentration using the Synergy HT plate reader.

  6. Assess a randomly selected set of samples to determine the size of the library using the Fragment Analyzer.

  7. Pooling the barcoded libraries using equimolar ratio.

  8. Determine the size of the pooled library using the Fragment Analyzer.

  9. Clean the pool with Ampur beads if necessary.

  10. Determine the pool concentration using Qubit and qPCR.

  11. Sequencing.

Prices and Quotes

Contact for Financial Inquiries and Quote Requests

Please email Kim and Elizabeth at ggbc@uga.edu, for financial inquiries or to request a quote. Be as specific as possible, so that they can more quickly assist you.

Table 1. Illumina compatible library preparation fees

llumina Compatible Library Type (submitted in 96 well plate)UGA FeeNon-UGA FeeCommercial Fee
Amplicon specific primers with overhang (if necessary)$150.00$177.00$188.00
DNA/PCR product clean up (per half plate)$98.00$116.00$123.00
DNA/PCR product clean up (per plate)$191.00$226.00$239.00
Modified Amplicons (16S/ITS/Custom)(up to 48 samples per plate)$457.00$540.00$572.00
Modified Amplicons (16S/ITS/Custom)(49 to 96 samples per plate)$681.00$804.00$852.00

Table 2. Library pooling and pre-sequencing QC fees

Service DescriptionUGA FeeNon-UGA FeeCommercial Fee
Library Pooling up to 2-24 samples $60$71$75
Library Pooling up to 25-48 samples $105$124$132
Library Pooling up to 49-96 samples$162$192$203
Library Pooling up to 97-144 samples$234$277$293
Library Pooling up to 145-192 samples $306$362$383
Library Pooling up to 193-288 samples$436$515$545
Library Pooling up to 289-384 samples$565$667$707
Pre-Sequencing QC (Qubit, FA, Kapa)$120$142$150

Table 3. Illumina run types

Run TypeExpected number of reads passing filter
Expected total number of
UGA FeeNon-UGA FeeCommercial Fee
MiSeq (500 Cycles) (v2) flow cell; PE25024-307.5-8.5 Gb$1,932$2,280$2,415
NextSeq2000 (600 Cycles) flow cell (P1); PE30020060 Gb

NextSeq2000 (600 Cycles) flow cell (P2); PE300600180 Gb



References for further information

Peer reviewed articles –

Broad review of metagenomics:

Xu, Jianping. 2006. Microbial ecology in the age of genomics and metagenomics: concepts, tools, and recent advances. Molecular Ecology. 15:1713-1731.

Details and development of the targeted amplicon sequencing method on which our and Illumina’s protocols are based:

Bybee, S.M, et. al. 2011. Targeted Amplicon Sequencing (TAS): A Scalable Next-Gen Approach to Multilocus, Multitaxa Phylogenetics. Genome Biology and Evolution. 3:1312-1323.

Validation of suitability and accuracy of Illumina platforms for community amplicon sequencing:

Caporaso, J.G., et. al.. 2012. Ultra-high-throughput microbial community analysis on the Illumina HiSeq and MiSeq platforms. The ISME Journal. 6:1621-1624.

Primer evaluation for 16S studies:

Klindworth, A. 2012. Evaluation of general 16S ribosomal RNA gene PCR primers for classical and next-generation sequencing-based diversity studies. Nucleic Acids Research. 41:1

Review of metagenomics focused on public health and clinical microbiology:

Forbes, J.D., et.al. 2017. Metagenomics: the next culture-independent game changer. Frontiers in Microbiology. 8:1069

Other good sources of information –

Illumina’s 16S protocol