Microbiome Service

Summary

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.

Contact for Genomics and Bioinformatics Consultation

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

Contact for PacBio Technical Assistance

For technical questions about existing or new Illumina projects, please contact the following Lab Managers;

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.

Ideally, submitting 30uL at 25ng/uL of clean DNA extracted with a Qiagen kit or other high-quality protocol gives us the best starting material for targeted amplicon sequencing. However, this is not always possible with environmental or microbiome samples, and attempting to further clean or concentrate rare or arduously acquired samples may be risky. Since our protocol relies on traditional PCR, with some optimization it can be adapted successfully to work with samples of variable qualities and concentrations. If your samples are of low quality or concentration, please consult with us before submitting and submit at least 20uL of each sample. Additionally, please submit extra volume of a few samples for us to use to optimize the protocol. A good test to see if your samples will work with our protocol is to simply test them with a standard PCR using the marker specific primers.

GGBC has several primers that are appropriate for various taxa. If you would like to use a marker/primer that are not listed below, please provide us with the following: 1) the sequences of the primers you would like to use, 2) some extra volume or samples for optimizing our protocol with the new primers, and 3) expect a short amount of extra time for us to work on your project due to optimization and ordering the primers.

Microbiome Service

llumina Compatible Library (submitted in 96 well plate)UGA FeeNon-UGA FeeCommercial Fee
16S/ITS/Custom (up to 48 samples per plate)$432$510$648
16S/ITS/Custom (49 to 96 samples per plate)$728$859$1,092

 

If you are are submitting 24 or more samples, please submit samples in a 96-well plate and include an Excel file with the sample layout in your order.

PLEASE USE V BOTTOM PLATES AND NOT ROUND/FLAT BOTTOM PLATES.

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 and Pre-Sequencing QC fees

llumina Compatible Library Type (submitted in 96 well plate)UGA FeeNon-UGA FeeCommercial Fee
Amplicons (16S/ITS/Custom)(up to 48 samples per plate)$607$717$911
Amplicons (16S/ITS/Custom)(49 to 96 samples per plate)$903$1066$1355
Pre-sequencing QC$61.35$72.45$92.05
PCR reactions and clean up optimization$100.00$118.00$150.00

Table 2. MiSeq run types

Run TypeMaximum number of reads passing filter
(million)a
Maximum total number of
basesa
UGA FeeNon-UGA FeeCommercial Fee
MiSeq Nano (500 Cycles) (v2) flow cell; PE2502500 Mb$723$854$1,085
MiSeq (500 Cycles) (v2) flow cell; PE25024-307.5-8.5 Gb$1,578$1,863$2,367
MiSeq (600 Cycles) (v3) flow cell; PE30044-50 13.2-15 Gb$1,954$2,306$2,931
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