Homogenized Stool Samples

The 5 sets of homogenized human stool samples were prepared by The BioCollective and stabilized using DNA Genotek’s OMNIgene•GUT Solution following the steps outlined below.

  1. Donor Selection and Sample Collection
    1. Collected at least a 200 g sample from each of the 5 donors using The BioCollective’s BioCollector whole stool collection kit.
    2. The donors were provided with BioCollector whole stool collection kits and requested to provide a sample and take the sample to FedEx on the same day. Samples were shipped to the lab for processing overnight via FedEx Express, stabilized at 4C.
  2. Sample Processing for homogenization
    1. Data sheets printed, relevant data recorded, and sample transferred to a heavy duty specimen bag with corner cut off for dispensing.
    2. Sample transferred from the heavy duty specimen bag into specimen jars in aliquots of roughly 30 g each; sample weights recorded; samples; samples stored frozen at -80°C.
  3. Tube labeling
    1. Prepared 800 labeled tubes before starting sample prep.
  4. Sample Homogenization
    1. Used Ninja blender to homogenize sample in a biological safety cabinet.
      1. Added dry ice to blender first to create powder. Added dry ice at 1-2X the amount of sample (150-300 g).
      2. Added 150 g fecal sample and homogenized to a powder. In order to end up with 700 samples preserved for DNA, we assume 10% loss at homogenization step and 20% loss at the pipetting step.
      3. Placed blender vessel in -20°C freezer overnight to allow dry ice to sublime.
  5. Addition of Sample Preservative
    1. Sanitized 1 L beaker, graduated cylinder, spatula and stir bar in 10% bleach. Rinsed with sterile water and allowed to dry.
    2. Added 900 ml OMNIgene•GUT Solution (OGS) to beaker on a stir plate. Gradually added frozen stool powder to OGS while mixing. This is a 1:10 dilution of the stool sample, resulting in 100 mg sample per 1 ml aliquot. Samples are now stable at ambient and elevated temperatures and are protected from freeze-thaw degradation.
    3. Mixed on stir plate until sample is completely uniform and particles dissolved as much as possible. This may have required mixing overnight.
  6. Addition of Spike-in bacteria
    1. Grew Aliivibrio fischeri (gram negative) and Leifsonia xyli (gram positive) to an approximate density of 109 cells/ml.
    2. Measured actual cell concentration by Optical Density. Performed plate count as confirmation.
    3. Centrifuged cells to concentrate if needed and resuspended in OGS.
    4. Added synthetic spike-in organisms, Leifsonia xyli and Aliivibrio fischeri, at 108 CFU per 100mg/stool and 107 CFU per 100mg/stool respectively, a few minutes before dispensing into tubes. Continued to mix poo-stew throughout process.
    5. Dispensed 750 x 1 ml aliquots into labeled tubes.
    6. Set aside 1 sample per every 75 to be used for QC sequence analysis.
    7. Stored samples at -80°C until ambient temperature shipping.

NIST mixed microbial DNA samples

The Complex Microbial Systems Group at the National Institute of Standards and Technology (NIST) is currently developing microbial genomic and metagenomic reference materials that will allow users to assess the analytical sensitivity and specificity of NGS-based microbial metagenomic measurements.

For the Mosaic Standards Challenge, NIST has contributed two different research materials (700 aliquots of each) that will serve as “ground truth”. Both of these research materials are mixtures of purified genomic DNA (gDNA) from less than 20 unique strains of bacteria. This material was designed to assess the qualitative and quantitative biases that are introduced by different methods in the measurement pipeline; excluding, of course, the DNA extraction method.

To prepare the NIST DNA research material, we extracted total gDNA from pure cultures of each organism. Each purified gDNA was normalized to ~100 ng/µl (based on UV adsorption and dsDNA-specific fluorescence measurements). Final mixtures were made by combining pre-determined volumes of purified gDNA (100 ng/µl) from each organism into a single tube (pool).

Purified gDNA extracted from each organism was sequenced using PacBio and Illumina technologies (~200x and ~500x coverage, respectively). These data were used to generate high-quality genome assemblies for each organism via Canu, Spades and Pilon. Genome assemblies were shared with DNAnexus for inclusion in reference genome databases. Additionally, the same raw genome sequence data was analyzed using several different metagenomic profiling tools to assess the “purity” of each gDNA preparation (identify trace-levels of organismal contamination).

For quantitative analysis of the NIST DNA material, we designed strain-specific digital droplet PCR (ddPCR) assays to determine the absolute abundance (concentration) of each organism (in units of genome copies/µl). These individual genome concentrations were then used to calculate the relative abundance of each genome in the two NIST materials.

Note that the NIST DNA material were designed, in part, to challenge the limits-of-detection of NGS-based metagenomic measurements. To accomplish this, the relative abundance of each genome/strain was staggered in the two DNA mixtures. The relative abundance of each strain/genome can vary from 40% to 0.01% within a mixture. Please consider this fact when deciding how much sequence data (number of reads) to generate as low coverage (fewer reads) may thwart the detection and identification of some of the organisms that are present at relatively low abundance.

Upon arrival, we highly recommend storing the NIST DNA research material indefinitely at 4o C. Avoid repeated freeze/thaw cycles as this can degrade the DNA. The NIST DNA material is in 1 x TE buffer (pH=8) at a concentration of ~100 ng/µl and contain approximately 20 µl (2 µg) of DNA per tube. We recommend that participants re-confirm the concentration of DNA in their own laboratories using their preferred method(s) prior to processing the samples (and please report the results in the metadata form when uploading your sequence data).

The content of the NIST DNA research material (strains and their relative abundance) will be revealed to Challenge participants after we finalize our quantitative assessment (ddPCR) of each organism’s relative abundance. We estimate that these results will be made available by end of Summer 2018.