TCR/BCR repertoire analysis

repertoire analysis FAQ

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Bacterial Flora Analysis

Flora Analysis FAQ

TCR/BCR repertoire analysis Q&A

Basic information

What is immune repaertoire?
T cells and B cells, the main types of immune cells, have individually different specific receptors, which express TCR and BCR on the cell surface as they can react to various antigens. A collection of lymphocytes characterized by TCR and BCR is called a TCR/BCR repertoire. “Repertoire” is a French word, synonymous with “Repertory.”

What is immune repaertoire?

Where was repertoire born from?
The immune system, to react to various antigens, have a mechanism to create a diverse TCR or BCR by gene rearrangement and somatic hypermutation, and a repertoire is formed through these mechanisms. Gene reconstruction mainly occurs in lymphoid tissue (bone marrow for B cells and the thymus for T cells).
What is the scale of repertoire?
It is estimated to be up to 1018 for TCR and 1014 for BCR.

Analysis technology

What is repertoire analysis?
The repertoire analysis is conducted to clarify the specificity and diversity of TCR or BCR by examining the frequency of V and J genes and the nucleotide sequence of the CDR3*, consisting of TCR or BCR. To determine the nucleotide sequence of the TCR or BCR gene, we use an mRNA for gene amplification and unbiased gene amplification method originally developed in our company. In addition, we can conduct the most exhaustive and accurate repertoire analysis.
* CDR3: Complementarity Determining Region 3. CDR3 is particularly diverse region which binds directly to an antigenic peptide by TCR and to an antigen by BCR. What os our repertoire analusis?
What targets are analyzed?
Our repertoire analysis targets are α, β, γ, and δ chains for T cell receptor (TCR), and heavy chain; μ (IgM), γ (IgG), α (IgA), δ (IgD), ε (IgE) and light chain; λ (IgL), κ (IgK) for B cell receptor (BCR).
Please tell us about Adaptor-Ligation PCR (AL-PCR).
AL-PCR adds the adapter sequence at the 5′ end of DNA, which is then subjected to PCR amplification using a pair of primer sets consisting of an adapter primer and a constant region (C region) specific primer. Thereby, for all TCR or BCR genes, we can amplify it without bias. Please check the technology page for more information.
Are there any advantages in comparison with other technologies?
In principle, AL-PCR, adopted by our company, does not cause the PCR bias at the time of gene amplification. Thus, it correlates well with the results of the repertoire analysis obtained using techniques such as flow cytometry (FACS). In contrast, for a generally used Multiplex PCR method which uses several primers specific for the V and J genes, PCR bias occurs by the difference in amplification efficiency between the primers. In addition, for BCR, because somatic hypermutation occurs frequently in the V and J genes, mismatches between the designed primers and templates is inevitable and thus PCR amplification failure occurs in some parts of the gene. In AL-PCR, because it does not require a designed primer that corresponds to the region where mutations can occur, more accurate repertoire can be obtained, which as advantageous.
What is the difference between FACS and your analyses?
Repertoire analysis reagents commercially available for use in FACS analysis today include a panel of antibodies against human TCRVβ chain. This antibodies panel supports ~70% of the current Vβ chain, but very few are against the Vα chain. In addition, in FACS, because numerous cells are required, very few types of the samples can be analyzed; in addition, FACS is not suitable analyzing cells infiltrating tissues. In our repertoire analysis, because the total RNA extracted from the sample is used, analysis of various samples is possible. By a large scale of sequencing using the next-generation sequencing, homology search with a dedicated database, translation of CDR3 to amino acid sequence, and aggregation of the number of reads using special software can be performed. Therefore, frequency analysis of all the V and J genes and ranking analysis of all clones with CDR3 amino acid sequence will be possible. In addition, we have confirmed that the results of our repertoire analysis correlate well with those of FACS analysis. Therefore, customers having repertoire data analyzed by FACS can use our service with confidence as well.
Please tell us about the sequencer you are using.
We use a MiSeq manufactured by Illumina.
How do you analyze the sequence data?
After sequencing is complete, it is automatically analyzed using a repertoire analysis software (Repertoire Genesis), originally developed by our company. A homology search is performed in each sequence read with the database of the V and J genes; furthermore, the amino acid sequence in the CDR3 is specified. The search aggregates the reads with a common combination to determine the level of the clones present in the specimen. We can also generate a 3D graph for cross-tabulation in the V and J genes. For useful CDR3 length analysis to analyze clonality, using the CDR3 sequences obtained from the next-generation repertoire analysis, it is also possible to reproduce as digital CDR3 Spectratyping.
We do not know TCRV chain gene name well.
There are multiple nomenclatures for TCRV chain, and each of them have different orders. Please check the comparison table by IMGT. Although notation such as “Vβ” was previously used for FACS analysis, IMGT classification, which is a numbering system based on the location of the gene on the genome (referred to as TRAV or TRBV), is commonly used now. We also use the IMGT classification for notation.
Is analysis software original?
We have developed a dedicated, fully automatic repertoire analysis program called Repertoire Genesis to process the data output from the next-generation sequencing at high speed and accuracy.

