Microbiome Core Facility Sample Requirements

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 10 ul Illumina 250 PE
16S V4 10 ul Illumina 300 PE
18S 10 ul Illumina 250 PE
ITS 10 ul Illumina 300 PE
16S full length 10 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)

 - 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