TrueBac ID User Guide
Bacterial Identification 101
Bacterial identification is the process of assigning an unknown bacterial strain to a known species or subspecies (note that only a few species have subspecies). Species is a basic unit of taxonomy or classification of living organisms, and yet, it is not evident that ‘species’ as a discrete entity exists in the real bacterial world. Nevertheless, we still need the concept of species for practical reasons when classifying and identifying bacteria. Therefore we must also have an acceptable working practice when defining bacterial species. Bacterial identification is a routine process for many clinical and environmental microbiology laboratories, as well as those in the biotechnology and food processing sectors where quality control is extremely important. In recent years, big advances in bacterial identification have followed big advances in analytical chemistry and molecular biology, particularly, high-throughput DNA sequencing. There are now numerous methods that can be used to identify bacteria, and still, accuracy, turn-around-time (TAT), and cost are major concerns for laboratories that need to do this routinely. The accuracy of bacterial identification methods in particular, or rather inaccuracy, can lead to costly mistakes in industrial problem solving, and more importantly, costly mistakes in medical diagnoses and treatment.
Taxonomy consists of three basic interconnected processes: classification, nomenclature, and identification [Learn more]. Most academic, clinical and industrial microbiology laboratories focus on identification. Much fewer still fulfill the other processes of classification and nomenclature; these scientists are called “taxonomists”.
Two Categories of Identification Methods
All methods for bacterial identification fall into two categories:
- Pattern-based identification: A bacterial cell is made of DNA, RNA, proteins and other complex molecules that can be extracted and detected as various patterns. Patterns that are specific to a known species, can be used to identify them. Various types of phenotypic, chemical, molecular and immunological patterns are used to assign bacterial isolates to known species. The quality of the identification is highly dependent on the quality of known patterns used to represent the true and broad diversity of the species. If new patterns have arisen in a target species (e.g. new variant), then identification can likely fail.
- Species concept-based identification: In this method, each bacterial isolate is compared to the type strains of known species using the criterion that is used for defining bacterial species. Since the same method used to classify new species, is used to identify isolates, in theory, every identification should be successful and accuracy should be guaranteed. The only pitfall is that the cost and TAT of this method are generally higher than those of pattern-based methods.
Pattern-based identification systems
|Method||Pattern to detect|
|Biochemical profiles||Presence of specific enzymes and metabolic pathways|
|PCR||Presence of specific DNA sequences|
|Gene sequencing||The sequence of a gene (e.g. 16S rRNA gene, gyrB)|
|Immunological||Presence of a specific antigen|
|MALDI-TOF||Mass spectra of whole cells. Patterns are mainly from ribosomal proteins that are most abundant in cells. Seng et al. 2009|
|Chemical||Profile of chemical components (e.g. cellular fatty acids, whole proteins). MALDI-TOF is one of these.|
Species Concept-based Identification
The bacterial species concept is now based on a direct comparison of genome sequences, so a species concept-based identification scheme can similarly be built using genome sequence data. This process involves two steps: (1) selection of phylogenetically close species using a fast search engine and (2) calculation of average nucleotide identity (ANI) to the chosen species. The generally accepted ANI cutoff for species boundary is 95~96 %. These general standards and procedures for taxonomic purposes have been proposed and should give a good overview of the species concept-based identification scheme. [Chun et al., 2018].
The EzBioCloud team / Last edited on Mar. 25, 2018
About TrueBac TM ID
The TrueBac TM ID system is ChunLab’s cloud service for bacterial identification using whole genome sequences. It is designed to give you the true identity of bacterial isolates.
How does TrueBac ID differ from other bacterial identification systems?
TrueBac ID-Genome uses genome sequence data whereas other bacterial identification systems employ various aspects of phenotypes and genotypes. Because modern bacterial taxonomy defines the species by directly whole comparing genome sequences [Learn more], identification based on genome sequence data is, in theory, always correct and definitive. All other methods detect various types of partial phenotypic or genotypic patterns in bacterial cells, which can give an incorrect identification or is not able to recognize the novel species. When the genome sequence and other data give conflicting identification results, one should always trust the former, since formal bacterial classification is based on the genome data.
