Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Updating Urinary Microbiome Analyses to Enhance Biologic Interpretation

doi: 10.3389/fcimb.2022.789439

Figure Lengend Snippet: Schematic depicting the 16S rRNA gene and approximate locations of variable regions (V1-V9) that can be selected for amplicon-based sequencing. Samples assessed for this study underwent polymerase chain reaction (PCR) to amplify the V1-V3 region using PCR primers A17F and 515R. When forward and reverse reads are merged, the entire V1-V3 region spans 513 ± 22 base pairs with approximately 87 base pairs of overlapping sequence. Samples also underwent PCR to amplify the V4-V6 region using PCR primers 515F and 1114R. When forward and reverse reads are merged, the entire V4-V6 region spans 581 ± 2 base pairs with approximately 19 base pairs of overlapping sequence. For this study, forward and reverse reads were generated on an Illumina MiSeq platform, which creates sequencing reads of approximately 300 base pairs in length. The initial and final portions of each sequencing read tend to contain lower quality sequence (i.e., lower confidence scores with nucleotide assignment) that could be adjusted or truncated in a DADA2 processing pipeline. As such, paired end reads without a substantial amount of overlapping sequencing may not be able to be merged. Created with BioRender.com .

Article Snippet: It is unclear if the reads were merged or unmerged in the original analysis which used the Illumina BaseSpace 16S Metagenomics App for sequence processing.

Techniques: Amplification, Sequencing, Polymerase Chain Reaction, Generated