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Relative species abundance distribution
In order to evaluate sample quality, we analysed both the Relative Species Abundance distribution (RSA) and the rarefaction curves. For each sample, we compared the RSA derived by shotgun and 16S sequencing. RSA histograms in logarithmic scale show that the distributions obtained by shotgun and 16S have similar shape at phylum level (Fig. 1a, b). In Fig. 1b, the 16S sample is characterized by a more patchy distribution, having identified less phyla. At phylum level, both strategies produce positively skewed samples in the log2-transformed distributions, except for 16S outliers, because none of the phyla is significantly rare (Fig. 2a).
Figure 1

RSA histograms in logarithmic scale (Preston plots 21) of bacterial abundances in one sample selected as anexample (caeca25): (a) genera sampled by shotgun sequencing, (b) genera sampled by 16S rRNA sequencing, (c) phyla sampled by shotgun sequencing and (d) phyla sampled by 16S sequencing.

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Figure 2

Box plot of the RSA skewness of bacterial communities at (a) phylum level and (b) genus level. Bacterial communities are sampled with (left) shotgun sequencing and (right) 16S sequencing.

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On the other hand, at genus level, the two strategies display different shapes (Fig. 1c, d, Supplementary Fig. S1, S2, S3, S4). Indeed, the log2-transformed distributions derived by shotgun sequencing generally have a skewness closer to zero compared to those obtained by 16S, i.e. are more symmetrical (Figure 2b): a paired Student’s t-test on the skewness shows a significant difference between them (P = 8·10–6). This indicates that shotgun samples are characterized by a higher sampling size. According to Preston, left-skewed shapes of the RSA can be explained as artefacts of small sample size21,22, since insufficient sampling of the original space produces a truncation of the left tail of the RSA, increasing its skewness.
In shotgun samples, the RSA skewness at genus level is related to the total number of reads (Supplementary Fig. S5): the shotgun samples with the lowest total number of reads have the largest skewness. Specifically, Supplementary Figure S5 shows that shotgun samples cluster in two groups, one characterized by a low number of reads (# reads  500,000, 50/78 samples) and a less skewed RSA.
Noticeably, the high-skewness group includes all 9 samples from 1st day, all 15 crop samples from 14th day and 4 out of 18 crop samples from 35th day. The samples collected at day 1 were very poor in terms of biomass and the crop samples contained more feed residues than caecal samples, making the DNA extraction less efficient both in terms of DNA quantity and quality. For the comparative analysis we removed samples with less than 500,000 reads being characterized by a low quality. This choice was corroborated by the analysis of the rarefaction curves, showing that shotgun samples with less than 500,000 reads do not reach a plateau in terms of identified genera (Supplementary Fig. S6). All the 50 samples included in the comparative analysis have a total number of reads  > 500,000 and a skewness lower than the median of 16S samples, indicating a good sampling depth. Since included samples were characterized by a high microbial load, we are confident to extend the results of the following analyses only to samples with few contaminant DNA and low cross-contaminations. Nonetheless, we have shown that shotgun samples have a RSA similar to 16S samples when a low number of total reads is available, thus hypothesizing that in differential analyses carried on samples with a low microbial load regime, shotgun sequencing could perform similarly to 16S sequencing or even worse.
For a balanced comparison, also 16S samples corresponding to the discarded shotgun samples were removed.
Differential analysis for the experimental conditions
Since in many situations a metagenomic analysis is used to discriminate between different experimental conditions, we compared the results of differential analysis performed on reads obtained by the two strategies. To this aim, we analysed the fold changes of genera abundances between compartments of the GI tract and between sampling times (Fig. 3 for caeca vs crop, Supplementary Fig. S7 for 14th vs. 35th day) common to both sequencing strategies (288 genera for caeca vs crop, and 246 for 14th vs. 45th day). Comparing the genera abundances between caeca and crop, 16S identified 108 statistically significant differences (adjusted P  More

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Phylogenetic analysis
A 1444 bp fragment of the 16S rRNA gene was amplified from the P. eucalypticola strain NP-1 T, sequenced and the sequence deposited in GenBank under accession number MN 238,862. A similarity search with this sequence was performed using EzBioCloud. Thirty valid species belonging to P. fluorescens intrageneric group (IG) proposed by Mulet et al.15 exhibited at least 97% similarity with NP-1 T, and these include P. vancouverensis ATCC 700688 T (98.8% similarity), P. moorei DSM12647T (98.8% similarity), P. koreensis Ps9-14 T (98.8% similarity), P. parafulva NBRC16636T (98.5% similarity) and P. reinekei Mt-1 T (98.5% similarity). The similarities with the other 25 species are provided in Supplementary Table S1. A phylogenetic tree based on the 16S rRNA sequence was constructed and is shown in Fig. 1. Strain NP-1 T forms a weakly supported cluster with P. kuykendallii NRRL B-59562 T, but both strains are situated on separate branches. Strain NP-1 T grouped in none known group or subgroup within P. fluorescens lineage, and it clusters of the outer edge of a much larger group containing several Pseudomonas groups/subgroups. However, Pseudomonas species cannot be identified based only on 16S rRNA analysis.
Figure 1

