AMPlify your immune repertoire sequencing

By Kiri Burrow, Ph.D. on Mon Aug 14, 2017


Advantages of AMP for IR sequencing

  • The accuracy of clone frequency quantification does not depend on uniform PCR amplification; rather, it is driven by ligation event counting.
  • Molecular barcodes (MBCs), in conjunction with Archer® Analysis error correction software, correct for sequencing and PCR-derived errors, thereby reducing the risk of over-estimating your sample’s diversity and enhancing detection of low-abundance clones.
  • Single gene-specific primer amplification prevents clone dropout due to IgHV somatic hypermutation and enables CDR3 capture from degraded samples, such as FFPE specimens.
  • Interrogation of RNA allows for high sensitivity by amplifying more template molecules and enriching for activated immune cells.
 

The immune repertoire (IR) can be analyzed for various applications, such as:

  • Monitoring the adaptive immune response to disease
  • Following therapeutic interventions
  • Vaccine development
  • Organ transplantation research
  • Detection and characterization of tumor infiltrating lymphocytes (TILs)
  • The IR is characterized by analyzing clonal diversity and quantity, which are measured by sequencing the complementarity-determining region 3 (CDR3) in the T-cell receptor (TCR) and B-cell receptor (BCR) genes. The gold standard for IR analysis is sequencing TCR- or BCR-enriched libraries by next-generation sequencing (NGS).

    Challenges with IR sequencing:

    Multiple laboratories have reported errors when conducting IR analysis using NGS. Nguyen, et al. (2011) reported an error rate of up to 6% using traditional, single-end CDR3 sequencing of TCR genes, causing inflation in the diversity of the sample. T.A. Khan and colleagues (2016) found that PCR bias resulted in antibody frequencies with only 42-62% accuracy. Additionally, they reported that sequencing and PCR errors resulted in an overestimation of clonotype diversity by up to 5000-fold. Bolotin et al. (2012) also reported significant amplification errors in addition to low-quality sequencing reads, causing artificial diversity and information loss. These inaccuracies occurring during library preparation and sequencing can be detrimental to interpreting the state of the adaptive immune response.

    PCR and sequencing errors, as well as amplification bias, often plague NGS sequencing results. However, these issues are mitigated by Archer technology. Archer’s error correction technology (described in more detail below) removes erroneous point mutations and insertions & deletions (indels) that are introduced during PCR and sequencing and returns accurate representations of diversity and clone frequencies. Amplification biases are mitigated simply and elegantly with Archer’s unidirectional gene-specific primer amplification strategy (also described below), which ligates a universal primer binding site to the input nucleic acid fragments. By only requiring one sequence gene-specific primer (GSP) binding site in the input molecule, degraded FFPE samples are more likely to be amplified with Archer’s technology, unlike with traditional amplicon-based approaches where input fragments might be lost. This unidirectional GSP-driven target enrichment strategy improves the sensitivity of CDR3 and V segment detection of TCR and BCR genes over opposing primer-based library preparation techniques.

    PCR and sequencing errors, as well as amplification bias, often plague NGS sequencing results. However, these issues are mitigated by Archer technology.

    AMP-powered amplification:

    Archer Immunoverse™ TCR and BCR kits employ targeted NGS based on Anchored Multiplex PCR (AMP™) enrichment chemistry to most accurately quantify clonotypes. Many traditional amplicon-based methods require large primer panels to capture the extensive combinatorial diversity exhibited by the IR. However, AMP enrichment chemistry uses molecular barcoded (MBC) adapters and a minimal set of unidirectional primers to amplify the CDR3 sequence of interest from highly variable and fragmented RNA inputs. The MBC adapters contain universal primer binding sites, allowing for open-ended amplification and sequencing throughout the entire V(D)J region. Since amplification is not restricted to opposing primer sets, information from smaller degraded fragments may still be captured. This results in increased coverage of CDR3 and reduced error rates. MBC adapters provide the additional benefit of enabling unique molecule identification for accurate ligation event counting and mitigation of PCR bias.

    Anchored Multiplex PCR (AMP™) enrichment chemistry is ideal for immune repertiore profiling

    Molecular barcodes (MBCs) enable error correction:

    A molecular barcode is a unique 8-bp sequence that is embedded within the Illumina® P5 adapter and then ligated to the cDNA or enzymatically fragmented genomic DNA prior to amplification. Because each input molecule is assigned a unique tag, PCR duplicates can be easily identified and error-corrected during analysis. This method minimizes artificial diversity and allows for accurate clonotype sequencing regardless of its arrangement. As such, MBC adapters used in conjunction with Archer Analysis error correction algorithms result in an error rate that is lower than the lowest published error rate reported by Bolotin, et al. (2012).

    Molecular barcodes enable single-molecule counting, de-duplication and error correction A molecular barcode is a unique 8-bp sequence that is embedded within the Illumina® P5 adapter and then ligated to the cDNA or enzymatically fragmented genomic DNA prior to amplification.

    RNA interrogation for characterization of the expressed IR:

    Archer Immunoverse™ TCR and BCR kits use RNA as the input material, which has multiple advantages over DNA input when conducting NGS-based IR analysis. As RNA is more abundant than DNA, less input is required to detect the expressed immune receptor genes. Furthermore, activated immune cells typically show higher receptor expression, meaning that repertoire measurements from mRNA will reflect the dynamics of the antigen response. This results in a more robust representation of the functional status of the IR by amplifying more template molecules and enriching for activated clonotypes. Finally, because of the intron separating the CDR3 from the C-region in IGH, DNA-based chemistries generally fail to link an IGH isotype to a given CDR3.

    Summary:

    AMP enrichment chemistry and Archer Analysis software reduce PCR bias and minimize PCR and sequencing errors that plague amplicon-based approaches by delivering reliable BCR and TCR sequencing. Immunoverse TCR and BCR kits enable quantitative and sensitive clonotype detection, with sequences detectable at fractions as low as one in a million. Coupled with the fully automated Archer Analysis software and user-owned data, a wide variety of research applications are available to you. Click to learn more and to read our latest poster presented at Immunology 2017.

    References


    D.A. Bolotin et al., Next generation sequencing for TCR repertoire profiling: Platform-specific features and correction algorithms. Eur. J. Immunol. 42:3073-3083 (2012).

    T.A. Khan et al., Accurate and predictive antibody repertoire profiling by molecular amplification fingerprinting. Science Advances. 2(3):e1501371. (2016).

    P. Nguyen et al., Identification of errors introduced during high throughput sequencing of the T cell receptor repertoire. BMC Genomics. 12:106 (2011).


    About Kiri Burrow, Ph.D.

    Kiri earned her Ph.D. in Behavioral Genetics and Neuroscience from the University of Colorado at Boulder. Her research involved identifying novel genetic mutations associated with addictive behaviors. Her interest in cancer genomics led her to join the ArcherDX team in May, 2017. In her free time, Kiri enjoys hiking with her dog, Frankie, and spending time with her family.

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    For Research Use Only. Not for use in diagnostic procedures. For Research Use Only. Not for use in diagnostic procedures.