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ZymoBIOMICS™ Microbial Community Standards

A mock microbial community of well-defined composition.
Ideal for the validation and optimization, along with the quality control of microbiomics or metagenomcis workflows.
Perfect for assessing bias of DNA extraction methods since it contains a mixture of both tough and easy-to-lyse microbes.

Product Size Catalog # Price Qty
ZymoBIOMICS™ Microbial Community Standard 10 Preps D6300
$250.00
ZymoBIOMICS™ Microbial Community DNA Standard 200 ng D6305
$104.00
ZymoBIOMICS™ Microbial Community DNA Standard 2000 ng D6306
$208.00

About ZymoBIOMICS™ Microbial Community Standards

ZymoBIOMICS™ Microbial Community Standard is a mock microbial community consisting of eight bacterial and two fungal strains. It includes 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) (Table 1). Seven of these strains are known human pathogens and have been fully inactivated with DNA/RNA Shield™ (Cat. No. R1100-50). The GC content of the contained genomes covers a range from 15% to 85%. It was constructed by pooling pure cultures of the 10 microbial strains. The cells and DNA content of each pure culture were quantified before pooling. Cultures were mixed to a predetermined composition (Table 1). The actual microbial composition was measured using Next-Generation sequencing techniques (Table 1) after extracting the genomic DNA with the ZymoBIOMICS™ DNA Mini Kit. The ZymoBIOMICS™ Microbial Community Standard was used to assess bias associated with several of the most cited methods for DNA extraction methods, and found that each suffered from over representation of easy-to-lyse gram negative organisms. The ZymoBIOMICS™ DNA Mini Kit was the only method that achieved unbiased isolation (Figure 1). 

ZymoBIOMICS™ Microbial Community DNA Standard is a mixture of genomic DNA extracted from pure cultures of eight bacterial and two fungal strains. Genomic DNA from each pure culture was extracted and quantified before mixing. The GC content of the containing genomes covers a range from 15% to 85%. The microbial standard is accurately characterized and contains negligible impurities (< 0.01%). This enables it to expose artifacts, errors, and bias in microbiomics or metagenomics workflows. The DNA Standard is ideal for assessing biases and errors in processes of library preparation, sequencing, and bioinformatics analyses. It serves perfectly as a microbial standard for benchmarking the performance of microbiomics or metagenomics analyses and to serve as a control in inter-lab studies. This standard is also ideal to help users construct and optimize workflows, e.g. controlling PCR chimera rate (Figure 2) and noise in the library preparation (Figure 3) of 16S rRNA gene targeted sequencing, and assessing GC bias in sequencing coverage of shotgun metagenomic sequencing (Figure 4). 

Details regarding the ten microbial strains (including species name, genome size, ploidy, average GC content, 16S/18S copy number, phylogeny) can be found in Table 2. The 16S/18S rRNA sequences (fasta format) and genomes (fasta format) of these strains are available here. Feel free to contact us if you need help analyzing the sequencing data generated from this standard.


Source A mixture of genomic DNA of ten microbial strains.
Composition

Table 1. Well defined and well characterized standards composed of 5 Gram-Positive and 3 Gram- Negative bacteria plus 2 yeast species with wide GC range (15%-85%) for evaluating and optimizing microbiomics workflows. Impurities less than 0.01%

For lot to lot composition data click below:

ZymoBIOMICS™ Microbial Community Standard
ZymoBIOMICS™ Microbial Community DNA Standard
Impurity level

Table 2. Microbial composition was profiled with shotgun metagenomic sequencing (178 million reads). Taxonomy identification was performed with mOTU (http://www.bork.embl.de/software/mOTU/)


Figure 1. Benchmarking DNA extraction processes with ZymoBIOMICS™ Microbial Community Standard. DNA was extracted from ZymoBIOMICS™ Microbial Community Standard using the four different DNA extraction methods (ZymoBIOMICS™ DNA Mini Kit, Human Microbiome Project fecal DNA extraction protocol, a DNA extraction kit from Supplier M, and a fecal DNA extraction kit from Supplier Q) and analyzed using 16S rRNA gene sequencing. 16S rRNA genes were amplified with primers targeting v3-4 region and the amplicons were sequenced on Illumina® MiSeq™ (2x250bp). Overlapping paired-end reads were assembled into complete amplicon sequences. The composition profile was determined based on sequence counts after mapping amplicon sequences to the known 16S rRNA genes of the eight different bacterial species contained in the standard. Only ZymoBIOMICS™ DNA Mini Kit provides unbiased profiles in this comparison.

Figure 2. PCR chimera increases with increasing PCR cycle number in the library preparation process of 16S rRNA gene targeted sequencing. 20 ng ZymoBIOMICS™ Microbial Community DNA Standard was used as a template. The PCR reaction was performed with primers that target v3-4 region of 16S rRNA gene. Chimera sequences were identified with Uchime (http://drive5.com/usearch) and using the 16S rRNA gene of the 8 bacterial strains contained in the standard as reference.

Figure 3. Controlling noise/rare biosphere in 16S rRNA gene targeted sequencing with ZymoBIOMICS™ Microbial Community DNA standard. The pie chart on the left is microbial composition profile of the standard determined by a regular workflow of 16S sequencing using primers targeting 16S v3-4 region. The pie chart on the right is the profile of the same standard determined using the same primer sets but with an optimized in-house workflow of 16S sequencing. Noises observed on the left panel were mainly caused by PCR chimera, process contamination and reagent contamination, which were controlled in the optimized workflow.

Figure 4. Assessing GC bias of two different library preparation methods in shotgun metagenomic sequencing. Library preparation for shotgun metagenomic sequencing was performed in two different ways: one by Illumina Nextera® XT kit and one by an in-house method. Shotgun sequencing was performed on MiSeq™ with paired-end sequencing (2x150 bp). Raw reads were mapped to the 10 microbial genomes to evaluate the potential effect of GC content on sequencing coverage. Normalized coverage was calculated by normalization with the average sequencing coverage of each genome. The coverage profiles of two selected genomes, Staphylococcus aureus and Pseudomonas aeruginosa, were picked to demonstrate as they cover a wide range of GC content, 15%-85%. While the in-house method shows little/no GC-bias, the Nextera® XT kit has reduced representation for both low GC and high GC areas.

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