Detection and enumeration of Lak megaphages in microbiome samples by endpoint and quantitative PCR

Summary Lak megaphages are prevalent across diverse gut microbiomes and may potentially impact animal and human health through lysis of Prevotella. Given their large genome size (up to 660 kbp), Lak megaphages are difficult to culture, and their identification relies on molecular techniques. Here, we present optimized protocols for identifying Lak phages in various microbiome samples, including procedures for DNA extraction, followed by detection and quantification of genes encoding Lak structural proteins using diagnostic endpoint and SYBR green-based quantitative PCR, respectively. For complete details on the use and execution of this protocol, please refer to Crisci et al., (2021).


SUMMARY
Lak megaphages are prevalent across diverse gut microbiomes and may potentially impact animal and human health through lysis of Prevotella. Given their large genome size (up to 660 kbp), Lak megaphages are difficult to culture, and their identification relies on molecular techniques. Here, we present optimized protocols for identifying Lak phages in various microbiome samples, including procedures for DNA extraction, followed by detection and quantification of genes encoding Lak structural proteins using diagnostic endpoint and SYBR green-based quantitative PCR, respectively. For complete details on the use and execution of this protocol, please refer to Crisci et al., (2021).

BEFORE YOU BEGIN
The protocol below describes specific steps for processing and screening fecal, digesta and mucosal samples for Lak phages, but can be adapted for different sample types. We have also implemented the protocol for use with liquid samples (e.g., jejunal and rumen fluid) and mucosal brushes suspended in sodium-magnesium (SM) buffer.

Sample collection and storage
Timing: 30 min-2 h 1. Avoid cross-contamination between samples and environmental contaminants.
a. Change gloves between samples. b. Use clean, sterile collection equipment. i. For mucosal samples, dissection utensils can be sterilized in 100% ethanol.
ii. For fecal/digesta samples, use sterile, disposable equipment. c. Collect fecal samples from the centre of the stool where possible.
CRITICAL: Use appropriate personal protective equipment and take care when collecting biological samples which may contain pathogens. Alternatives: Purchase pre-prepared TE buffer (e.g., Invitrogen TM , Cat. no. 12090015)

Deoxynucleotide triphosphates (dNTPs)
Preparation of dNTPs is essential for the proposed PCR assay. Prepare solution as follows: Alternatives: Purchase pre-prepared dNTPs (e.g., Thermo Scientific TM , Cat. no. R0181)

STEP-BY-STEP METHOD DETAILS
DNA extraction from microbiome samples

Timing: 1 h
Validated procedure for DNA extraction using PowerFecal Pro DNA kit (Qiagen, Hilden, Germany) on a range of microbiome samples, including feces/solid digesta, mucosal tissues and fluid digesta. Follow manufacturer instructions with adaptations as below: 1. Lysis. a. Mix solid samples in collection tubes with a sterile spatula. b. Place approximately 0.25 g of solid sample in a bead tube with 800 mL lysis buffer. i. For liquid samples, use 200 mL of sample with 600 mL lysis buffer. c. Heat tubes at 65 C for 10 min, using a water bath or heat block.
Note: Heating will aid disassembly of the phage capsid and the release of genetic material. d. Using a vortex adapter, vortex tubes for 10 min at maximum speed.

Inhibitor removal and washing (as specified in manufacturer instructions)
3. Elution.
a. Use 60 mL of elution buffer and incubate spin column at room temperature (20 C-22 C) for 10 min. i . Centrifuge at 13,000 3g for 1 min.
ii . Once eluted, pass the same 60 mL eluate back through the same spin column (without incubation) and repeat centrifugation.
4. Measure DNA concentration by UV spectrophotometry. 5. Store DNA at À20 C until analysis.

Amplification of Lak phage structural protein-coding genes by PCR
Store at À20 C.

