Aliquoting stool, oral samples
- Samples should be collected and stored in format that preserves the microbiome
- Omnigene or Norgen tubes
- stool samples at -80
- swabs at -80
- aliquotting is performed in hood to minimize background contamination
- please contact us if you have questions on best practices for sample collection and storage
Nucleic Acid extraction
- Standard DNA extractions will be done using Qiagen Powersoil Pro kit on the Qiacube HT
- Provide at least 0.1g of tissue in eppendorf tube stored at -80C
- Provide at least 250ul of liquid (blood, water or body fluid) stored in -80C
- Pelleted cells in 1.5ml eppendorf tube stored in -80C
Microbiome Profiling
NGS library prep for amplicon sequencing from extracted DNA in eppendorf tubes or 96 well plates.
All sequencing is performed by the Duke Sequencing and Genomic Technologies Core. Sequencing requests and cost estimates can be made through SeqLims. For amplicon libraries and DNA extractions prepared by the core, we will prepare sequencing submissions for you.
| amplicon | minimum volume | flow cell suggested |
| 16S V4 | 20 ul | Illumina 250 PE |
| 16S V4 | 20 ul | Illumina 300 PE |
| 18S | 20 ul | Illumina 250 PE |
| ITS | 20 ul | Illumina 300 PE |
| 16S full length | 20 ul | PacBio Revio |
| metagenomic | contact Sequencing core for details |
| NanoString Nucleic Acid Sample Input Recommendations | ||
| Analyte | Amount Needed (max volume 5uls) | Sample Preparation Guides |
| Unamplified Total RNA from Fresh Frozen | 100 ngs (~20ng/ul) | - Preparing RNA and Lysates from Fresh/Frozen Samples |
| Unamplified Total RNA from FFPE | 300 ngs (~60ng/ul) |
- Provide 4uls of sample in eppendorf tubes that are clearly labeled
- DNA must be stored at -20C
- RNA must be stored at -80C