Archer® Immunoverse™ FAQs

How deeply should I sequence my Immunoverse™ TCR or BCR libraries?

How deeply you sequence your Immunoverse libraries depends on your application. Deeper sequencing will allow for the identification of more clonotypes; however, there is a plateau where the user will not see many additional clonotypes despite deeper sequencing. This plateau trends with library diversity, which is driven by input amount, input quality, immune cell content and other biological factors.

For dominant clone identification, the user may only need to sequence to 100,000 reads, while diversity or low abundance applications may require more input and deeper sequencing. A recommended starting point is 1.5 M reads with 400 ng of total RNA to capture most of the sample diversity in the sequencing data.

Panel Application RNA input recommendations* Sequencing read depth
TCR B/G Clonality or dominant clones 25-400 ng 250,000
TCR A/D Clonality or dominant clones 25-400 ng 250,000
TCR All Chains Clonality or dominant clones 25-400 ng 500,000
TCR B/G Thorough characterization of repertoire or rare clonotype identification 400-2000 ng 1.5 M
TCR A/D Thorough characterization of repertoire or rare clonotype identification 400-2000 ng 1.5 M
TCR All Chains Thorough characterization of repertoire or rare clonotype identification 400-2000 ng 3 M
BCR IGH Clonality or dominant clones 100-400 ng 250,000
BCR IGK/L Clonality or dominant clones 100-400 ng 250,000
BCR All Chains Clonality or dominant clones 100-400 ng 500,000
BCR IGH Thorough characterization of repertoire or rare clonotype identification 400-6000 ng 1.5 M
BCR IGK/L Thorough characterization of repertoire or rare clonotype identification 400-6000 ng 1.5 M
BCR All Chains Thorough characterization of repertoire or rare clonotype identification 400-6000 ng 3 M

* Depending on sample T and B cell content. See the following resources:

The rarefaction analysis below was derived from Immunoverse TCR libraries generated from varying amounts of high-quality input from a PBL sample and illustrates the relationship between input amounts, sequencing depths and clonotype count for a diverse sample type.

 


 

How much input should I use with my Immunoverse™ TCR or BCR libraries?

Generally, more input will allow for identification of more clonotypes at any level of sequencing. The TCR assay will tolerate between 25 ng - 2 ug of high quality input. Inputs between 100 ng and up to 6 ug of high quality diverse PBL RNA have been tolerated in the BCR assay. For diversity applications, we recommend using as much available input as possible to increase the total number of clonotypes identified. For dominant clonotyping applications, less input can be used but we recommend starting with 200-400ng if possible.

Panel Application RNA input recommendations* Sequencing read depth
TCR B/G Clonality or dominant clones 25-400 ng 250,000
TCR A/D Clonality or dominant clones 25-400 ng 250,000
TCR All Chains Clonality or dominant clones 25-400 ng 500,000
TCR B/G Thorough characterization of repertoire or rare clonotype identification 400-2000 ng 1.5 M
TCR A/D Thorough characterization of repertoire or rare clonotype identification 400-2000 ng 1.5 M
TCR All Chains Thorough characterization of repertoire or rare clonotype identification 400-2000 ng 3 M
BCR IGH Clonality or dominant clones 100-400 ng 250,000
BCR IGK/L Clonality or dominant clones 100-400 ng 250,000
BCR All Chains Clonality or dominant clones 100-400 ng 500,000
BCR IGH Thorough characterization of repertoire or rare clonotype identification 400-6000 ng 1.5 M
BCR IGK/L Thorough characterization of repertoire or rare clonotype identification 400-6000 ng 1.5 M
BCR All Chains Thorough characterization of repertoire or rare clonotype identification 400-6000 ng 3 M

* Depending on sample T and B cell content. See the following resources:

The Rarefaction analysis below was derived from Immunoverse TCR libraries generated from varying amounts of high-quality input from a PBL sample and illustrates the relationship between input amounts, sequencing depths and clonotype count for a diverse sample type.

 


 

What Illumina sequencing chemistry should I use for my Immunoverse™ kits?

MiSeq v2 chemistry and NextSeq is suitable for all Immunoverse TCR kits, however, MiSeq v3 chemistry (2x300 bp reads) is recommended for TCR alpha/delta panel and is required for all BCR panels, since it allows for longer sequencing reads and thus more coverage of the CDR3 regions and subsequent identification of more clonotypes. MiSeq v3 chemistry (2x300 bp reads) is also compatible with the TCR beta/gamma panel.

 


 

What yield should I expect from my Immunoverse™ TCR and BCR libraries?

Yields vary by input type and amount, and other factors. Typical yields for TCR positive input range from 10s to 100s of nM. Users should expect to see low yields from input without lymphocytes that express TCR or BCR. For example, the MCF-7 input generally results in low (<10 nM) yields.

 


 

What negative controls should I use with my Immunoverse™ kit?

Any non-lymphocyte cell lines (eg. MCF-7) can be used as a negative control. The library yield of negative controls depends on the input amount and can exceed > 4 nM. In non-lymphocyte cell lines that do not express TCR or BCR, no or very few clonotypes are called.

 


 

What positive controls should I use with my Immunoverse™ Kit?

There are many commercially available controls containing T lymphocytes (for the TCR assays) or B lymphocytes (for BCR assays) that could be used for positive controls depending on experiment design. We recommend mixing Jurkat cell line RNA with peripheral blood leukocyte RNA to generate a positive control for the TCR assay, and the Raji cell line RNA mixed with peripheral blood leukocyte RNA to generate a positive control for the BCR assay. Both cell lines have a known clonotype that can be used in dilution experiments to assess low abundance clones.

 


 

What sample types are appropriate for Immunoverse™ kits?

The Immunoverse kits support RNA extracted from the following sample types:

  • Sorted B (for the BCR assay) or T (for the TCR assay) cells
  • Peripheral blood mononucleated cells (PBMCs)
  • Whole blood
  • Fresh frozen tissue containing either B or T cells -- depending on which assay is being used
  • Formalin fixed paraffin-embedded (FFPE) tissue containing either B or T cells -- depending on which assay is being used

 


 

What indications in the data suggest a failed library prep?

  1. Library preps that use 25 ng - 2 ug input RNA from a sample type that contains T-cells (e.g. PBLs, bone marrow, lymphoid organs), should generally produce greater than 4 nM final library, and up to hundreds of nM when using the Immunoverse TCR kits. Due to generally lower B-cell content in PBL, inputs of 100 ng - 3 μg RNA input from PBL should result in final library concentrations > 4 nM. If a library does not result in sufficient yield, the user should confirm the input contains RNA derived from T-cells or B-cells.
  2. After analyzing the sequencing results with Archer Analysis, there are several metrics that indicate a successful library prep and sequencing run. A user should expect that the % Reads Post Adapter Trimming is > 75% and that clonotypes are reported by the analysis pipeline.

 


 

Can I use my Immunoverse™ TCR and BCR kits with FFPE samples?

Yes, the Immunoverse Kit can be used with FFPE samples. We have detected TCR and BCR clones within FFPE input samples using the Immunoverse kits. FFPE samples tend to be more degraded and produce fewer read-pairs across the CDR3 region. Complexity of the library and robust clonotype identification with Archer Analysis is driven by input quality, quantity, and B/T-cell content. Users should consider these factors in their experimental design -- especially when selecting RNA extraction procedures which affect the quality of the RNA. Please refer to the “Getting Started” section in the protocol for extraction recommendations.


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