The MUGTC provides a targeted amplicon approach for evaluating and understanding complex bacterial communities. Universal primers (U515F/806R) designed to the V4 hypervariable region of 16S ribosomal gene. The pipeline provides a streamlined process for the analysis of 96 samples simutaneously with the used of unique dual indexes.
Universal primers (ITS1-30F/ITS1-217R) which target the internal transcribed spacer (ITS) region for analyzing fungal diversity in environmental samples are also available.
In additon to these pre-designed primer sets, a custom target approach is availalbe that uses researcher designed primers in conjunction with a 2-step PCR process to perform targeted sequencing on any region. The approach has been successfully used as a survey technigue for amphibians, viruses in collected eDNA.
The Bioinformatics and Analytics Core can provide an analysis of the data using the Qiime pipeline, http://qiime.org/. This will provide graphical and tabular summaries of the taxa and OTUs identified and their relative proportional representations.
Core staff are available to assist with experimental design and to answer questions related to next-generation sequencing technology. Arrangements for a meeting to discuss new or existing projects can be made by sending an email to Nathan Bivens, Genomics Core director.
All projects must be initiated with a quote before samples are submitted. Send a quote request to the general core email mugenomicscore@missouri.edu.
General Considerations:
The preferred 96-well plate for submitting targeted amplicon samples is from MedSupply Partners (877-633-7779, http://www.medsupplypartners.com/); catalog # 15-3590. The core recommends the use of adhesive sealing sheets from Thermo Scientific (cat. #AB-0558).
Submission Requirements:
| Input Range | Submission Concentration |
Recommended Buffer |
Special Instructions |
|
| Target amplicon (16S & ITS) | 210 ng(3.5 ng/ul)volume = 60 ul | 10 mM Tris-Cl (pH 8.5) | We strongly recommend DNA be quantified by Qubit assay. | |
| Custom amplicon (2-step PCR) | 60 ng (1 ng/ul)volume = 60 ul | 10 mM Tris-Cl (pH 8.5) | We strongly recommend DNA be quantified by Qubit assay. | |
| Amplicon | Sequence Depth/Coverage* | Read Type | Index Type | Read Length | |
| 16S V4 | 100,000 reads/sample | Paired end | Unique dual index | 300 bases | |
| ITS | 100,000 reads/sample | Paired end | Unique dual index | 300 bases | |
* minimum read count recommended; increased sequencing depth may be desired for some studies
Standard amplicon primer sets:
| Target Region | Primer Name | Primer Sequence | Amplicon Length (nt)* | ||
| 16S V4 | U515F | 5'-GTGCCAGCMGCCGCGGTAA-3' | 430 | ||
| 806R | 5'-GGACTACHVGGGTWTCTAAT-3' | ||||
| ITS1 | ITS1-30F | 5'-GTCCCTGCCCTTTGTACACA-3' | 535 | ||
| ITS1-217R | 5'-TTTCGCTGCGTTCTTCATCG-3' | ||||
Custom amplicon primer design:
| PCR Round | Primer Name | Primer Sequence | |||
| Round 1* | Forward Primer | 5'-acactctttccctacacgacgctcttccgatct NNNNNNNNNNN-3' | |||
| Reverse Primer | 5'-gtgactggagttcagacgtgtgctcttccgatct NNNNNNNNNNN-3' | ||||
| * First round amplification to be performed by the researcher. | |||||
| Round 2 | i5 Index Primer | 5'-aatgatacggcgaccaccgagatctacac[i5]acactctttccctacacgacgctcttccgatct-3' | |||
| i7 Index Primer | 5'-caagcagaagacggcatacgagat[i7]gtgactggagttcagacgtgtgctcttccgatct-3' | ||||
Design considerations:
- Lowercase letters denote Illumina adapter sequences necessary for binding to the flow cell
- Underlined lowercase are binding sites for the Illumina sequencing primers
- [i5] and [i7] refer to dual indexing sites; 8 bases per index
- Sequence of uppercase "N's" are region specific portion of round 1 primers