The SNV-microbe-metabolite trios were assessed using the causal inference test (CIT)57 to test the possibility that SNV causally influences microbe abundance and then the microbe metabolite. The enriched SNVs and their associated microbes, and the differential metabolites between ASD and TD group were subjected to the test. Briefly, the CIT has statistical tests for four conditions, all of which must be met for the microbe -mediated causal classification: (1) SNV and microbe are associated; (2) SNV is associated with microbe after adjusting for metabolite; (3) microbe is associated with metabolite after adjusting for SNV; and (4) SNV is independent of metabolite after adjusting for microbe. The CIT p-value was defined as the maximum of the component test p-values, and multivariate linear regression was used in the four-component tests.

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