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Appendix C
Standardize Sample Preparation with ZymoBIOMICS
™
Microbial Community Standards
The
ZymoBIOMICS
™
Microbial Community Standard (Cat. No. D6300)
is a mock microbial community of defined and well characterized
composition making it the perfect control for all microbiome profiling and metagenomics analyses.
It is ideal for assessing bias of DNA extraction methods since it contains three easy-to-lyse Gram-negative bacteria (
e.g.
Escherichia coli
),
five tough-to-lyse Gram-positive bacteria (
e.g
.
Listeria monocytogenes
), and two tough-to-lyse yeasts (
e.g.
Saccharomyces cerevisiae
).
Bead Beating Device Calibration Protocol:
Zymo Research suggests calibrating bead beating devices with the ZymoBIOMICS
™
Microbial Community Standard in order to ensure bias
free microbial extraction. For Disruptor Genie
â
, vortex adapters, and vortex lysis we suggest a time course ranging from 10-45 minutes with
the vortex at maximum speed. For high speed cell disruptors such as the MP FastPrep -24
â
we suggest a time course at maximum speed
with a range of 3-10 minutes. The resulting DNA should be evaluated by quantifying DNA yield and changes in microbial profile at each
time point. The bead beating time that yields a profile that closely matches the theoretical composition should become standard operating
procedure for the bead beating device.
ZymoBIOMICS
™
Microbial Community DNA Standard (Cat. No. D6305)
is a mixture of genomic DNA extracted from pure cultures of
eight bacterial and two fungal strains. Genomic DNA from each culture was quantified before mixing. The ZymoBIOMICS
™
Microbial
Community Standard
allows for assessment of bias from library preparation, sequencing, and bioinformatics analysis.
It serves perfectly as a microbial standard for benchmarking the performance of microbiomics or metagenomics analyses, including those
provided by a 3
rd
party.
Species
GC
%
Gram
Stain
gDNA
Abun.
(%)
Pseudomonas
aeruginosa
66.2
-
12
Escherichia coli
56.8
-
12
Salmonella enterica
52.2
-
12
Lactobacillus fermentum
52.8
+
12
Enterococcus faecalis
37.5
+
12
Staphylococcus aureus
32.7
+
12
Listeria monocytogenes
38.0
+
12
Bacillus subtilis
43.8
+
12
Saccharomyces
cerevisiae
38.4
Yeast
2
Cryptococcus
neoformans
48.2
Yeast
2
gDNA by Shotgun Sequencing
16S Counts by 16S Sequencing
Accurate composition for reliable use to evaluate shotgun seq. and 16S rRNA seq.
Figure 8.
Characterization of the microbial composition of the two ZymoBIOMICS
™
standards with shotgun metagenomic sequencing (left panel) and 16S rRNA gene
targeted sequencing (right panel). The measured composition of the two standards agrees with the theoretical/designed composition. “DNA Standard” represents
ZymoBIOMICS
™
Microbial Community DNA Standard (DNA version) and “Microbial Standard” represents ZymoBIOMICS
™
Microbial Community Standard (cellular
version). Genomic DNA composition by shotgun sequencing was calculated based on counting the amounts of raw reads mapped to each genome. 16S composition
by 16S rRNA gene targeted sequencing was calculated based on counting the amount of 16S raw reads mapped to each genomes.