Prerequisites for accurate genome-based bacterial identification
The science behind genome-based bacterial identification is simple. Two conditions are required:
- Each known bacterial species has a type strain whose genome sequence has been determined for comparison to other genomes.
- If the genome sequence of a bacterial isolate is sufficiently similar to that of the type strain of a known species, then the strain is identified to that species.
For requirement (a), a genome sequence database of type strains should be established. Such a database should contain quality-assured genome sequences that are taxonomically correct. Also, ideally, it should cover most, if not all, species.
The scientific background of (b) is well established; if the average nucleotide identity (ANI) value between the type strain of a known species and an isolate is ≥ 95~96%, the latter is identified as a strain of the former species [Learn more].
TrueBac TM database consists of highly curated, quality-controlled genome and 16S sequences of type and reference strains, that cover most clinically and commercially important bacterial species.
TrueBac TM DB
TrueBac TM DB is the ChunLab’s proprietary database of 16S gene, core genes and whole genome sequences of type and reference strains [Learn more] and used by “TrueBac ID-Genome“ and “TrueBac ID-16S“ services.
Databases operated by ChunLab, Inc.
|EzBioCloud DB||TrueBac TM DB|
|Data sources||Public domain||Public domain + in-house generated data|
|Curation||Semi-automated curation||Extensive manual curation|
|Contents||All available genome sequences (excluding metagenomic assemblies)||Taxonomically relevant genome and 16S sequences|
|Services||EzBioCloud 16S Identify (Free for academia/non-profit)||TrueBac TM ID-16S for 16S-based ID TrueBac TM ID-Genome for genome-based ID truebacid.com|
At present, TrueBac DB contains over qualified whole genome data for 9,400 species/sub-species, which have been subject to stringent quality-control, and manually curated and taxonomically validated (taxonomic authenticity was checked). TrueBac TM ID-Genome is the only identification system that can provide the identification for over 9,400 bacterial species with almost perfect accuracy.
The EzBioCloud team / Last edited on April 24, 2018
TrueBac TM ID algorithm
Algorithm for identification of a bacterium using its 16S rRNA gene sequence
The most critical measurement for 16S-based species identification is pairwise sequence similarity. However, different sequence alignment algorithms may produce different similarity values. Therefore, it is important to use a taxonomically valid algorithm for alignment and similarity calculation. It is ideal if we calculate all similarities between the isolate and all type strains of the known species. This is doable, but not efficient as it will take very long for computing all pairs (>70,000) while we only need the values that are close enough (i.e., species with >97% similarity). For this reason, a two-step approach is devised for the TrueBac ID-16S service. It is the same as the one used on our public [Identify] service (www.ezbiocloud.net), except that the reference database used in TrueBac ID-16S is more stringently curated. [Learn more].
TrueBac ID-16S algorithm (the above figure) performs the following steps:
- The query sequence is chopped into three fragments of equal length. If the length of the query sequence is < 1000 bp, the query is chopped into two fragments. If the length of the query sequence is < 500 bp, the query will not be chopped.
- The original full-length query and the fragmented sequences, four sequences in total, are used as the query sequence for a BLASTn-based search against the TrueBac 16S Database. Using the different parts of the query sequences in the BLASTn search ensure the correct identification of all potentially similar reference sequences. Fifty hits are collected from each of the four BLASTn searches and combined. Because there are always duplicated hits, the final hit list contains much less than 200 hits.