Neighbor-joining phylogenetic tree based on the 16S rRNA gene of Pseudomonas eucalypticola NP-1T and phylogenetically close members of Pseudomonas. The evolutionary distances were computed using the Jukes-Cantor method. The optimal tree with a sum of branch length = 0.23535266 is shown. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) is shown next to the branches. Cellvibrio japonicus Ueda107T was used as outgroup.

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The MLSA approach based on the concatenated sequences of the partial 16S rRNA, gyrB, rpoB and rpoD genes, has been demonstrated to greatly facilitate the identification of new Pseudomonas strains16. According to the 16S rRNA alignment, 33 species from P. fluorescens IG and one species from P. pertucinogena IG were selected for MLSA. The concatenated sequences of the type strains of each selected species comprised a total of 3813 bp (Supplementary Table S2) and were used for phylogenetic tree construction. The analysis of concatenated gene sequences indicated that strain NP-1 T belongs to the P. fluorescens lineage, and this finding was supported by a bootstrap value of 91% (Fig. 2).However, NP-1 T still cannot be determined which group belongs to17.
Figure 2

Neighbor-joining phylogenetic tree based on concatenated 16S rRNA, gyrB, rpoB and rpoD gene partial of Pseudomonas eucalypticola NP-1T and the type strains of other Pseudomonas species. The evolutionary distances were computed using the Jukes-Cantor method. The evolutionary distances were computed using the Jukes-Cantor method e optimal tree with the sum of branch length = 1.37677586 is shown. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) is shown next to the branches.

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For further identification of NP-1 T, a phylogenomic tree inferred with GBDP was constructed by using Type (Strain) Genome Server (TYGS)18, and all reference type strains and their genome sources are listed in Supplementary Table S3. The result showed the presence of an independent branch supported by a bootstrap value of 88% that can be differentiated from the other Pseudomonas species type strains (Fig. 3) and revealed that NP-1 T clustered with P. coleopterorum LMG 28558 T and P. rhizosphaerae LMG 21640 T which affiliated with P. fluorescens IG, but does not belong to any group. Strain NP-1 T was not be affiliated with any previously described Pseudomonas species and can thus be considered to represent a novel species. Based on above-described the results, P. coleopterorum, P. rhizosphaerae, P. graminis and P. lutea were selected for further analysis with NP-1 T.
Figure 3

Phylogenomic tree of strain NP-1T and related type strains of the genus Pseudomonas available on the TYGS database. The tree inferred with FastME 2.1.6.1 based on GBDP distances calculated from the genome sequences. The branch lengths are scaled in terms of the GBDP distance formula d5. The numbers above the branches show the GBDP pseudo-bootstrap support values  > 60% from 100 replications, and the average branch support is 94.6%.

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General taxonomic genome feature
The draft genome assembly of strain NP-1 T contains 6,401,699 bp. The genome of NP-1 T, which consists of one chromosome and one plasmid, has been deposited in GenBank under the accession numbers CP056030 and CP056031, respectively. The genome has a G + C content of 63.96 mol%, as determined from the complete genome sequence, and 83.45% of the genome is coding and consists of 5,788 genes. The similarity of the genome of P. eucalypticola NP-1 T to other publicly available genomes of closely related Pseudomonas species was determined using ANI, digital DDH and G + C mol %5,6,7,8,9. Each of these comparisons yielded different ANIm and ANIb values, but the highest ANIb and ANIm values of 78.7 and 86.5 were obtained for NP-1 T and P. rhizosphaerae LMG 21640 T. The similarity between P. coleopterorum LMG 28558 T and NP-1 T was higher than that between P. graminis DSM 11363 T and P. lutea LMG 21974 T (Table 1). All ANIb and ANIm values obtained from the comparisons of NP-1 T with the other tested species were below 95%, which confirmed that strain NP-1 T belongs to an independent species. The TETRA frequencies between NP-1 T and the other tested type strains were lower than 0.99, which is the recommended cutoff value for species (Table 2). The digital DNA-DNA hybridization (dDDH) comparison with the draft genome of the type strain NP-1 T yielded low percentages ( More

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