Table 1.

Important remarks for analyzing the microbiome of anaerobic reactors for biogas production.

Method	16S rRNA amplicon seq.	Shotgun DNA	Shotgun RNA
Number of reads needed for accurate taxonomic analysis.	Low (>10,000). All the sequences target the 16S rRNA gene and this allows reliable investigation of the main taxa with few reads.	Very high (>1,000,000). Number of reads assigned to the 16S rRNA gene is low.	Intermediate (>100,000). Loss of reads determined by the presence of transcripts other than 16S rRNA gene is quite limited.
Possible suggestions.		Increase the number of clade-specific marker genes other than 16S rRNA using dedicated software (e.g. MetaPhlAn)
Hypervariable regions.	Analysis targets one or two selected regions. This can reduce accuracy in calculating abundance of specific taxa (e.g. Peptostreptococcus, Tepidimicrobium and Acetivibrio).	Analysis targets all the hypervariable regions. This can increase both the efficiency of taxonomic analysis and the evaluation of abundance for most taxonomic groups.	Same as shotgun DNA.
Possible suggestions.	Increase the number of hypervariable regions under investigation with longer reads (e.g. using PacBio SMRT technology) or analyzing more than one amplicon. V1-V2 regions seem particularly promising to improve taxonomic results.
Universal primers.	Universal primers introduce biases (e.g. Sphaerochaeta and Methanoculleus) due to inability of hybridizing on all the 16S rRNA molecules.	No amplification step needed, this reduces biases in taxonomic investigation.	Same as shotgun DNA.
Possible suggestions.	Perform accurate check for potential biases in 16S rRNA gene amplification. Use more than one couple of universal primers.
Transcriptional activity.	This approach targets genomic DNA, transcriptional activity cannot be monitored and expression level of the 16S rRNA gene does not influence analysis.	Same as 16S rRNA amplicon seq.	This approach targets RNA molecules and provides insights in activity of specific taxa. Analysis can be inaccurate in determining abundance of taxa characterized by high or low activity.
Possible suggestions.	Combine different sequencing approaches to gain insights both on microbial abundance and on their activity.	Same as 16S rRNA amplicon seq.	Same as 16S rRNA amplicon seq.