6 Knowledge Points Must Know in Microbiology Research

Microbial Technology Channel News: What is the meaning of microbial sequencing setting biological repetition? In general, how many repetitions are set appropriate? Biological repetition refers to sample repetition, such as 3 mice, doing the same treatment at the same time, is three biological repetitionsBiological repetition is very important for the experimental design of sequencing and subsequent information analysis, and the main role of setting biological repetition is the following:1) can eliminate intra-group error s: biological repetition can measure the degree of variation; 2) Enhance the reliability of the results: the more samples sequenced, the more you can reduce the background difference, thus increasing the credibilityof the results; 3) Detection of outlier samples: the existence of abnormal samples, will seriously affect the accuracy of sequencing results, through the follow-up information analysis can be found abnormal samples, to exclude themgenerally, in the natural environment (e.gsoil, roots, plants, etc.) and model animals (e.grats, mice, etc.) recommend at least 5 biological repetitions per group, generally recommended 10 biological repetitions; It is worth noting that if you mix multiple samples together to build a library sequencing, multiple samples become one sample, which is still non-biologically repetitiveTips: It is generally recommended to do more biological repetition, if you encounter a large difference between individuals you can choose to reject individual samples, to avoid the late sample size is not enough, late retransmission of the case of poor repeatingWhat databases are commonly used16S sequencing species annotation s? what are the characteristics (or pros and cons) of these databases?microecological research, is to study the relationship between microorganisms and the environment, the main research subjects include fungi, bacteria, ancient bacteria and virusesFor the large number of 16S rRNA sequencing sequences obtained, reliable species classification results are closely related to a comprehensive databaseThe databases commonly used for 16S sequencing species are RDP, SILVA, Greengenes, NT-16S (NT Library Extraction and Finishing 16S Sequence Database), etc., and the details of these databases can be found in the following table: table: 16S notes common database listNote: first column database name;RDP database is the most commonly used comparison and annotation reference database, the version update is faster, 16S sequence information is complete SILVA database is one of the most popular reference databases available because it is updated in a timely manner Greengenes database relative to RDP, SILVA database, long time not updated, currently longer for the insert removal reference database, and 16S function prediction-PICRUST software is based on Greengenes gg_13_5 developed, so to do PICRUST analysis must also rely on the Greengenes database for comparison NT-16S database is based on NT library extraction and finishing 16S database, the database is the characteristic spool database, because the RDP database can only be commented to the genus level, based on the RDP comment results, further comment the sequence, get a kind of level of comment results 16S species taxonomic notes, often get UNCLASSIFIED, NO_RANK, OTHERS and other meanings? Although theoretically all microbial sequences should be able to be identified at the seed or even strain level, but due to the wide variety of microorganisms, the above-mentioned common database is still difficult to cover all-encompassing Coupled with the limitation of second-generation sequencing read length, it is often only necessary to select 16S rRNA 1-3 high variable zones (currently commonly used amplification fragments V3-V4/V4 zones) as amplification fragments for sequencing, the accuracy of classification is limited (high variable regions of certain species) It may also be very similar, and the ability to distinguish their specific sequence fragments may not be in the amplification area and therefore subject to the length of sequencing during the identification process), so that in the actual analysis process, not all OTU-represented sequences are able to obtain taxonomic information at genus or levels (i.e., the corresponding taxonomy level is not yet "Unclassified") In addition, it is always possible to encounter some of the more novel, not yet fully studied microorganisms (at higher classification levels, such as the door level identified as "Unclassified"), which is normal No_Rank indicates that there is no clear classification information or classification name (related to the database) at a classification level Others is generally self-defined in the mapping, such as when doing heat or bar analysis, generally select the abundance of species (such as the first 20 species of abundance) to show, the remaining species are defined by Others, and The abundance of Others is the sum of the abundance of all remaining species What is OTU ? What to choose from 16S Diversity Sequencing PrimeRs? oTU (operational taxonomy unit) operating classification unit, in micro-ecological research, for the sake of easy analysis, artificially given a classification unit (strain, species, genus, etc.) set the same mark Simply put, The OTU is similar to a species, based on the high variation region sequence comparison, generally the similarity greater than 97% of the sequence group into a class, called the same OTU typically, all sample sequences from the same environment are combined to classify the similarity between the sequences greater than 97% (equivalent to the sequence difference between species classification levels) as an OTU, which theoretically corresponds to a different 16S rDNA sequence, i.e each OTU corresponds to a different species Through OTU clustering analysis, the microbiodiversity in the sample can also be obtained by simplifying the data structure, as well as the abundance of different microorganisms alpha diversity and BETA diversity in microbial detection ? the study of community diversity and ecological diversity is mainly used to compare diversity indices, which include alpha diversity index and beta diversity index alpha diversity refers to the diversity within a particular environment or ecosystem, with a primary focus on the number of species in the uniform habitat of the territory and is therefore called diversity in living territories There are four common indices for alpha diversity: Observed Species, Chao1, Shannon, Simpson the Observed species index refers to the number of OTUs actually included in the sample, and the chao1 index refers to the number of OTUs in the estimated sample, both of which reflect the number of OTUs (species) in the sample, and the higher the two indices indicate the higher the species richness in the sample The Shanon index is used to describe the disorders and uncertainties that occur in the OTU, the higher the uncertainty, the higher the diversity index, the Simpson index refers to the probability of taking two random sequences from the sample, which belong to different OTUs, if there is only one OTU in the sample, the Simpson index is 0, the lowest diversity The Shannon and Simpson indices reflect not only the number of species in the sample (i.e abundance) but also the abundance distribution (i.e homogeneity) of each species in the sample In summary, the more OTU in the sample, the more evenly the Distribution of OTU abundance and the higher the diversity index beta diversity refers to the opposite sex of species composition between different habitat communities along the environmental gradient or the rate of replacement of species along the environmental gradient, also known as habitat diversity, which mainly measures differences between communities The significance of beta diversity is that it can reflect the extent of habitat change or indicate the extent to which habitats are divided by species ; 2) Beta diversity can be used to compare the diversity of different habitats in general, alpha diversity is primarily concerned with species diversity in a particular community, while beta diversity is primarily concerned with differences in species diversity between communities what to choose from 16S Diversity Sequencing PrimeRs? the most commonly used primers in traditional methods are 27F and 1492R, which can almost amplify the complete full-length sequence of 16S rRNA genes, but due to the limitations of second-generation high-throughput sequencing, the primer is clearly not suitable for high-throughput sequencing platforms Given the reading length limitations, only one segment or two variable segments of 16S rDNA can be sequenced In general, environmental microbiome is commonly used in the highdegree sequencing region, V3-V4, V4-V5, or a single V4 region The common primer of the V3-V4 zone, which is commonly used , is 338F/806R, in the following sequence: