Immune Repertoire Analysis

Using RNA-Seq data as input, the Immune Repertoire Analysis tool can be used to characterize either the T or B cell receptor repertoire.

The tool requires a reference data sequence list (Image seq_list_nucleotide) containing reference sequences for the V and J segments.

Whether the tool identifies T or B cell receptors depends on the types of V and J segments present in the provided sequence list. The tool does not accept sequence lists containing reference sequences for both TCR and BCR.

Primers are often designed to target the C segment. This part of the read should be trimmed prior to the analysis. This can be done using the Trim Reads tool and a trim adapter list, see Trim adapter lists are provided as part of the reference data set or imported for trimming at the 5'. For protocols where the C segment is at the 3' end of the read, a protocol-specific trim adapter list should be used instead, see Import Immune Reference Segments.

The Reference Data Manager (see Reference Data Management) offers two QIAGEN set for this tool. Each set contains a sequences list for Immune Repertoire Analysis as well as a trim adapter list for trimming the reads prior to the analysis:

If reference data is needed for BCR or for a different species than those above, Import Immune Reference Segments can be used to import reference data, see Import Immune Reference Segments.

The tool assumes that one read spans both V and J in order to successfully recover the CDR3 region. It is therefore recommended to collapse overlapping paired-end reads using Merge Overlapping Pairs, see

Identification of clonotypes

Clonotyping a read consists of identifying which V and J segments from the reference data are used and extracting the CDR3 region found between the conserved amino acids.

V segments are rather long ($ >200$ bp), whereas J segments are relatively short ( $ \approx 50-70$ bp). The identification of V and J segments is therefore performed using two different strategies.

For V segments, the Map Reads to Reference tool is used internally. A read with multiple segments matches will provisionally have all these segments assigned and in a subsequent merging step, a read may be assigned a specific segment.

As J segments are short, a strategy similar to IMSEQ [Kuchenbecker et al., 2015] is used. First, a pairwise alignment with a 15 bp subsequence of the full segment called a Segment Core Fragment (SCF) is performed to find candidates for full pairwise alignments. If the pairwise alignment of an SCF to the read has a sufficiently small number of errors, it is nominated as a candidate. A full pairwise alignment is then made for all the segments corresponding to the candidate SCFs. If there is a sufficiently good match among the full alignments it will be assigned to the read.

Merging of clonotypes

After the initial identification of clonotypes, some clonotypes are merged to reduce false positives due to sequencing errors and resolve ambiguities, i.e. multiple assigned segments. Clonotype merging is performed in two steps.

The first step tries to resolve ambiguous segment assignments. Some of the reference V and J segments have a large degree of sequence identity, e.g. in mouse a recent duplication event has resulted in multiple paralogue TCR V segments with a sequence identity of more than 97%. If a sequencing read does not cover the regions where paralogue segments differ, the segment cannot be unambiguously identified. In these cases all possible V and/or J segments will be listed using a comma for separation of the different options. However, there might be reads with the same CDR3 nucleotide sequence where the segment can be uniquely determined. It is unlikely that two different clonotypes would share the same CDR3 and have almost identical V and J segments. We thus merge clonotypes with ambiguous segments into clonotypes with unambiguous segments provided they share CDR3 sequence and their V and J segments overlap.

The second merging step tries to correct sequencing errors in the CDR3 region, where a highly expressed clonotype would result in multiple clonotypes being reported if not corrected for. In this step, clonotypes are merged if their V and J segments are identical and the CDR3 region is sufficiently similar. For two CDR3 regions to be deemed sufficiently similar, two types of errors are considered: errors occurring in positions of low quality and errors occurring anywhere within the CDR3 region.

Running the tool

To run Immune Repertoire Analysis go to the Toolbox and select:

        Tools | QIAseq Panel Expert Tools (Image qiaseq_expert_folder_closed_16_n_p) | Immune Repertoire Expert Tools (Image immune_rept_folderclosed_16_n_p) | Immune Repertoire Analysis (Image immune_rept_tool_16_n_p)

This opens a dialog where the trimmed reads can be selected. Click Next to configure the execution (see figure 8.8):

Image ImmuneRepertoireAnalysisToolWizard
Figure 8.8: Options for configuring the execution of Immune Repertoire Analysis.

Output from the Immune Repertoire Analysis tool

Two different outputs are produced by the Immune Repertoire Analysis tool:


The report includes the following information:

Note that the V segment is identified first and the identification of the J segment is performed only for those fragments for which a V segment was found. Therefore, the number of fragments for which a J segment was identified will always be at most the same as the number of fragments for which a V segment is identified.

The remaining information in the report is given per chain type and only for those chain types for which clonotypes have been identified.

Note. All plots can be opened in table view by double-clicking on the plot and clicking on the table icon in the lower left corner.


The TCR clonotypes (Image tcr_table_16_n_p) / BCR clonotypes (Image bcr_table_16_n_p) output includes the following columns:

The clonotypes are sorted by count in decreasing order.