OPEN ACCESS
Amplification of Lak phage major capsid protein (MCP), portal vertex protein (PVP) and tail sheath monomer (TSM) gene fragments can be used for detection of Lak phages in biological samples. These signature genes were selected from metagenomic datasets, due to their specificity and conservation across Lak phage genomes. The general screening primers are specific to Lak phages found in humans, primates, pigs and most other animals. An additional set was designed to detect the divergent $660 kbp variant discovered in a racehorse microbiome (Crisci et al., 2021). Primer sequences are reported in Table 1. 6. On ice, prepare master mixes for each assay containing the following: 11. Presence of Lak phage in microbiome DNA can be visualized using agarose gel electrophoresis. 12. Purified PCR products can be sequenced for confirmation and comparison. We recommend using either QIAquick PCR purification or gel extraction kits (Qiagen, Hilden, Germany) prior to sequencing. To maximise yield, follow manufacturer instructions with adaptations as below: a. Use 30 mL of elution buffer and incubate spin column at room temperature for 10 min. b. Centrifuge at 13,000 3g for 1 min. c. Once eluted, pass the same 30 mL eluate back through the same spin column (without incubation) and repeat centrifugation. 13. Measure DNA concentration by UV spectrophotometry.
CRITICAL: PCR purification of Lak MCP gene amplicons is a prerequisite in generating standards for the following qPCR assay.
qPCR quantification of Lak phage major capsid protein-coding gene This protocol has been validated for use with the QuantiNova SYBR Green PCR kit (Qiagen, Hilden, Germany), and PikoReal TM real-time PCR system (Thermo Fisher Scientific, MA, USA) (Crisci et al., 2021). However, the following procedure can be adapted for any SYBR green-based qPCR kit and compatible instrument. As Lak phage genomes are generally AT-rich, the MCP gene was selected due to its conservation and higher GC content ($41%) than other genes, which provides better thermal stability in qPCR reactions.
CRITICAL: The use of PCR product as qPCR standard is expected to yield an efficiency over 100% (Crisci et al., 2021). Efficiencies between 90 and 110% are considered acceptable for quantification. It is advisable to calculate amplification efficiencies using standards and primers prior to qPCR quantification. e.g. (À1+10 À1/slope ) 3 100

Prepare standards for qPCR
Timing: 30 min Lak phage MCP gene amplicons (MCP: LakMC581-F/LakMC1053-R and MCP_660: LakHMC185-F/ LakHMC984-R) are used as qPCR standards. The qPCR target regions are within the regions amplified by the endpoint PCR primers (Table 1). Once a PCR product is obtained, prepare qPCR standards as follows.
a. In the first tube, adjust Lak major capsid PCR product to 5 ng/mL using nuclease-free water or 13 template dilution buffer. b. Then, add 2 mL from the first tube to 18 mL nuclease-free water or 13 template dilution buffer, mix by pipetting up and down, and discard the tip. i. Repeat for remaining serial dilutions. For example:

Prepare DNA dilutions for qPCR
Timing: 30 min 15. Adjust sample DNA to 10 ng/mL using nuclease-free water or 13 template dilution buffer (so that 1 mL provides 10 ng DNA per 10 mL qPCR reaction). attributed by host DNA. For fecal samples and fresh digesta, $70 ng mL À1 to $400 ng mL À1 DNA was obtained.

Expected PCR outcomes: endpoint PCR results
The present endpoint PCR method using phage structural proteins is effective for detection of Lak phages in a variety of microbiome samples. Figure 1 shows positive PCR results for the Lak MCP assay, derived from pig digesta samples.
Expected qPCR outcomes Figure 2 shows representative standard curves and efficiencies obtained using the proposed qPCR assays for Lak phages (based on most known genomes; Figure 2A), and for the $660 kbp variant ( Figure 2B).
Note: Acceptable qPCR primer efficiencies for reliable quantification should be between 90 and 100%. If efficiencies outside of this range are encountered, see troubleshooting.
Figure 3 shows representative melt curves with no non-specific binding using the proposed qPCR assays for Lak phages (based on most known genomes; Figure 3A), and for the $660 kbp variant ( Figure 3B).
Quantification results obtained using the present qPCR assays for Lak phages are published in Crisci et al. (2021). For pig gut luminal contents and mucosal tissues (using Lak_MCP) and horse faeces (using Lak MCP_660), Log copies ranged from $2 to $7.

