How to use the ZymoBIOMICS® Microbial Community Standards
ZymoBIOMICS® Microbial Community Standard and DNA Standard can be used as a defined input to assess microbiomics workflows. The standards aim to help assess how accurately the microbial composition is measured. They can be used in two major applications: (1) the establishment and optimization of an accurate and reliable microbiomics workflow, and (2) the routine quality control of an established microbiomics workflow.
Microbial Community DNA Standard
The ZymoBIOMICS® Microbial Community DNA Standard can be used to determine difference between library preparation protocols by fixing other variables, such as sequencing platforms and bioinformatics analysis methods. The DNA standard is also ideal at optimizing conditions in the library preparation process, e. g. PCR cycle numbers, PCR annealing temperature, 16S primers etc. To use the ZymoBIOMICS® Microbial Community DNA Standards simply thaw the standards and use the recommended amount of DNA input for you library preparation process. We recommend first time Microbial Community Standards users to start off with this kit to optimize and validate your sequencing and analysis methods before addressing bias in extraction protocols.
Microbial Community Standard
While it is highly suggested to use the standards in conjunction with each other, they can be used independently and serve two separate purposes. The ZymoBIOMICS® Microbial Community Standard, which is a mock microbial community containing know quantities of ten different microbes, is used to measure the accuracy of a DNA extraction method. To measure the accuracy of a DNA extraction method, the next time you are extracting DNA from your microbial samples, take 75 ul of the microbial community standards and treat it as if it were just another one of your actual microbial sample. After extracting the DNA from your samples and the microbial community standard, send the DNA through your pre-optimized library preparation, sequencing and data analysis process. We have provided the theoretical composition of the microbial community standard to compare against the sequenced DNA of microbial community standard that went through your extraction protocol. If there are any major discrepancies between the theatrical standard and your sequenced standard, you will be able to determine the flaws within extraction protocol.
How to establish an accurate microbiomic workflow in your lab
How can the ZymoBIOMICS® Microbial Community and DNA Standards help establish an accurate microbiomics workflow? Let’s assume two cases: User A wants to establish a microbiomics workflow in a new lab and User B wants to optimize an existing microbiomics workflow. We will recommend both users apply the ZymoBIOMICS® Microbial Community DNA Standard first to determine best practices in the workflow after DNA extraction step. To determine which library preparation protocols to use, you can compare different library preparation protocols by fixing other variables, such as sequencing platforms and bioinformatics analysis methods. For example, in the case of 16S sequencing, you can use the standard to compare different library preparation protocols, such HMP protocol and EMP protocol. Zymo will soon release a library preparation kit for targeted sequencing. You can also use the ZymoBIOMICS® Microbial Community DNA Standard to optimize conditions in the library preparation process, e. g. PCR cycle numbers, PCR annealing temperature, 16S primers etc.
After you determine the best practices for library preparation, you can start to optimize the DNA extraction step; this requires the use of ZymoBIOMICS® Microbial Community Standard, cellular format. This kit enables you to compare different DNA extraction protocols or commercial kits with the microbial standard Post extraction, DNA samples isolated with different DNA extraction methods can go through the same, pre-optimized library preparation process, sequencing and data analysis. It is a good idea to include a sample of the DNA standard, so that you can compare the results between microbial community standards and the DNA standard. If the results of the DNA standard and the microbial community standard match each other, this means that there is minimal bias in your DNA extraction step; and if they both agree well with theoretical values of the standard, this means that there is minimal bias throughout your entire workflow.
After the best practices for the entire workflow are determined, the ZymoBIOMICS® Microbial Community Standard can be used as a quality control for the whole workflow. For example, it is a good practice to include a positive control (with ZymoBIOMICS® Microbial Community Standard) and a negative control (blank control) in each batch of the DNA extraction. The positive control will show you how consistently and accurately your workflow performs. The negative control can help you assess the total bioburden (or contaminations) of your workflow. Including a negative control is critical to the analysis of low-biomass samples, such as skin swabs.
Analyzing 16S sequencing data of the ZymoBIOMICS® Standard
When sequencing the ZymoBIOMICS® standards analyze them using your regular 16S analysis pipelines, such as Qiime and Mothur. You can compare the measured composition with the theoretical composition of the standard. Questions that should be kept in mind during this comparison include: (1) whether your measurement covers all strains with the proper taxonomy assignment and with correct abundance, (2) whether your measurement indicates the presence of foreign taxa with significant abundance. Taxonomy assignment might be incorrect or improper because of problems in the reference database. Abundance estimation might be off because of bias in DNA extraction, bias in library preparation, poor quality of MiSeq runs, etc. The presence of foreign taxa might indicate process contamination, poor sequencing quality, PCR chimera in library preparation, defects in bioinformatics analysis, defects in reference database, etc. Both the ZymoBIOMICS®Microbial Community Standard and the DNA Standard are certified to have low impurities levels, <0.01% by DNA abundance. So any foreign taxa with abundance higher than 0.01% are derived from artifacts in your workflow.
Analyzing shotgun metagenomic data of the ZymoBIOMICS® Standard
In terms of the accuracy of the measurement of microbial composition, we found shotgun metagenomic sequencing is generally more accurate than targeted sequencing, including 16S sequencing. This increase in accuracy can be attributed to shotgun sequencing library preparation protocols requiring less PCR cycles or can be PCR-free while 16S library preparation is solely PCR based. With that being said, shotgun metagenomic sequencing is not impervious to bias and can experience bias. The ZymoBIOMICS® Microbial Community DNA Standard can easily help you capture this bias. For example, using the ZymoBIOMICS® Microbial Community DNA Standard, we found that the shotgun library preparation kit of Nextera XT (Illumina, CA, US) resulted in lower sequencing coverage for both low GC content regions and high GC content regions.