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Table 1 Recommended best practices during sequenced-based microbiome studies

From: Identifying biases and their potential solutions in human microbiome studies

Processing step Recommended best practices
Sample collection - 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.
Sample storage - 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.
DNA extraction - 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).
PCR amplification - 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