Microbiome/Amplicon 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.

Consultation and Assistance

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 Microbiome Technical Assistance

For technical questions about existing or new Illumina projects, please contact the following Genomics Professional;

Kateryna Stoianova (kstoianova@uga.edu)

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 1^st, 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 2^nd PCR step) as follows:

5’ TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-Forward-Specific-Primer-Sequence 3’

5’ GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG-Reverse-Specific-Primer-Sequence 3’

We have several sets of primers in the lab and are willing to provide aliquots to people who wish to test things out in their own labs.

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:

Clean the initial PCR product provided by the researcher, if necessary.
Check the concentration with the Synergy HT plate reader.
Performing the second PCR to add the barcoded Illumina adapters to all samples.
Clean the second PCR products using Ampure Beads.
Determine the concentration using the Synergy HT plate reader.
Assess a randomly selected set of samples to determine the size of the library using the Fragment Analyzer.
Pooling the barcoded libraries using equimolar ratio.
Determine the size of the pooled library using the Fragment Analyzer.
Clean the pool with Ampur beads if necessary.
Determine the pool concentration using Qubit and qPCR.
Sequencing.

*Under special circumstances we will accept projects that need to be started from gDNA and taken through the PCR1 target region amplification. Please contact us for further information.

Primers Currently Available at GGBC

Primer Name	Literature Names	Target Region	Expected size	Specific Primers	Reference
Klindworth 16S V1V3	27F For. Over	16S: V1-V3	~527	AGAGTTTGATCMTGGCTCAG	Klindworth et al., 2013
Klindworth 16S V1V3	518 Rev. Over	16S: V1-V3	~527	GTATTACCGCGGCTGCTGG	Klindworth et al., 2013

Klindworth 16S V4	F S-D-Bact-0564-a-S-15	16S: V4	~253	AYTGGGYDTAAAGNG	Klindworth et al., 2013
Klindworth 16S V4	R S-D-Bact-0785-b-A-18	16S: V4	~253	TACNVGGGTATCTAATCC	Klindworth et al., 2013

Bradley 18S V4	18S_EukV4F	18S: V4	418	CCAGCASCYGCGGTAATTCC	Bradley et al. 2016, Zhao et al. 2019
Bradley 18S V4	18S_EukV4R	18S: V4	418	ACTTTCGTTCTTGATYRA	Bradley et al. 2016, Zhao et al. 2019

Quince 16S V4V5	16S_Bac515F-Y	16S: V4-V5	400-500	GTGYCAGCMGCCGCGGTAA	Quince et al., 2011; Parada et al., 2016
Quince 16S V4V5	16S_Bac926R	16S: V4-V5	400-500	CCGYCAATTYMTTTRAGTTT	Quince et al., 2011; Parada et al., 2016

16S V3V6	16S-338F	16S:V3-V6???		ACTCCTACGGGAGGCAGCAGT
16S V3V6	16S-1052R	16S:V3-V6???		CGAGCTGACGACAYCCATGCA

Taylor ITS2	18S_5.8S-Fun	ITS2	267-511	AACTTTYRRCAAYGGATCWCT	Lee Taylor et al., 2016
Taylor ITS2	18S_ITS4_Fun	ITS2	267-511	AGCCTCCGCTTATTGATATGCTTAART	Lee Taylor et al., 2016

Caporaso 16S V4	515f forward	16S: V4	300-350	GTGYCAGCMGCCGCGGTAA	Caporaso et al., 2011
Caporaso 16S V4	806rB Reverse	16S: V4	300-350	GGACTACNVGGGTWTCTAAT	Caporaso et al., 2011

H9 1862R	H9			TTACCTGGTCCGGACATCAA
H9 1862R	Reverse Primer-1862R			ATTGTAGCGCGCGTGCAG

Stoek 18S V9	Euk1391F	18S: V9	~260	GTACACACCGCCCGTC	Amaral-Zettler et al. (2009) and Stoek et al. (2010)
Stoek 18S V9	EukBr	18S: V9	~260	TGATCCTTCTGCAGGTTCACCTAC	Amaral-Zettler et al. (2009) and Stoek et al. (2010)

Caporaso 16S V4	16s-515FB forward	16S: V4	300-350	GTGYCAGCMGCCGCGGTAA	Modified from Caporaso et al. (2011)
Caporaso 16S V4	16s-806RB Reverse	16S: V4	300-350	GGACTACNVGGGTWTCTAAT	Modified from Caporaso et al. (2011)

White ITS1	ITS1-F Forward	ITS1	200-600	CTTGGTCATTTAGAGGAAGTAA	White et al. (1990)
White ITS1	ITS2-R Reverse	ITS1	200-600	GCTGCGTTCTTCATCGATGC	White et al. (1990)

Kindworth V3V4	S-D-Bact-0341-b-S-17 (for)	16S: V3-V4	~464	CCTACGGGNGGCWGCAG	Kindworth et al (2013), Illumina
Kindworth V3V4	S-D-Bact-0785-a-A-21 (rev)	16S: V3-V4	~464	GACTACHVGGGTATCTAATCC	Kindworth et al (2013), Illumina

