Levan

For bacterial sequences, paired–end sequences were assembled using FLASH^{16 (link)} v 1.2.7, demultiplexed by barcode, and low quality reads (Q–score < 30) were discarded in QIIME¹⁷ 1.8. If three consecutive bases were < Q30, then the read was truncated and the resulting read retained in the data set only if it was at least 75% of the original length. Sequences were checked for chimeras using UCHIME^{18 (link)} and filtered from the dataset prior to OTU picking at 97% sequence identification using UCLUST^{19 (link)} against the Greengenes database^{20 (link)} version 13_5. Those sequence reads that failed to cluster with a reference sequence were clustered de novo. Sequences were aligned using PyNAST^{21 (link)}, and taxonomy assigned using the RDP classifier and Greengenes reference database version^{20 (link)} 13_5. To de–noise the OTU table, taxa with less than 5 total sequences across all samples were removed. A bacterial phylogenetic tree was built using FastTree^{22 (link)} 2.1.3.
Fungal sequences were quality trimmed (Q–score < 25) and adapter sequences removed using cutadapt²³ prior to assembling paired–end reads with FLASH^{16 (link)}. Sequences were de–multiplexed by barcode and truncated to 150 bp prior to clustering using USEARCH vers. 7 pipeline, specifically the UPARSE²⁴ function, and chimera–checked using UCHIME. Taxonomy was assigned using UNITE^{25 (link)} vers. 6.
To normalize variation in read depth across samples, data were rarefied to the minimum read depth of 202,367 sequences per sample for bacteria (n = 298) and 30,590 for fungi (n = 188). To ensure that a truly representative community was used for analysis for each sample, sequence sub–sampling at these defined depths was rarefied 100 times. The representative community composition for each sample was defined as that which exhibited the minimum average Euclidean distance to all other OTU vectors generated from all sub–samplings for that particular sample. Investigators at UCSF were blinded to sample identity until microbiota datasets underwent the aforementioned processing and were ready for statistical analyses.

Stool samples from 308 infants were extracted using a modified cetyltrimethylammonium bromide (CTAB) buffer based protocol^{13 (link)}. Briefly, 0.5 ml of modified CTAB extraction buffer were added to 25 mg of stool in a 2 ml Lysing Matrix E tube (MP Biomedicals, Santa Ana, CA) then incubated (65 °C, 15 min). Samples were bead–beaten (5.5 m s⁻¹, 30 sec) in a Fastprep–24 (MP Biomedicals, Santa Ana, CA) followed by the addition of 0.5 ml of phenol:chloroform:isoamyl alcohol (25:24:1). Following centrifugation (14,000 rpm, 5 min), the supernatant was added to a heavy phase–lock gel tube (5 Prime, Gaithersburg, MD) and chloroform (v:v) was added. Samples were centrifuged (14,000 rpm, 5 min); resulting supernatants were added to fresh tubes, followed by addition of 1 μl of linear acrylamide prior to PEG–NaCl (2v:v). Samples were incubated (21 °C, 2 h), washed with 70% EtOH and resuspended in 10 mM Tris–Cl, pH 8.5.