Results
The sequence data recovered from INSDC databases ranged in average read length, between 200 bp (animal microbiome) to 300 bp (aquatic microbiome) of average read lengths and pre-processing steps (Figure 1). Notably, the aquatic microbiome data was archived with merged paired ends, for a total average length of 600 bp. On average across datasets, read qualities remained high until 200 bp, but were lowest for the soil microbiome data (Figure 1). Among the 15 samples included for each dataset, the number of reads per sample varied between 14,088 and 97,218 reads per sample animal, between 73,058 and 31,237 reads per sample for aquatic, and between 196,518 and 51,092 reads per sample for soil microbiomes.
Processing sequence data across a gradient of read lengths had consistent effects across datasets, with the average number of reads conserved per sample decreasing gradually with read length in data processed without chimera checking (Figure 2, top), consistent with the quality profile (Figure 1). Importantly, for lower quality sequences, trimming reads below 100 bp resulted in the removal of a large proportion of the original reads during chimera checking, but this was dependent on sequence quality (Figure 2, bottom). In the soil data, chimera checking removed over 25% of the original reads when trimming resulted in reads of 50-80 bp in length, but removed only 11.9 ± 8% if the reads were trimmed to 100 bp. In the higher quality animal microbiome dataset, chimera checking reads that were trimmed below 60 bp in length resulted in a loss of 10.9±2%, while chimera checking reads that were 70 bp or longer only resulted in a loss of 5.1% of the original reads.
The percentage of taxonomically unclassified reads decreased sharply with read length. As expected, this effect was most pronounced at the lower taxonomic levels, but depended on the diversity and/or prior characterization of the system (Figure 3). For the less diverse and more well characterized animal microbiome, read lengths beyond 70 bp achieved minimal improvements in taxonomic classification, while aquatic and soil microbiomes achieved minimal improvements in taxonomic classification beyond 90 and 110 bp respectively. Genus-level classification followed a similar pattern, but required longer reads to reach saturation: in the animal microbiome, 87.1% of the community was classified on average when reads were 120 bp or longer, while in aquatic and soil microbiomes, trimming to 200 bp achieved a classification of 78.8% and 69.3% of the community, respectively.
The number of ASVs detected across the 5 control samples in each dataset was assessed given the same read depth and increasing read length (Figure S1). With increasing read depth, all datasets detected higher numbers of ASVs. Trimming reads to 50 bp resulted in the detection of 180 animal, 228 aquatic, and 367 soil ASVs, which increased to 1029, 1833, and 5105 ASVs, respectively, when trimming to 200 bp.
To determine how trimming affected the detection of diversity, the richness and inverse Simpson index in the 5 control samples of each dataset were assessed. Within each dataset, alpha diversity increased with read depth, but saturated more rapidly in less diverse environments (Figure 4). Richness estimates exhibited a hump with increasing read length in soil samples, decreasing after 160 reads in line with the decrease in read quality (Figure 1). Importantly, while similar patterns were observed for both alpha diversity estimates, Inverse Simpson’s index was more robust to read lengths, saturating with 70, 100, and 160 bp in the animal, water, and soil samples.
The extent to which shorter read lengths affected the variance in compositional metrics (i.e., beta diversity) was assessed by measuring the pairwise Sorensen and Bray-Curtis dissimilarities between the five control samples in each dataset. For all datasets, increasing read lengths resulted in gradual, but saturating increases in dissimilarity. The point of saturation depended on the expected diversity in each system: animal and soil microbiomes approached saturation with 60 and 90 bp reads, respectively (Figure 5). Bray-Curtis dissimilarities were less variable (standard deviation of 0.03, 0.04, and 0.03 for animal, aquatic, and soil microbiomes, respectively) across read lengths than Sorensen dissimilarities (standard deviation of 0.03, 0.09, and 0.05 for animal, aquatic, and soil microbiomes, respectively). To further examine information loss from shorter read lengths, Mantel tests between the communities resulting from each read length and the dataset trimmed to 200 bp (i.e., the most information-rich version, Figure 6) were performed using Sorensen and Bray-Curtis dissimilarities. While the strength of Sorensen-based correlations increased with read length, Bray-Curtis dissimilarities were more robust, and exhibited little deviation from the 200 bp dataset. Importantly, this pattern was consistent regardless of whether a chimera-checking step was included (Figure S2).
Finally, the extent to which shorter read lengths recovered differences in alpha and beta diversity between the control and disturbed samples (1 day after disturbance) was evaluated for each dataset (Figure 7) using Wilcoxon rank sum tests and PERMANOVAs. In general, longer read lengths were able to better discriminate between the alpha diversity of control and disturbed samples, both in terms of richness and Inverse Simpson diversity (Figure 7, top). Above 100 bp, only marginal differences in discrimination between the richness in the control and disturbed treatments were detected with increasing read length for both metrics, with p-values ranging between 0.095-0.055, 0.012-0.010, and 0.151-0.309 for aquatic, animal, and soil microbiomes. respectively. Inverse Simpson diversity was more robust to decreasing lengths, and discrimination did not change with reads greater than 90 bp (p=0.008 all the comparison between control and disturbed in all microbiomes). The discrimination of beta diversity between control and disturbed samples exhibited similar patterns, with the exception of the water dataset evaluated with Sorensen dissimilarities, which indicated that samples became more similar with longer reads (Figure 7, bottom). Sorensen dissimilarity between the treatments in the aquatic communities decreased linearly with read length and discrimination decreased, from p=0.074 with 50 bp data to p=0.456 with 200 bp data). The abundance-weighted Bray-Curtis dissimilarities were more robust to read lengths, exhibiting low variation in general.