Computational protocol: Development of Human Breast Milk Microbiota-Associated Mice as a Method to Identify Breast Milk Bacteria Capable of Colonizing Gut

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Protocol publication

[…] The 16S rRNA gene sequences obtained from the DGGE bands and bacterial isolates were blasted against the nr database of Genbank using the basic local alignment search tool (BLAST), and their closest relative bacteria were determined. A neighbor-joining phylogenetic tree containing the sequences and their relatives was constructed with the Molecular Evolutionary Genetics Analysis package (MEGA5) with the Jukes-Cantor algorithm. The phylogenetic robustness was assessed by bootstrap analysis with 1000 replicates using the same software. The taxonomy of the bacterial isolates and bacteria represented by the DGGE bands was determined based on the position of their sequences in the phylogenetic tree.For the raw data of Illumina sequencing of 16S rRNA gene V3–V4 amplicons, both the forward and reverse ends of the same read were truncated at the first base where the Q value became no more than 2. Using USEARCH v8.0.1623, individual pairs of reads were merged into a complete read only if they had a minimum overlap of 50 bp. The merged reads that were longer than 399 nt with an expected error of no more than 0.5 were kept for further processing. Quality-filtered reads were delineated into unique sequences and then sorted by decreasing abundance, and singletons were discarded. OTUs were clustered de novo with Uparse () at 97% similarity level. Reference-based chimera detection was performed using UCHIME () against the RDP classifier training database (v9) (). The OTU table was finalized by mapping quality-filtered reads to the remaining OTUs with the Usearch () global alignment algorithm at a 97% cutoff. Sequence data were rarefied to 25,000 reads per sample (1,000 permutations) to avoid bias caused by the difference in sequencing depth. Representative sequences for each OTU were subjected to the RDP classifier to determine the taxonomy with a bootstrap cutoff of 80% (RDP database version 2.10). […]

Pipeline specifications

Software tools BLASTN, MEGA, USEARCH, UPARSE, UCHIME, RDP Classifier
Applications Phylogenetics, 16S rRNA-seq analysis
Diseases Escherichia coli Infections