- A robust pairwise sequence alignment (Myers and Miller, 1988) is carried out between all pairs, that is, the query sequence against all BLASTn hit species identified in the previous step. The alignment algorithm used in TrueBac ID-16S service is same as the one used in defining the 16S cutoff (98.7%) for species definition (Kim et al., 2014) and used in the highly cited EzBioCloud (formerly EzTaxon) service. For more details about 16S similarity calculation, please read this article. Please note that BLASTn identity values are not used for taxonomic purposes [Learn more].
- The completeness(%) of the query sequence is calculated [Learn more]. For example, 50% completeness means that the query sequence covers only half of the full-length 16S gene. The taxonomically meaningful 16S sequence similarity was proposed on the basis of full-length sequences. Therefore, similarity values based on partial sequences should be interpreted carefully.
- Finally, the hit species are sorted by the 16S similarities and displayed as a table and stored. The completeness (%) of the query sequence is also provided.
Interpretation of 16S similarity values should be carefully done. For example, Bacillus cereus shows >99.8% 16S similarity to about ten species [Learn more], implying that very similar 16S sequence does not always mean that the isolate belongs to the hit species.
Algorithm for identification of a bacterium using it’s whole genome sequence
If strain 1 belongs to species A, the necessary conditions are:
- The genome sequence of the type strain of species A must be available.
- The average nucleotide sequence identity (ANI) value between the genomes of the strain 1 and type strain of species A should be higher than the proposed cutoff for bacterial species definition, i.e., 95~96%.
Ideally, ANI values are calculated for the genome sequence of the isolate against the genome of type strains of all known species. This would require an enormous computing resource and is not efficient as we are only interested in the species identification. In other words, only closely related species would matter. For this reason, TrueBac ID-Genome adopted a two-step approach where, first, the potential hit species are identified using four-way searches which are then used to compute ANI values with the query genome. Sometimes, a good-quality sequence cannot be extracted from the final genome assembly. Therefore, in addition to the 16S gene, the recA and rplC genes are used for searching the potential neighboring species. The RplC is a ribosomal protein whereas RecA is not. They are members of the recently revised bacterial core gene set [Learn more].
The TrueBac ID-Genome algorithm (figure above) performs the following steps:
- The query genome is used to find the potential hit species against the TrueBac DB using the following search engines:
1.1. If the query genome contains 16S sequence, it will be used to search against the TrueBac DB using BLASTn program.
1.2. If the query genome contains recA gene sequence, it will be used to search against the TrueBac DB using BLASTn program.
1.3. If the query genome contains rplC gene sequence, it will be used to search against the TrueBac DB using BLASTn program.
1.4. Additionally, at least one search algorithm that utilizes whole-genome information is used. At present, the MASH tool (Ondov et al., 2016) is included in our pipeline.
- The potential hit species identified in the step 1 are pooled and used for computing average nucleotide identity using the MUMmer tool (ANIm; Richter & Rosselló-Móra, 2009)
- The decision for species-level identification is made by considering ANIm and 16S similarity values.
The TrueBac ID-Genome algorithm (figure above) decision making process:
- If there is a known species (species with valid name or genomospecies) with ≥95 % ANI, the decision is made as “Identified correctly to that species.”
- If there are no known species with ≥95 % ANI, the decision is made as “sp. nov.” (meaning novel species).
- If there is an ambiguity in making an identification call, the decision is made as “sp.” (e.g., Bacillus sp.). It includes the following cases.
3.1. The genome sequence(s) are not available for the type strains of some of the potential hit species. In this case, the result of identification can be updated later when TrueBac DB is updated with that missing genomes in future.
3.2. The query genome shows identical or very similar ANI values to two or more species. In most cases, the latter species belong to the same species. In other words, they are synonyms but the necessary taxonomic change (i.e., combining them into a single species) has not proposed yet.