QUANTIFICATION AND STATISTICAL ANALYSIS
Depending on the users' requirements, statistical analysis of qPCR data will vary. However, for comparison of Lak phage abundance between different treatments or sample groups, we briefly describe steps employed by Crisci et al. (2021). The proposed method for statistical analysis is compatible with JMPâ Pro 14.1 (SAS Institute Inc., NC, USA, 2019), but can be adapted to virtually any statistical package.
1. Prepare data for analysis. a. Collate data into a table, including headings for all known variables. b. Calculate averages for technical replicates, and log-transform. Note: Log transformation simplifies the analysis of data that does not usually conform to normality.
2. Analyse distribution. a. Perform distribution analysis by group (e.g., treatment, or body site). b. Identify outliers and remove as required.
Note: Lak phage abundance may vary beyond the normal distribution for a given population. JMPâ identifies outliers as 1.5*IQR (interquartile range).
3. Design appropriate statistical model. a. If feasible, use least squared mean comparisons or ANOVA. b. Identify covariates so that they can be accounted for in the statistical model.

LIMITATIONS
It should be considered that Lak phage genomes which are genetically distinct to those described by Crisci et al. (2021) and Devoto et al. (2019) may not be detected using the proposed methods. Given that qPCR assays yield efficiencies over 100% (due to use of PCR amplicons as standards), quantification results should be considered as estimations, useful for comparison purposes, rather than definitive quantities.  Table 1 for primer details. A version of this figure has been published previously by Crisci et al. (2021).

OPEN ACCESS
TROUBLESHOOTING Problem 1 DNA concentration too low for PCR or sequencing (step 4).

Potential solution
During DNA extraction, prepare multiple lysates for each sample, but pass all through the same spin column for washing and elution into one tube.

Potential solution 1
To confirm whether negative PCR results are true or due to the absence of Lak phages, it is recommended to use a positive control alongside unknown samples. Once Lak phages are confirmed within sample DNA, the sample should be used thereon as a positive control.

Potential solution 2
During DNA spectrophotometry, check 260/280 and 260/230 ratios, as this can indicate contamination which may interfere with PCR. Furthermore, check that microbiome DNA is not degraded prior to use by electrophoresing 5 uL on a 1% agarose gel with 63 loading dye. If DNA is contaminated, it can be diluted or re-extracted.
Thus far, the present PCR and qPCR primers have not resulted in nonspecific binding. However, should nonspecific binding or secondary structures arise (e.g., PCR amplicon sizes not as expected), the following potential solutions can be implemented.

Potential solution 1
It is advisable to sequence PCR products and perform a BLASTN (Altschul et al., 1990) search for confirmation. Published Lak genomes are deposited in NCBI (Crisci et al., 2021;Devoto et al., 2019).

Potential solution 2
New primers can be designed using published Lak phage genes. Before synthesis, check primer sets for secondary structures (e.g., OligoAnalyzer; Integrated DNA Technologies Inc., Iowa, USA), and validate the assay using methods described in this protocol.

Problem 4
Insufficient PCR product yield for sequencing (step 12).

Potential solution
Repeat the PCR, using a larger reaction volume until a sufficient product yield is obtained.

Problem 5
Lak phage abundance below that reliably detected using the standard curve (step 15).

Potential solution
If Cq values for sample DNA are outside of those plotted in the standard curve (e.g., lower than highest standard dilution), the starting concentration of DNA may be increased.

Problem 6
Inhibition of qPCR (step 15). If Lak phages are detected in sample DNA, but are not quantifiable by qPCR, impurities in the DNA preparation may be accountable.

Potential solution
Use further dilutions of sample DNA to reduce inhibition (e.g., reduce template DNA concentration to less than 10 ng).
Problem 7 qPCR primer efficiencies outside of an acceptable range (step 16).
For efficiencies below 90% or above 110%, the following potential solutions can be implemented.

Potential solution 1
Check primer concentrations are correct, prepare fresh master mix, and repeat the experiment.

Potential solution 2
Use a larger qPCR reaction volume (e.g., greater than 10 uL), as this will decrease the propensity for pipetting errors.

RESOURCE AVAILABILITY
Lead contact Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Joanne M. Santini (j.santini@ucl.ac.uk).

Materials availability
This study did not generate any new unique reagents.

Data and code availability
The original/source data for figures in the paper are available (Crisci et al., 2021).