Microbiome Core Facility Sample Requirements

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)

 - Preparing Nucleic Acid from FFPE Samples

 - Smear Analysis of FFPE Samples for NanoString

  • 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