Aliquoting stool, oral samples
- Samples should be collected and stored in format that preserves the microbiome
- Omnigene-GUT (stable at room temperature)
- stool samples at -80
- swabs at -80
- aliquotting is performed in hood to minimize background contamination
Nucleic Acid extraction
- Specify appropriate Qiagen kit for sample type
- 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
| Library type | Library prep kit | Input type | Recommended volume | Recommended concentration | Recommended input amount | Product size | Maximum number of samples pooled per lane | Recommended sequencing |
| 16S EMP | EMP protocol (V4) | DNA | 2ul | 5ngs/ul | 10ngs | ~390bp | 384 | 250bp PE MiSeq lane |
| Illumina DNA seq for bacteria | Kappa Hyper Prep | DNA | 25ul | 20ngs/ul | 500ngs | insert size ~500bp | contact sequencing@duke.edu | contact sequencing@duke.edu |
| Illumina RNA seq for bacteria | Stranded RNA-Seq + Ribo-zero, Epidemiology; human-mouse-rat bacteria | total RNA | 10ul | 50ngs/ul | 500ngs | contact sequencing@duke.edu | contact sequencing@duke.edu |
- Provide DNA and RNA samples in eppendorf tubes
- DNAseq and RNAseq requests are made through DUGSIM
- EMP library prep refers to the Earth Microbiome Project
| 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
- Email microbiomesr@duke.edu with the library prep platform/protocol
- Provide suggested Covaris Shearing protocol, if possible
- Provide desired fragment length (bp) in online submission form
- Provide 1.5 - 3ug of genomic DNA or as directed by the library prep protocol in 1.5mL tubes
- Provide 50ul or 130ul in an eppendorf tube for microTube (for shearing sizes between 2,000 - 5,000 base pairs)
- Provide 1,000ul in an eppendorf tube for the milliTube