免疫组库的测序 常见问题解答

基本知识

在体内免疫T细胞和B细胞表面上有许许多多识别特异抗原的受体,即T细胞受体和B细胞受体(抗体)。这些受体是由病毒细菌或肿瘤抗原诱导和产生的,可以发挥免疫防御机制。免疫组库是机体内T淋巴细胞和B淋巴细胞多样性的总和,反应着特定时期机体内的免疫系统应对内外界刺激应答的能力。

What is immune repaertoire?

免疫组库的多样性是怎样产生的?
B细胞主要在骨髓组织,而T细胞在胸腺组织产生。在这些组织中各种不同的抗原刺激免疫系统,启动了基因重排机制,同时发生体细胞超突变,从而产生多种多样的T细胞受体和B细胞受体。
免疫组库多样性的规模有多大?
据预测B细胞受体约有1014而T细胞受体则可达到1018

分析技术

什么是免疫组库测序?
免疫组库测序主要检查V和J基因的表达频率,分析TCR或BCR的CDR3 的核苷酸序列。其目的是阐明TCR或BCR的特异性和多样性。 为了确定TCR或BCR的基因序列,我们把mRNA作为模板进行无偏差基因扩增,测序以后,进一步进行全面详细准确的生物信息分析。

* CDR3:是指互补决定区3。 CDR3在受体基因的多变区,是T细胞受体或抗体直接与抗原结合的部位。CDR3的氨基酸序列是决定受体或抗体特异性的最重要的信息。

What os our repertoire analusis?

免疫组库测序的目标基因都有那些?
我们可以分析所有T细胞受体和抗体基因序列。其中T细胞受体包括α,β,γ和δ链; B细胞受体(抗体)则包括重链的μ(IgM),γ(IgG),α(IgA),δ(IgD),ε(IgE)和轻链的λ(IgL),κ(IgK)。
请介绍贵公司的衔接子连接PCR (AL-PCR)
在TCR或BCR的dsDNA的5’末端添加衔接子,利用该衔接子设计一个特异性引物,在3’C区设计另外一个特异性引物。这样只利用一对引物进行PCR, 可进行无偏差扩增基因,保证了高精度免疫组库测序的目的。如果想了解更多,请您能查看我们的技术页面。
贵公司的AL-PCR和其他扩增技术比较有什么优点?
可想而知,我公司采用的AL-PCR在基因扩增时不会引起PCR扩增偏差。 用此方法经过高通量序列分析后,把我们的测序结果和流式细胞法的结果做了比较,发现两者之间具有显著的相关关系。 相比之下,在通常使用的多重PCR方法,由于使用V和J基因特异的多对引物而且其各引物的扩增效率有差异,常常导致不均匀的PCR扩增。 除此之外,在BCR的V和J区的基因由于频繁发生体细胞超突变,所设计的引物和模板之间的错配是不可避免,这又是常常造成PCR扩增失败的原因。 AL-PCR扩增法则不受其基因发生体细胞超突变的影响,因此可以获得更精确的分析结果,这对准确的测定抗体的序列是非常有益处的。
贵公司的免疫组库测序法和FACS分析法有什么不同?
市售可用于FACS分析的试剂盒十分有限。这些试剂包括一些抗人TCRVβ链抗体,而这些抗体只能分析约〜70%的Vβ链,而可用于分析Vα链的抗体则更少。此外,FACS分析需要很多细胞,常常不适合分析莫些细胞,也不能分析浸润组织的免疫细胞。我们的免疫组库测序是用少量细胞样品提取总RNA后测序的,因此可以对各种样品进行分析。通过高通量测序后可进行同源性搜索,确定CDR3的氨基酸序列,归纳所有V和J基因的频率。此外,我们的免疫组库测序结果与FACS分析结果高度正相关。因此,一直用FACS分析的研究人员也可以安心利用我们的分析方法。
请告诉贵公司在免疫组库测序时用哪个高通量测序仪
我们用Illumina公司的MiSeq高通量测序仪。
贵公司是怎样分析测序数据的?
免疫组库基因测序后,我们使用我们自己开发的专用分析软件Repertoire Genesis,可以自动进行分析数据。利用所读取的序列信息在V和J基因的数据库上进行同源性检索,确定CDR3中的氨基酸序列。综合分析各基因读长的组合频率,确定样克隆性和多样性。 我们还可以简便的制作V和J基因的交叉3D图。 利用组库分析获得的CDR3序列也可以再现数码CDR3 Spectratyping,分析克隆性。
我们不知道T细胞V链的名称,怎么办才好?
TCRV链有几种命名法,各个命名法都有各自不同的顺序号码。详细内容请参考IMGT上的比较表。以前在FACS分析中使用过诸如Vβ的符号,但现在通常多使用IMGT的分类法。该分类法是按照基因在基因组上的位置编排号码的,比如TRAV,TRBV 等等。 我们也使用IMGT分类法的符号。
请介绍一下贵公司的免疫组库分析软件
我公司独自开发了专门用于免疫组库分析的全自动程序,我们命名为Repertoire Genesis。该软件可以快速,准确地处理从高通量测序得来的序列数据。