Please note that the TrueBac DB contains >2,000 genomospecies [Learn more]. If your query genome is identified as one of these, it means that you have a novel species. Genomospecies are included in the database and are being expanded constantly to provide users the way of tracking isolates belonging to novel species. For example, CP015110 (a genome sequence deposited in NCBI) represents a novel species in the genus Acinetobacter so it was named the genomospecies CP015110_s in TrueBac DB. If a user isolates multiple strains belonging to this species, they will be identified as “CP015110_s” instead of “Acinetobacter sp. nov.” by TrueBac ID-Genome service. In this way, most, if not all, isolates can be properly classified and organized easily according to the species.
The EzBioCloud team / Last edited on May 01, 2018
How to identify a bacterium using TrueBac TM ID
Bacterial taxonomy now fully embraces genomics. Bacterial species can now be objectively and precisely defined using a comparison of whole genome sequences. Bacterial identification at the species level can, in theory, be always accurate if genome sequences are used. In fact, it has been shown that the substantial portion of clinical isolates are either misidentified or belong to hitherto undescribed/unrecognized species, named genomospecies when genome sequencing is used for the identification at the species level(Roach et al., 2015). In routine microbiology laboratories where bacterial identification is carried out on a regular basis, the scheme described in the proposed minimal standard for bacterial taxonomy (Chun et al. 2018) can be applied to ensure accurate identification using a combination of 16S rRNA gene (16S) and whole genome sequences.
16S and whole genome sequences for use in bacterial identification
The use of 16S and whole genome sequences for the purpose of bacterial identification has its pros and cons:
|Feature||16S rRNA gene sequence||Whole genome sequence|
|Length of sequence||~1,500 bp||0.5~16 Mbp|
|Cutoff used in bacterial identification||98.7% sequence similarity. Be aware that having >98.7% similarity does not guarantee the correct species identification.||95~96% average nucleotide identity (ANI)|
|Database coverage||Almost all species with the valid name were covered in TrueBac DB.||Some species are not covered for whole genome information. However, most of the clinically important species were included in TrueBac DB.|
|Definitive species identification||Not possible||Possible|
|Sequencing time||Hours||<1 hour or hours depending on NGS platform|
Both 16S and whole genome sequencing are valuable tools in routine microbiology laboratories. In most cases, the genome sequence contains at least one 16S sequence, so specific 16S amplicon sequencing is not necessary. Here are three possible scenarios for routine bacterial identification by sequencing:
1. Whole genome sequencing only
Bacterial isolates are subjected to whole genome sequencing using one of available NGS platforms. Assemble raw data to generate contigs and upload the resulting genome assembly to EzBioCloud’s TrueBac ID-Genome for the definitive identification. TrueBac ID-Genome also provides the search result of the 16S sequence included in the genome assembly. If your isolate exhibits the average nucleotide identity (ANI) value of ≥95% to a known species, it is considered successfully identified. Otherwise, it is likely a member of a hitherto unknown novel species. Finding a novel species in either environmental or clinical laboratories is not a rare event.
2. 16S sequencing only
The 16S sequence is determined from an isolate using a Sanger sequencing instrument (e.g., ABI 3700). The final 16S sequence should be edited manually to ensure the highest quality possible. Upload the sequence into EzBioCloud’s TrueBac ID-16S service. If 16S of the isolate shows <98.7% sequence similarity to all known species, your isolate likely belongs to a novel species. However, the opposite is not always true. Many closely related species show 16S similarity of ≥98.7%, even identical 16S sequences in some cases. If you want to achieve an accurate and definitive identification at the species level, you should use whole genome sequencing instead of 16S sequencing.
3. 16S sequencing, and then whole genome sequencing
To reduce cost (but not time), 16S amplicon sequencing can be performed first to see if the isolate belongs to a novel species. Otherwise, the whole genome is then sequenced. This strategy was proposed by Chun et al. (2018). However, this sequential approach does require extra time.
