Metagenome Sequencing Q&A

  • General Questions

  • What are the differences between metagenome and microbial diversity?
    • (1) Experimental difference: Microbial diversity mainly targets the amplification and sequencing of ribosomal small subunit gene sequences (16s rDNA or 18s rDNA), while metagenome sequencing only requires fragmentation of DNA obtained by extraction.
      (2) Analytical differences: Microbial diversity sequencing requires OTU clustering and can only study the class and composition ratio of species, while metagenome sequencing requires sequence assembly and evaluation, and can be analyzed and studied at species, gene and function levels at the same time. In summary, microbial diversity mainly tells us what microorganisms are in the environment, while metagenomes mainly tell us what microorganisms in the environment can do.
  • How to choose between metagenome sequencing or diversity analysis?
    • The choice is based on the purpose of your study. If you only focus on the community composition of the environment, including which dominant and non-dominant genera, then microbial diversity is sufficient. If you need to further explore deeper functional information based on community composition, then metagenome sequencing is recommended. Alternatively, a large sample size can be used for microbial diversity analysis, and suitable samples can be selected for metagenome sequencing based on the results of diversity analysis to explore the functional properties of community species in a deeper way.
  • What is the difference between metagenome and metatranscriptome?
    • Metagenome is the study of DNA of all microorganisms in the environment, and metatranscriptomic is the study of RNA of all microorganisms in the environment. Metagenome tells us what all microorganisms in the environment can do, and metatranscriptome tells us what all microorganisms in the environment are doing.
  • How to perform metagenome assembly?
    • After getting the metagenome sequence, in order to remove the connectors and low-quality sequences, it is necessary to perform quality control analysis before assembling the gene sequence. Assembly is the process of splicing a series of short sequences obtained by sequencing into a continuous long sequence, i.e., it is the process of splicing from short sequences to long Scaffolds sequences.
  • Why perform metagenome assembly?
    • 1) Less computational time to perform sequence comparisons after assembly.
      2) The genome can be reconstructed.
      3) The current sequence read length for second-generation sequencing is shorter.
  • What factors are associated with the effectiveness of metagenome assembly?
    • The effect of metagenome assembly is mainly related to the amount of sequencing data of samples, the diversity of species and the distribution of species abundance, which can cause metagenome assembly to be more difficult than that of single species such as bacteria, and this is the focus of the current metagenome research to be broken through.
  • In metagenome assembly, why can't all sample data be combined together for assembly?
    • The high abundance species in different samples are very different, if all samples are mixed together for assembly, it will greatly increase the complexity of the data and the assembly effect may be worse.
  • In the assembly process, is it that the common high-abundance genes can be assembled, but the individual-specific low-abundance genes cannot be assembled?
    • 1) Due to the influence of sequencing depth and sequencing cost, in the current metagenome article, the amount of sequencing data is generally chosen to be 6G, which can detect most of the microorganisms in the sample, but for some low-abundance species, it is indeed likely that they cannot be assembled because of the sequencing depth;
      2) In metagenome analysis, also generally focus more on the composition of higher abundance species, if you want to do special analysis for low abundance species, you generally need to increase the sequencing data volume, or go through some special treatment in the pre-extraction process to enrich as much low abundance species as possible, and then do sequencing analysis.
  • What is the recommended amount of sequencing for different samples?
    • Sequencing volumes generally vary depending on the complexity of the environmental sample. For sample types with complex microbial composition, such as soil samples, sequencing volume of 12G is recommended, while for samples with relatively simple microbial composition, such as intestinal samples, sequencing volume of 6G is generally recommended.
  • Can metagenome sequencing detect information about microorganisms such as bacteria, fungi, protozoa, plankton and viruses?
    • Yes, because the DNA extracted by metagenome sequencing is the total DNA of the organisms in the environmental sample, which can be sequenced and analyzed to detect the information of bacteria, fungi, protozoa, plankton and viruses (DNA viruses) in the environment at the same time.
  • For metagenomic projects, how to exclude host contamination?
    • 1) When taking samples, try not to take them close to the tissue.
      2) Use the appropriate kit when extracting.
      3) If there is a reference genome, the host genome contamination can be removed by comparative analysis.
  • Can metagenome sequencing be performed if there is a large amount of host contamination and there is no reference sequence of the host genome?
    • No, if the host DNA contains a large amount of environmental DNA, after sequencing, there will be serious contamination, and there is no known host genome sequence for comparison and decontamination, which will affect the accuracy of the subsequent analysis and the amount of available data will be small; however, if the amount of contamination of the host genome is small during the extraction process, the contamination can be ignored for the later data analysis.
  • Which genes are annotated by KEGG?
    • All genes annotated by our analysis can be annotated, and you can also select the parts you are interested in.
  • Do I need to have biological replicates for Meta-analysis?
    • It is better to have 5 or more biological replicates, and more than 10 biological replicates are recommended for human and animal samples due to their individual specificity.

  • Sample Preparation

  • What are the considerations for metagenomic soil sample preparation?
    • Soil samples: When sampling, remove the surface floating soil, dig the soil layer of 5-20 cm underground using ethanol fire shovel, after removing the visible roots, the soil is passed through 2 mm sieve, each sample is collected from 3 sampling sites and mixed, where the sample volume is 5g, save the collected soil samples in sterile centrifuge tubes, put them below 0℃, after the samples are collected, transport them back to the laboratory for DNA extraction, and use them for DNA extraction. If you cannot experiment immediately, please freeze the soil samples quickly in -20℃ refrigerator.
  • What are the considerations for metagenomic manure sample preparation?
    • Stool samples: Stool can be stored at -80°C. As a rule of thumb, it is best to take internal stool for genome extraction, but it is not easy to handle. Due to the peculiarities of feces itself, it is recommended to collect and store it at any time to achieve optimal DNA extraction.
For research purposes only, not intended for clinical diagnosis, treatment, or individual health assessments.
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