For repertoire analysis request

How much amount of specimen is needed?
In the repertoire analysis, it is assumed that sufficient number of lymphocyte (the targets for analysis) is included in the specimen. For a healthy individual, 5 mL of whole blood (i.e., 5 x 106 of peripheral blood mononuclear cells) and ~100 mg of tissue are generally required, but for sites other than lymphoid tissues (e.g., skin tissue), more amount of specimen is required. If you sort a certain cell populations including CD4+ or CD8+ cells, it will be possible to analyze by 104-106 cells. If you consult us for the purpose of analysis or regarding the amount of specimen required, we will individually convey an appropriate way.
Can you conduct repertoire analysis with RNA extracted by a customer?
Although this is possible, before we begin the repertoire analysis, we conduct a quality check using Agilent TapeStation at our company. If RINe value is extremely low (1–5) (and favorable results may not be obtained), we contact the customer before analysis. If you cancel the analysis, the customer is charged only for the cost of quality check.
Can you conduct a repertoire analysis using genomic DNA?
Our repertoire analysis is only suitable for total RNA. Unfortunately, we cannot analyze genomic DNA.
Please tell me how to store the tissue for analysis.
We recommend that you store the tissue immersed in RNA stabilization reagent (RNAlater). For more information, please check our protocol of tissue collection method.
Can the submitted specimen be returned after analysis?
In principle, we cannot return the specimen. With regard to the total RNA extracted from the sample, we can nevertheless return the remainder to you.
How do I send a specimen to your company?
Please send it in accordance with our sample shipping protocol.
What is the delivery time required?
We generally deliver the results in 1–2 months. We estimate the delivery time after we receive your specimen, and let you know the analysis schedule; however, the schedule may be changed depending on the schedule of routine analysis with sequencing equipment. If you require the results earlier (e.g., for a conference or because of paper submission deadline), please contact us.
Is it possible to occupy the sequencer?
Because numerous reads can be obtained in the next-generation sequencing, tag sequences (index sequence and MID sequence) are added for multiple samples before sequencing. Therefore, if the number of specimens is less than that required for a single run, we basically sequence multiple samples. If you want to occupy the analyzer, please consult us. We will provide the estimate individually.

Evaluation of the analysis results

How many reads are required?
Required number of reads differs depending on the object for analysis. For highly diverse sample, to prove that a particular TCR content is less than 0.001%, more than 100,000 effective reads are required, and the actual required total number of reads is 1.5–2.0 times more than the number of the effective reads. On the other hand, if you want to determine TCR content from the established T-cell clone, ~1,000 reads are considered to be sufficient. Although just a standard at our company, the target value is 100,000 effective reads and 10,000 effective reads for the MiSeq and GS Junior, respectively. Analysis may be adjusted if you consult us for the target at the time of order.
Please tell me about how to read the analysis results
The 2D graph shows what each V and J gene is being used for TCR and BCR in the specimen. Among the control samples, comparing the frequency of gene usage and finding the outliers is possible. The 3D graph of VJ gene shows the frequency of usage of the combination of each VJ gene in TCR and BCR. Hence, you can see the bird’s eye view of the entire repertoire. From the increase of specific VJ gene, it is also possible to detect the change in clonality. Aggregate ranking shows a rank table ordered by the copy number of unique reads of TCR or BCR present in the specimen. In the case of specimens with high diversity, it consists of only the low copy number of the reads, whereas with a high clonality specimen, a small number of high copy of the reads is detected. Using the CDR3 sequence of the reads of interest, it is possible to conduct various comparisons as per the analysis target and purpose (e.g., whether a particular clone is present in the other samples or whether it increased or decreased). Please consult us if you have any further questions.
Why is the peak higher in the 3D graph than in the top ranking?
The 3D graph of VJ gene shows the frequency of genes with the same combination of VJ gene. Even the same combination of the VJ gene contains genes with different CDR3 sequence; therefore, the high peak in the 3D graph does not necessarily indicate the high presence of a clone. If you want to evaluate clonality, please also confirm the results of the final ranking.
How should I perform the comparison between specimens?
You can compare the frequency of usage of the V and J genes between samples using the 2D or 3D graph. You can efficiently track the clone of interest in ranking data by searching the CDR3 sequence on the entire sequence reads in the analyte of interest. Please consult us if you have any questions.

