Analysis of colony-based PCR 16s rRNA data

Sanger sequencing of colony-based PCR products will result in chromatogram files and sequence (or fasta) files.

Chromatogram files in .ab1 format can be viewed using the Teal Trace Analysis Viewer (https://www.gear-genomics.com/teal/) and fasta files trimmed in a text editor.

Bacterial species (or more typically genera) can be identified using a BLAST search on Genbank. For more details, see our Student handout on BLAST analysis of sequencing data. More accurate identification is possible using a curated database of 16s rRNA sequences, such as Silva (https://www.arb-silva.de/aligner/). The sequences for each individual colony can be submitted separately or the fasta files for each colony can be combined into a single fasta file first.  For directions on how to combine fasta files (which are text files) in Windows, see https://www.computerhope.com/issues/ch001376.htm. To combine fasta files on a Mac, navigate to the folder with the files in the Terminal.  Then, type “cat *.fasta > combined.fasta”.  The extension for the sequence file might be .seq, .fasta, or .fa, and you will need to change the command accordingly.

Analysis of MiSeq Data

For bioinformatic analysis of MiSeq data, we suggest using Purple line in DNA Subway (https://dnasubway.cyverse.org/). The Purple line is a web-based implementation of the QIIME2 pipeline. Use our DNA Subway Tutorial and DNA Subway Video Tutorial for guidance on how to use the Purple line.

Community analysis of MiSeq Data

To conduct community analysis of the level-5 (family-level) taxonomy data that are generated by the Purple line on DNA Subway, we developed the BeanBeetleMicrobiome app (https://beanbeetles.shinyapps.io/BeanBeetleMicrobiome/). The app allows students to identify core and unique taxa, view rarefaction curves, visualize taxonomic diversity, and explore alpha and beta diversity. Our BeanBeetleMicrobiome app Tutorial provides additional guidance.