First, we demonstrated the necessity of performing MID sub-clustering to eliminate erroneous sequences. Here, we describe using an MID Clustering-based IR-Seq (MIDCIRS) method to quantitatively study TCR RNA molecule copy number and clonality in T cells. This limited the application of TCR repertoire sequencing (TCR-seq) in clinical settings, such as detecting minimal residual disease in lymphoid malignancies after treatment, evaluating effectiveness of vaccination and assessing degree of infection. However, evaluating the sensitivity to detect rare T cells and the degree of clonal expansion in IR-seq has been difficult due to the lack of knowledge of T cell receptor (TCR) RNA molecule copy number and a generalized approach to estimate T cell clone size from TCR RNA molecule quantification. A universal PCR is ultimately carried out to amplify the library and introduce platform-specific adapter sequences, as well as additional sample indices.Unique molecular identifiers (MIDs) have been demonstrated to effectively improve immune repertoire sequencing (IR-seq) accuracy, especially to identify somatic hypermutations in antibody repertoire sequencing. For enrichment, ligated cDNA molecules are subjected to targeted PCR using one TCR constant-region-specific primer and one universal primer complementary to the adapter. Target enrichment and final library constructionįollowing UMI assignment, target enrichment is performed to ensure that TCR cDNA molecules are sufficiently enriched in the sequenced library. In addition, this ligated adapter also contains the first sample index. Statistically, this process provides 4^12 possible indices per adapter, and each DNA molecule in the sample receives a unique UMI sequence. Prior to target enrichment and library amplification, each original cDNA molecule is assigned a UMI by ligating an adapter containing a 12-base fully random sequence (i.e., the UMI) to the ds-cDNA. This ds-cDNA is then end-repaired and A-tailed in a single-tube protocol. Subsequently, second-strand synthesis occurs, which generates double-stranded cDNA (ds-cDNA). RNA samples are first reverse transcribed into cDNA with TCR-specific RT primers. TCR reverse transcriptase and enrichment panel primers are provided, together with library reagents. The QIAseq Immune Repertoire RNA Library Kit relies on a highly efficient, TCR-specific cDNA synthesis, TCR gene-specific primer enrichment and molecular indexing for accurate and sensitive TCR clonotype and diversity assessment (see figure " QIAseq Immune Repertoire RNA Library workflow").
For data analysis, UMIs and Raw Reads are used to ensure high precision around each clonotype sequence identified. Even when present at only 0.01%, the Jurkat RNA is readily quantifiably identified. Table 1 shows the number of raw reads and the demultiplexed unique captures (UMIs) per Jurkat TCR-alpha and TCR-beta clonotype. Sensitive to at least 0.01% RNA from Jurkat cells was spiked into RNA extracted from peripheral blood mononuclear cells (PBMCs Precision Medicine) at 10%, 1%, 0.1% and 0.01% and used to make an RNA-seq library. The data analysis included with the purchase of the QIAseq Immune Repertoire T-cell receptor panels includes an online portal that seamlessly integrates with Illumina BaseSpace and provides primary read mapping, UMI demultiplexing and reports on sequencing performance, TCR chain usage, CDR3 peptide sequence and length distributions, together with rarefaction and V/D/J usage heat maps. This figure shows the major clonotype of the Jurkat cell, as well as the diversity of the PBMC background. Comprehensive view of the T-cell immune repertoire The heatmaps allow for easy identification of enriched clonotypes across the sample.
0 kommentar(er)