The EzBioCloud team / Last edited on May 5, 2018
TrueBac TM ID Service Types
TrueBac TM ID system for bacterial identification is provided as either API (Application Programming Interface), cloud or appliance.
|Usage||The service can be integrated into users’ existing pipelines||A user can upload 16S/genome data and browse the results using the web interface at www.ezbiocloud.net.||The service can be integrated into users’ existing pipelines|
|Installation of the system||Amazon cloud||Amazon cloud||On-premises with provided H/W|
|Availability||Available for beta-testing/free trial||Available for beta-testing/free trial||Please contact us at email@example.com|
TrueBac ID by subscription or using vouchers
TrueBac ID service can be provided as a subscription or using vouchers. The below video tutorial will show you how to use a voucher to carry out an identification using TrueBac ID-Genome. To get a voucher or for a free trial, please contact firstname.lastname@example.org.
Input data types
TrueBac TM ID system takes either sequence of contigs (after assembly) or raw NGS data.
|Manufacturers||Models||Assembled contigs||Raw NGS data|
|Pacific Biosciences||All models||FASTA/JSON||Under testing|
|Thermo Fisher Scientific||All models||FASTA/JSON||Under testing|
|Oxford nanopore||All models||FASTA/JSON||Under testing|
For a test drive, please contact us at email@example.com.
The EzBioCloud team / Last edited on June 13, 2018
TrueBac ID Demonstrations
TrueBac ID has been designed to definitively identify bacteria using whole genome sequence data. Here we have run TrueBac ID on some publicly available data to highlight the accuracy of the system. Because taxonomy and the TrueBac Reference Database are constantly being updated, the data presented here are those at the time of analysis.
Case #1: NCBI bacterial genome database
Input dataset contains 99,078 bacterial genomes from pure cultures (excluding metagenome and single cell assemblies). Contaminated genomes were also excluded by the ContEst16S tool. All identification results of TrueBac ID are provided at www.ezbiocloud.net. The identification was carried out on May 15, 2018.
Case #2: An unbiased collection of clinical isolates
A team at the University of Washington Medical Center published the genome data of >1,200 bacterial strains isolated from an Intensive Care Unit for a year (Roach et al., 2015; PLOS Genetics 11:e1005413). TrueBac ID was used to re-analyze the same dataset. The identification was carried out on May 15, 2018.
Identification of clinical isolates from ICU for a year using TrueBac ID-Genome
Detailed identification results are available here.
Case #3: Accurate identification of a gut bacterium fails using MALDI-TOF and other conventional methods
A team at Harvard University isolated a potential therapeutics strain from human gut. This strain could not be identified by MALDI-TOF or other conventional methods, so it was tentatively proposed as a novel species ‘Clostridium immunis‘ (Nature 2017; 14;552(7684):244-247) . TrueBac ID successfully identified this strain as Clostridium symbiosum by 98.48% Average Nucleotide Identity (ANI).
Categories of TrueBac ID results against the original species/subspecies designations in the database or publications
Further identified as the species level:
The original name of the genome has the correct genus name but does not contain specific epithet. In this example, Sulfitobacter sp. NAS-14.1 (GCA_000152645.1) is identified as Sulfitobacter pontiacus.
Further identified as the subspecies level:
The original name of the genome has the correct species name but does not contain subspecies name. In this example, Pasteurella multocida FDAARGOS_384 (GCF_002393385.1) is further identified to Pasteurella multocida subsp. septica.
Identified as a genomospecies:
Genomospecies is a potentially novel species and tentatively named in EzBioCloud/TrueBac databases [Learn more]. Actinomyces odontolyticus ATCC 17982 (GCF_000154225.1) is not a strain of Actinomyces odontolyticus but represents a novel species which we named a genomospecies (DS264586_s).
Not identified at the species level:
Because either it is a novel species or there is no sufficient reference genome data, the genome cannot be identified to a known species with confidence. Haemophilus parainfluenzae strain 1209_HPAR (GCA_001053035.1) is identified as a novel species. In this example, the closest known species is Haemophilus parainfluenzae.
The EzBioCloud team / Last edited on May 22, 2018