Computational protocol: Culturing of female bladder bacteria reveals an interconnected urogenital microbiota

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Protocol publication

[…] The isolates were grown in their preferred medium and pelleted. Genomic DNA was extracted from pelleted cells using a phenol–chloroform method. DNA was prepared and sequenced using the Illumina Hi-Seq platform with library fragment sizes of 200–300 bp and a read length of 100 bp at the Wellcome Sanger Institute, as previously described. Annotated assemblies were produced using the pipeline described previously. For each sample, sequence reads were used to create multiple assemblies using Velvet v1.2 and VelvetOptimiser v2.2.5 ( An assembly improvement step was applied to the assembly with the best N50 and contigs were scaffolded using SSPACE and sequence gaps filled using GapFiller. Automated annotation was performed using PROKKA v1.11. [...] Whole-genome metagenomic sequencing was performed on the Illumina HiSeq 2500 as described previously with human contaminating reads filtered by mapping to the Human reference genome (hg19) with bowtie2. Filtered sequences were compared at the genus and species levels using lowest common ancestor analysis previously described and by relative abundance at the sequence level by alignment using the bowtie2 algorithm to the complete bladder culture collection genome catalog. [...] The phylogenetic analysis was conducted by extracting amino acid sequence of 40 universal single copy marker genes, from bacterial collection using SpecI. The protein sequences were concatenated and aligned with MAFFT v. 7.20, and maximum-likelihood trees were constructed using FastTree with default settings. All phylogenetic trees were visualized in iTOL. ANI was calculated by performing pairwise comparison of genome assemblies using MUMmer. […]

Pipeline specifications

Software tools Bowtie2, MAFFT, FastTree, iTOL, MUMmer
Applications Phylogenetics, Metagenomic sequencing analysis, Nucleotide sequence alignment
Organisms Escherichia coli, Bacteria, Homo sapiens, Firmicutes, Streptococcus anginosus, Lactobacillus iners, Lactobacillus crispatus
Diseases Urinary Bladder Diseases