Metagenomics and Genomics Workflow Outline and Protocol

gDNA isolation 
- PURElink kit for gDNA – Slight variations to this protocol depending on bacteria 
- Base protocol
- Alterations for C. acnes
- Alterations for Staphylococcus
Hackflex prep – Contains workflows for Tagmentation, barcoding and library ampification PCR, Purification and size selection, and DNA quantification. Relevant steps can be pulled for your particular experiment.
- Hackflex 3.0 protocol
- Generating illumina barcoded stocks and barcode sequences are on the primers folder
Sequencing 
- Pooling: Add your barcoded libraries together in an even ratio at a target concentration 
- Pooling Samples Evenly
- Pooling Samples Evenly if you have very high and very low concentrations
  - Note: The minimum concentration you can use depends on your sequencer. You can convert to Molarity from ng/ul using your avg. Fragment size.
- Adding your amplified library to a tube using values calculated in the “pooling samples evenly” xls file. This is done using a python script that generates an html file you can open on an iPad – useful and worth it!
  - Copy the folder Pipette-Guide-96 into your private directory. Replace “volumes.csv” with the “output of pooling samples evenly.xls” and run “makehtml.py” in that subdirectory (from terminal). Open the new html file on a mobile device and tap through the wells as you pipet.

Lieberman Lab