Bacterial flora analysis Q&A

What are bacterial flora?
A great variety of bacteria exist in nature. Very few of these bacteria exist as a lone species in a given environment; instead there is a great variety of bacterial species. For example, it is known that in the feces, oral cavity, soil, and rivers, there exists fixed bacterial flora comprising more than 1,000 kinds of bacteria.
What is bacterial flora analysis?
This is an analysis method for specifying the distribution and species of the bacteria in a fixed environment.
What is the subject of this analysis?/dt>
We use a method that comprehensively and efficiently analyzes the bacterial species and distribution, whereby we extract the genomic DNA from bacteria in the sampled specimens, perform sequence analysis of the 16S rRNA -a characteristic sequence which is conserved across bacterial strains-, and clarify the bacterial flora based on the subtle differences between bacterial species.
Is this superior to other techniques?
In conventional methods, the specimen would be diluted and then plated on, for example, nutrient agar, to isolate bacteria individually. Then, various bacterial detection tests and 16S rRNA gene sequence analysis would be used to identify the individual bacteria. Another method was to amplify the 16S rRNA gene using PCR, digest with restriction enzymes, perform electrophoresis, and identify bacterial flora by differences in the position of the resultant bands. However, these methods had problems, such as the fact that the number of bacterial species that could be identified at once was limited from a few species to around 100 species and, while it was possible to indirectly analyze changes in the bacterial flora, it was not possible to identify bacteria definitively.
In “flora genesis” 16S rRNA bacterial flora analysis using next generation sequencers, it is possible to comprehensively analyze and identify from several thousand to several tens of thousands of bacterial species in specimens and test samples at once. Compared to conventional methods, it is possible to obtain orders of magnitude more data on the bacterial flora.
How is this different from meta-genome analysis?
The so-called meta-genome analysis is a method that uses next generation sequencers to analyze all the bacterial genomes within a sample simultaneously. While this gathers an exceptionally large amount of data, it increases the occupancy of sequencers for each specimen, and is thus very expensive. In the broader sense, 16S rRNA bacterial flora analysis is also a meta-genome analysis; however, since it is limited to the analysis of only part of a gene sequence, several specimens can be loaded for a single next generation sequence analysis, reducing the cost for this process. It is necessary to use appropriate methods to suit the aim of the study.
What region of the 16S rRNA is amplified?
In our company’s bacterial flora analysis, we mainly use the V3-V4 region as the target of our analysis. We can also analyze the V1-V2 regions, or other regions, tailored according to your needs.
What kinds of sample can be analyzed?
In principle, if the specimen contains bacteria, analysis is possible. Most specimens are feces or saliva, etc. Other specimens, such as bacterially infected human or animal tissues can also be analyzed; however, since mixtures containing host DNA are difficult to analyze, please contact us beforehand for such cases.
What methods are used to prepare and transport the samples?
>In bacterial flora analyses, we observe the distribution of the bacteria contained in the sample. Consequently, it is necessary to avoid conditions that allow for the proliferation of bacteria as much as possible. At our company, we generally recommend immediate freezing. Additionally, we also ask that shipping to our company be made via frozen delivery.
How much sample is required?
This depends upon the amount of bacteria in the sample. For feces, around 100 mg (about soybean size), and for saliva, around 500 µL are required. Since amounts vary depending on the specimen, for other specimens, please contact us.
What model of sequencers do you use?
Our company uses Illumina MiSeq.
How is the sequence data analysis performed?
Based on Fastq data obtained from sequence analysis, our in-house bacterial flora analysis software conducts primary analyses, including a dendrogramatic analysis, distribution mapping, and ranking.
What kind of data do you provide?
We provide the Fastq data obtained from the sequences and the primary analysis data from the bacterial flora analysis software. As an optional service, we also provide secondary analysis data to customers who request for them.
What kind of analyses can you provide as optional services?
We can provide diversity analysis, principal components analysis, and multivariate analyses. By conducting complex analyses, such as whether a relationship exists between clinical data and data from other analysis and the bacterial flora analysis, it is possible to extract significant data.
Is the analysis software original?
Yes, that is correct. The development was conducted in collaboration with BITS Co., Ltd., and the software can output consumer-friendly reports and perform association analyses between multiple factors including TCR/BCR repertoire analysis.
How long are the turn-around times?
The process generally takes 1-2 months. This is because multiple samples undergo sequence analysis at the same time, and varies depending on how busy the process is. We provide separate estimates for cases that need to be completed quickly or separately.
What are the read numbers for sequencing?
We conduct the sequence analysis with the aim of making 50,000-100,000 reads. Because multiple specimens undergo sequence analysis at once, it is not possible to specify a read number; however, it is possible to limit the read number during analysis.
Please provide an overview of the analysis results.
In addition to the client’s information and specimen information, the bacterial flora results report includes data on the bacterial flora of the particular sample, such as data on the detected bacterial distribution, and ranking data, as well as detailed information, such as comparative data that analyzes differences between samples.
How should I compare between samples?
For those who have requested multiple samples, we provide a range of distribution map representations for multiple samples. Additionally, we recommend % comparisons by bacterial species, based on the ranking data. These are conducted during the primary analyses; however, for complex, secondary analyses such as multivariate analysis, please contact us.