Beckers V5V7	799F-	16s: V5-V7	420	AACMGGATTAGATACCCKG	Beckers et al 2016; Bodenhausen et al., 2013
Beckers V5V7	1193R-	16s: V5-V7	420	ACGTCATCCCCACCTTCC	Beckers et al 2016; Bodenhausen et al., 2013

Vylgalys LSU	LSU-LRoR (forward)	LSU	550-700	ACCCGCTGAACTTAAGC	Vilgalys and Hester 1999
Vylgalys LSU	LSU-LR5 (reverse)	LSU	550-700	TCCTGAGGGAAACTTCG	Vilgalys and Hester 1999

Glass Bt2	Bt2a (forward)	Beta-tublin	~470	GGTAACCAAATCGGTGCTGCTTTC	Glass and Donaldson 1995
Glass Bt2	Bt2b (Reverse)	Beta-tublin	~470	ACCCTCAGTGTAGTGACCCTTGGC	Glass and Donaldson 1995

Hume ITS2	SYM_VAR_5.8SII	ITS-2	~400	GAATTGCAGAACTCCGTGAACC	Hume et al. 2013, 2015
Hume ITS2	SYM_VAR_REV	ITS-2	~400	CGGGTTCWCTTGTYTGACTTCATGC	Hume et al. 2013, 2015

Miya 12S	MiFish-U-F	12S	~160-180bp	GTCGGTAAAACTCGTGCCAGC	Miya et al., 2015.
Miya 12S	MiFish-U-R	12S	~160-180bp	CATAGTGGGGTATCTAATCCCAGTTTG	Miya et al., 2015.

LerayCOI	LerayCOI_FOR	Co1		GGATACATGGATTGAACATGTATTACTCCCTCC
LerayCOI	LerayCOI_REV	Co1		TAATCGACCTTCATCGGGAGTGATCGCCAGAAAGAACTCA

ZBJ COI	ZBJ-ArtF1c:	COI-Arthropods	157	AGATATTGGAACWTTATATTTTATTTTTGG	Zeale et al. (2011)
ZBJ COI	ZBJ-ArtR2c:	COI-Arthropods	157	WACTAATCAATTWCCAAATCCTCC	Zeale et al. (2011)

Braukman COI	MLepF1 (forward)	COI	407	GCTTTCCCACGAATAAATAATA	Braukman et al., 2019
	LepR1 (reverse)			TAAACTTCTGGATGTCCAAAAAATCA
	HCO2198 (reverse)			TAAACTTCAGGGTGACCAAAAAATCA

UniPlant ITS2	UniPlantF	ITS2	187-387	TGTGAATTGCARRATYCMG	Moorhouse-Gann et al., 2018
UniPlant ITS2	UniPlantR	ITS2	187-387	CCCGHYTGAYYTGRGGTCDC	Moorhouse-Gann et al., 2018

Zhao 18S V5V7	FW-F817	18S: V5-V7	379	TTAGCATGGAATAATRRAATAGGA	Zhao et al., 2019
Zhao 18S V5V7	REV-R1196	18S: V5-V7	379	TCTGGACCTGGTGAGTTTCC	Zhao et al., 2019

PacBio 16S	27F27F	16S		AGRGTTYGATYMTGGCTCAG	PacBio 16S protocol
PacBio 16S	14292R	16S		RGYTACCTTGTTACGACTT	PacBio 16S protocol

Gibson COI	ArF5	COI		GCICCIGAYATRKCITTYCCICG	Gibson et al. (2014)
Gibson COI	ArR5	COI		GTRATIGCICCIGCIARIACIGG	Gibson et al. (2014)

PDS	PDSABASGSP-F	Hybrid poplar		ATCCTTTCGYTCTTCTCCGC
PDS	PDSABASGSP-R	Hybrid poplar		TRAAACCATCTTGAGCCTCAACATA

GUX	GUX1ABASGSP-F	Hybrid poplar		ACAGRTGGATTTGGGGRGGA
GUX	GUX1ABASGSP-R	Hybrid poplar		GGATAAAAYCCYYTGGCAGA

TBL	TBL31ASGSP2,091-F	Hybrid poplar		GGTGGTTGCATGGAGCTTTG
TBL	TBL31ASGSP2,502-R	Hybrid poplar		ACCAGGTAAGGGCAGCTTTC

As we get more information or new primers, the table will be updated.

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 Fee	Non-UGA Fee	Commercial 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 Description	UGA Fee	Non-UGA Fee	Commercial 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 Type	Expected number of reads passing filter (million)^a	Expected total number of bases^a	UGA Fee	Non-UGA Fee	Commercial Fee
MiSeq Nano (500 Cycles) (v2) flow cell; PE250	2	500 Mb	$909	$1,073	$1,137
MiSeq (500 Cycles) (v2) flow cell; PE250	24-30	7.5-8.5 Gb	$1,932	$2,280	$2,415
MiSeq (600 Cycles) (v3) flow cell; PE300	44-50	13.2-15 Gb	$2,372	$2,799	$2,965
NextSeq2000 (600 Cycles) flow cell (P1); PE300	200	60 Gb	$2,328	$2,748	$2,910
NextSeq2000 (600 Cycles) flow cell (P2); PE300	600	180 Gb	$4,368	$5,155	$5,460

References

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