Table 1 Summary table of the metabarcoding partners’ individual performances compared to the results obtained in shotgun metagenomic bacterial profiling based on genus presence/absence, α- and β-diversity indexes. 0: poor performer, 1: medium performer, 2: good performer.

P1

2

2

2

2

0

0

1

0

9

2nd

Despite good performance to profile mock bacterial community samples, P1 identifies too high a of number of partner-exclusive bacterial genera at low abundances, which may be explained by the use of thresholds that are too low for genus-level sequence assignment for taxonomic annotation and/or outdated database, as Greengenes, for taxonomicannotation

P2

0

2

1

0

1

1

1

0

6

5th

Counting too high of a proportion of unclassified sequences in mock bacterial community samples, missing Escherichia, P2 also identifies too high of a proportion of partner-exclusive bacterial genera, which may be explain by use the taxonomic database SILVA, accounting for numerous sub-genera or intermediate genus taxonomical groups

P3

0

1

2

0

0

2

0

0

5

6th

Failing to identify numerous important members of the mock bacterial community and gut microbiota, P3 also provided the lowest genus richness and the highest proportion of unclassified sequences. Bacterial profiling remains as an outlier following use of a single bioinformatic pipeline. We make the assumption that the choice of primers for 16S rRNA amplification combined with the use of outdated database such as Greengenes greatly impacts the outcome of the analysis

P4

2

0

0

2

1

2

0

0

7

4th

Overestimates species diversity and genus richness, P4 also identifies a lower proportion of unclassified sequences, which may be due to the use of thresholds that are too low for genus-level sequence assignment for taxonomic annotation. P4 also identifies a higher proportion of partner-exclusive bacterial genera, which may be explain by use the taxonomic database SILVA, accounting for numerous sub-genera or intermediate genus taxonomical groups

P5

2

2

1

2

2

1

1

2

13

1st

Considering the bacterial profiles obtained compared to MGP, this partner performs the best compared to all other for almost all criteria expect the α-diversity, which appears to be slightly overestimated but representing a low proportion of bacterial genus relative abundance

P6

0

0

2

0

2

0

2

2

8

3rd

Despite a tendency for the identification of unclassified bacteria both in mock and fecal samples, which may be due to the scarcity of full-length 16S rRNA gene in the database used, P6 provides a good similarity with bacterial profiles as measured by shotgun metagenomics