关于免疫组库测序的要求

免疫组库测序需要多少样品?
我们假设在所要分析的样品里含有足够数量的淋巴细胞。 如果样品来自健康人,一般需要5mL全血(即5×106个外周血单核细胞)和〜100mg的组织就足够了。如果是皮肤等淋巴以外其他部位的组织则需要更多的量。 如果是CD4 +或CD8 T细胞等特定的细胞群,104-106个细胞数即可进行分析。 如果您有其他疑问,敬请联系我们,我们将在第一时间给您回复。
贵公司用我们提取的RNA可以进行免疫组库分析吗?
我公司可以用客户提取的RNA进行免疫组库测序,但在免疫组库分析之前,我们首先用Agilent TapeStation进行RNA的质量检查。 如果RINe值低于5,有可能难以得到理想的测序结果。这时候我们将联系客户进一步讨论决定是否继续进行测序。如果取消测序,我们只收取质量检查的所耗费用。
贵公司用基因组的DNA进行免疫组库分析吗?
我们只用RNA进行免疫组库分析,不做基因组的DNA的免疫组库测序。
请告诉怎样保存组织标本。
我们推荐您把样品浸入RNA稳定剂(RNAlater)中。如果需要更多的有关信息,请查参考我们的处理组织标本的方法。
免疫组库分析以后,可以还给我们标本吗?
原则上我们不返还客户的组织样品。 至于分析后剩下的总RNA,根据客户的要求我们可以还给客户。
我怎样寄送我的组织标本到贵公司呢?
请查参考我们的邮寄组织标本的方法。
免疫组库测序需要多长时间?
我们通常在1-2个月以内交付测序分析结果。 我们收到客户的样品以后,计划分析时间并通知客户交货时间。不过随着测序设备占用率的变化,交货时间也可能会有改变。 如果是因为会议或交稿截止日期等原因,您更快地需要分析报告书,请联系我们,我们将尽最大努力满足您的要求。
我们的组库分析可否和其他客户的样品不混合,独立进行测序?
高通量测序可以读取大量数据, 所以只要给样品添加标签序列(索引序列和MID序列),一次就可以分析很多样品。 如果样本数量少,我们一般同时把来自各客户的样品放在一起测序。 如果要求独立占用测序仪进行分析,请联系我们,我们将进一步讨论客户的特殊要求。
免疫组库分析需要多少读取数量?
所需的读取数量因分析对象和目的而不同。 对于高度多样化的样本来说,如果需要确认特异的TCR含量小于0.001%,需要超过100,000个有效读数,而实际总读取数量则需要有效读数的1.5-2.0倍。 如果要确定克隆T细胞的TCR含量,读取约1,000个读数应该是足够的。 这只是我们公司的标准,实际上我们用MiSeq读取10万个有效读数。 如果您有特殊要求,请联系我们咨询。

对免疫组库测序结果的评估

请告诉怎样理解免疫组库测序结果?
2D图分别揭示在样本中含有多少TCR或BCR的不同的V和J基因。通过对照样本,比较基因使用的频率,可以发现异常值。 VJ基因的3D图是个鸟瞰图,您可以比较容易地看出免疫组库测序的总体结果。它表示TCR或BCR中各种VJ基因组合的表达频率,从中可以判断特异性VJ基因的增加,也可以检测克隆性的变化。总体排行表则根据在样本中存在的TCR或BCR的特异基因序列的拷贝数多少依次排列的。在高多样性的标本其读取的拷贝数很低,而在高克隆性标本中只能检测到很多拷贝的读数。根据分析目标还可以比较CDR3的序列。比如,特定克隆是否存在于其他样品中,是否增加或减少。
为什么有些基因在3D图显示高峰,而在排行表去不在上位上?
VJ基因的3D图只表示示相同VJ基因组合的频率, 不反应CDR3序列的特异性。 在相同的VJ基因组合可以含有不同CDR3序列,所以3D图中的高峰基因根据CDR3的特异性还可以进一步区分出不同的类别,所以3D图中的高峰基因并不表示一定有高频度的克隆存在。 如果想确定克隆性,请您同时确认最终排行表的结果。
我怎样应该比较标本样品的结果?
您可以比较2D或3D图,去发现V和J基因的使用频率是否有无区别。 您还可以通过分析比较各个CDR3序列来判断是否特异的的克隆。 如果您有其他任何疑难的问题,请咨询我们。

Bacterial flora analysis FAQ

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.