cpn60 metagenomic amplicon library preparation for the Illumina Miseq platform

This protocol can be applied to determine the composition of a microbial community. The cpn60 gene (also known as groEL, hsp60) is present in almost all bacteria and a 552-558 bp region of the gene has been established as a barcode for species level identi�cation of bacteria. The primer cocktail used in this protocol ampli�es cpn60 barcode sequences from bacteria with a wide range of G+C content. Some species of Mycoplasma lack the cpn60 gene and therefore this method is not recommended to detect Mycoplasma. DNA sequences generated from this method could be compared to cpnDB, a public database of cpn60 sequences, for identi�cation. Library preparation involves cpn60 amplicon generation, PCR clean-up, index PCR, index PCR clean-up, library quanti�cation, normalization, pooling, library denaturation and loading. Time taken to complete depends on the number of samples included. If using 96 samples, the procedure takes 8 hours but there are several stages where the samples could be stored and continued the next day. Speci�c instructions are provided for the Illumina MiSeq platform, but the protocol could easily be adapted for other sequencing platforms.


Introduction
High throughput sequencing of ampli ed marker gene sequences (barcodes) is a simple and rapid method of assessing the taxonomic composition of a complex microbial community.The "metabarcoding" approach is often based on ampli cation of one or more variable regions of the 16S rRNA gene, however, this target often fails to provide species level resolution.The cpn60 gene (also known as groEL or hsp60) contains a 552-558 bp variable region (the cpn60 "universal target") that can be ampli ed with "universal" PCR primers and this sequence has been demonstrated to meet the criteria as a barcode for Bacteria [1].The sequence provides species level resolution and has been shown to be a good predictor of whole genome sequence similarities [2][3][4][5][6].Since cpn60 is conserved in Bacteria, Eukaryotes and some Archaea, cpn60 barcode sequencing can be used for simultaneous detection of multiple Domains of life [7].A public database of cpn60 sequences, cpnDB (www.cpndb.ca),facilitates the identi cation of cpn60 barcode sequences [8].As a result of these features, cpn60 meta-barcode sequencing has been applied to the characterization of a wide variety of host-associated and environmental microbiomes [7,[9][10][11][12][13][14].
Amplicon sequencing methods are subject to a variety of sources of bias, including ampli cation primer bias.To minimize this effect of cpn60 ampli cation, we developed a primer "cocktail" approach to ensure ampli cation of cpn60 sequences from genomes with a wide range of G+C content [15].Use of a single annealing temperature of 60 °C is appropriate for most samples, although practitioners may want to consider pooling PCR products produced across an annealing temperature gradient in some situations [16].
The protocol presented here is a modi cation of the Illumina 16S Metagenomic Sequencing Library Preparation (Part #15044223) [17] and describes the production of cpn60 amplicon libraries for sequencing on the MiSeq platform.The length of the amplicon prevents assembly of complete barcode sequences, but we have shown that as little as 150 bp from the 5' end of the barcode sequence is generally su cient for species level identi cation [18].We routinely use a 500 cycle sequencing kit but run it as 401x101 rather than 251x251 and use only the Read 1 sequences in downstream analysis.

Procedure
The procedure is presented in 5 steps: Evaluate the cpn60 PCR products by running the no template control and the positive control amplicons in a 1% agarose gel containing ethidium bromide and visualize using an imaging system.If there is a ~680 bp amplicon observed in the positive control and no ampli cation in the no template control (Fig. 1), proceed to amplicon clean-up step or store the amplicon PCR plate at 4 °C and proceed to PCR clean up the next day.

B. cpn60 PCR clean-up:
This step is carried out to remove primer-dimers and un-incorporated dNTPs and primers.
1. Bring the amplicon PCR plate and the NucleoMag beads to room temperature.
2. Centrifuge the amplicon PCR plate at 1000 x g at room temperature for 1 minute and remove the plate seal very carefully.
the index kit.9. Add 25µl of 2x KAPA HiFi HotStart Ready Mix each well.
10. Add a su cient volume of molecular biology grade water to a reagent reservoir (10 µL/well).
11. Add 10 µl of molecular biology grade water to each well using a multi-channel pipette, pipetting up and down 10 times to mix.
12. Cover the plate with a PCR plate sealer.
13. Centrifuge the plate at 1000 x g for 1 minute at room temperature.4. Vortex the NucleoMag beads for 1 minute to resuspend and then add the required amount of NucleoMag beads to a reagent reservoir.
5. Add 32.5µl of NucleoMag beads to each well using a multi-channel pipette, changing tips between samples.
6. Seal the PCR clean-up plate with a foil sealer and shake it in the plate shaker at 1800 rpm for 2 minutes.(Pipetting up and down 10 times using a multi-channel pipet is the alternative method in the absence of a plate shaker) 25. Evaluate the indexed cpn60 PCR product by running a randomly selected subset of NucleoMag cleaned indexed cpn60 amplicons in a 1% agarose gel (see Fig. 2).Alternatively, dilute selected amplicon libraries 1:50 and assess on a Bioanalyzer DNA High Sensitivity DNA chip (Fig. 4).If there is a ~730 bp amplicon observed with the absence of primer dimers (~200 bp), proceed immediately to the library quanti cation and pooling step (E) or store the cleaned-up indexed PCR products at -20 °C for up to 1 week.

E. Library Quanti cation, Normalization and Pooling
Bring the bead cleaned indexed cpn60 amplicon libraries from D to room temperature.
2. Determine the concentration of each amplicon library using the Qubit ds BR kit. 6. Store the remaining Indexed bead cleaned cpn60 amplicons at -20 °C.
6. Label a 1.7 ml microfuge tube as PAL (pooled amplicon library) 7. Add 5 µl from each diluted library to the PAL tube.
8. Follow the "Library Denaturation and MiSeq sample loading" section of the Illumina 16S metagenomics sequencing library preparation protocol.A 10 pM library with 5% phiX is recommended.

Troubleshooting
Ampli cation in the no template control: Repeat cpn60 PCR.Bio analyzer results for a cpn60 amplicon library.(A) Successful library with expected major peak with apparent size of ~730 bp, and without primer dimers (B) Small peak at ~200 bp is indicative of primer dimer contamination.This library is not suitable for sequencing.Repeat clean-up.

3 .
Determine the indexed PCR product size from the Bioanalyzer results from step D-25 4. Calculate the DNA concentration in nM using the below formula as shown in Illumina 16S metagenomics sequencing library preparation protocol: library concentration in nM = [(DNA concentration or Qubit value in ng/µl) x 10 6 ] / (660 g/mol x 748) 5. Adjust the concentration of each library to 4 nM with 10mM Tris pH 8.5 in 1.7 ml microcentrifuge tubes.

Figures Figure 1 of
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Figure 2 Results
Figure 2

Figure 3 Example
Figure 3