Supplemental Materials for: A standardized quantitative analysis strategy for stable isotope probing metagenomics DOI: https://doi.org/10.1128/msystems.01280-22

Table S1. E. coli AFE (%) in each treatment condition. 

Table S2. Characteristics of pre-centrifugation spike-ins.  To produce distinct distribution patterns along the density gradient, some spike-ins were artificially enriched with 13C through PCR by adjusting the ratio of unlabeled dNTPs and uniformly-labeled 13C dNTPs.  Theoretical AFE values are reported based on the ratio of labeled dNTPs, but actual AFE values were not experimentally confirmed. 

Table S3. Comparison of various abundance estimation strategies. All results were derived from the qSIP analysis method.  Sensitivity and specificity were averaged across the seven treatment conditions.

Table S4. Average total coverage across all fractions for E. coli in different treatment conditions after subsampling from 100% to 0.001% of the original E. coli sequence reads.

Table S5. Comparison of MW-HR-SIP and qSIP methods for detecting isotopic labeling of E. coli at different levels of total genome coverage across the density gradient. ‘True’ indicates E. coli was correctly identified to be isotopically labeled (true positive), and ‘false’ indicates E. coli was incorrectly identified as unlabeled (false negative). NA corresponds to the failure of the MW-HR-SIP algorithm with that dataset. 

Table S6. The impact of genome coverage levels on detecting isotope incorporation using the qSIP model.

Table S7. Comparison of MAGs retained and the number of false positives detected using the qSIP method after applying different minimum genome coverage thresholds. MAGs were retained if their average total coverage in the unlabeled controls exceeded the coverage threshold. E. coli was the only true positive and had a coverage of 1029X, thus no false negatives were detected using the coverage thresholds below.

Table S8. Comparison of specificity, sensitivity, and balanced accuracy of the qSIP method after applying minimum AFE thresholds. To be identified as isotopically labeled, the lower 95% CI interval of a genome’s estimated AFE must be greater than the minimum AFE threshold.

Table S9. Comparison of false positives MAGs identified by the MW-HR-SIP, qSIP, and ΔBD methods.  Names of the false positive MAGs are listed in each column.

Table S10. Comparison of E. coli AFE confidence intervals estimated using qSIP alone, qSIP after first applying MW-HR-SIP, and qSIP after first applying ΔBD method to identify a subset of putatively labeled MAGs. Condition B (“20pct_20ng”) was removed as it E coli was never identified as an isotope incorporator in this condition.