|Processing step||Recommended best practices|
- Collect biological and technical replicates when possible.|
- Use the same collection device and manufacturer.
- When possible, use the same collection personnel.
- Use aseptic techniques during sample collection.
- Make note of any variations during sample collection and include them in downstream analysis.
- When possible freeze samples at −80°C immediately upon collection.|
- Exposure of samples to room temperature conditions should be minimized.
- Preservatives should be used only when freezing of samples is infeasible (e.g., self-collected human samples).
- If used, all samples should be stored in the same preservative.
- Length of sample storage should be noted and included in downstream analysis.
- Extractions should be done using validated extraction kits or validated protocols such as those presented by the Earth Microbiome project or International Human Microbiome Standards.|
- All samples must be extracted with the same protocol.
- Extraction batches should be noted and used as covariates in downstream analysis.
- Extraction should include a mechanical lysis step (e.g., bead-beating).
- Extraction should be done using aseptic techniques and a biological safety cabinet to reduce the amount of possible contamination.
- A small pool of samples should be extracted during each extraction batch and sequenced to determine technical variation.
- Blanks should be carried through extraction to sequencing (critical for low-biomass samples).
- Use high-fidelity polymerases.|
- Minimize the number of required PCR cycles (preferably max. 20–25).
- Obtain as uniform amplification as possible for all samples.
- Primers should be chosen based on the microbes of interest and whether the work is to be compared against previous literature.
|Metagenomic library construction||
- Use equal amounts of template for library construction.|
- Avoid usage of discontinued Illumina Nextera XT kit.
- Note mechanical sonication can cause minor biases toward high-GC content sequences.
|Marker gene bioinformatics||
- Use denoising algorithms such as Deblur, DADA2, or UNOISE.|
- Use validated taxonomic classification methods such as QIIME2 feature classifiers or Kraken2.
- Taxonomic classifiers should be consistent between comparison studies.
- Use well-curated, up-to-date, comprehensive taxonomic databases such as SILVA, RDP, or NCBI.
|Metagenomic shotgun bioinformatics||
- Referenced-based analysis:|
◦ Use DNA-based KMER taxonomic assignment such as Kraken2 or taxMaps.
◦ Removal/filtering of low abundance taxa is recommended.
◦ Employ phylogenetic marker-gene-based strategies when examining low abundance taxa.
- Metagenomic assembly:
◦ Use well-validated assembly methods such as MEGAHIT or metaSPAdes.
◦ Use validated binning methods such as DASTool, MetaWrap, or MetaBat2.
- For all methods, reference databases should be well-curated and up-to-date such as:
◦ NCBI RefSeq
◦ Genome Taxonomy Database