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• Dahal-Koirala, Shiva; Balaban, Gabriel; Neumann, Ralf Stefan; Scheffer, Lonneke; Lundin, Knut & Greiff, Victor [Show all 9 contributors for this article] (2022). TCRpower: quantifying the detection power of T-cell receptor sequencing with a novel computational pipeline calibrated by spike-in sequences. Briefings in Bioinformatics. ISSN 1467-5463. 23(2), p. 1–14. doi: 10.1093/bib/bbab566. Full text in Research Archive Show summary

  T-cell receptor (TCR) sequencing has enabled the development of innovative diagnostic tests for cancers, autoimmune diseases and other applications. However, the rarity of many T-cell clonotypes presents a detection challenge, which may lead to misdiagnosis if diagnostically relevant TCRs remain undetected. To address this issue, we developed TCRpower, a novel computational pipeline for quantifying the statistical detection power of TCR sequencing methods. TCRpower calculates the probability of detecting a TCR sequence as a function of several key parameters: in-vivo TCR frequency, T-cell sample count, read sequencing depth and read cutoff. To calibrate TCRpower, we selected unique TCRs of 45 T-cell clones (TCCs) as spike-in TCRs. We sequenced the spike-in TCRs from TCCs, together with TCRs from peripheral blood, using a 5′ RACE protocol. The 45 spike-in TCRs covered a wide range of sample frequencies, ranging from 5 per 100 to 1 per 1 million. The resulting spike-in TCR read counts and ground truth frequencies allowed us to calibrate TCRpower. In our TCR sequencing data, we observed a consistent linear relationship between sample and sequencing read frequencies. We were also able to reliably detect spike-in TCRs with frequencies as low as one per million. By implementing an optimized read cutoff, we eliminated most of the falsely detected sequences in our data (TCR α-chain 99.0% and TCR β-chain 92.4%), thereby improving diagnostic specificity. TCRpower is publicly available and can be used to optimize future TCR sequencing experiments, and thereby enable reliable detection of disease-relevant TCRs for diagnostic applications.

• Risnes, Louise Fremgaard; Eggesbø, Linn Margrethe; Zühlke, Stephanie; Dahal-Koirala, Shiva; Neumann, Ralf Stefan & Lundin, Knut Erik Aslaksen [Show all 8 contributors for this article] (2021). Circulating CD103+ γδ and CD8+ T cells are clonally shared with tissue-resident intraepithelial lymphocytes in celiac disease. Mucosal Immunology. ISSN 1933-0219. 14(4), p. 842–851. doi: 10.1038/s41385-021-00385-8. Full text in Research Archive
• Yao, Ying; Zia, Asima; Neumann, Ralf Stefan; Pavlović, Milena; Balaban, Gabriel & Lundin, Knut Erik Aslaksen [Show all 8 contributors for this article] (2021). T cell receptor repertoire as a potential diagnostic marker for celiac disease. Clinical Immunology. ISSN 1521-6616. 222:108621, p. 1–7. doi: 10.1016/j.clim.2020.108621. Full text in Research Archive
• Dahal-Koirala, Shiva; Risnes, Louise Fremgaard; Neumann, Ralf Stefan; Christophersen, Asbjørn; Lundin, Knut E. A. & Sandve, Geir Kjetil [Show all 8 contributors for this article] (2021). Comprehensive Analysis of CDR3 Sequences in Gluten-Specific T-Cell Receptors Reveals a Dominant R-Motif and Several New Minor Motifs. Frontiers in Immunology. ISSN 1664-3224. 12:639672, p. 1–13. doi: 10.3389/fimmu.2021.639672. Full text in Research Archive
• Ting, Yi Tian; Dahal-Koirala, Shiva; Kim, Hui Shi Keshia; Qiao, Shuo Wang; Neumann, Ralf Stefan & Lundin, Knut Erik Aslaksen [Show all 10 contributors for this article] (2020). A molecular basis for the T cell response in HLA-DQ2.2 mediated celiac disease. Proceedings of the National Academy of Sciences of the United States of America. ISSN 0027-8424. 117(6), p. 3063–3073. doi: 10.1073/pnas.1914308117. Full text in Research Archive
• Dahal-Koirala, Shiva; Neumann, Ralf Stefan; Jahnsen, Jørgen; Lundin, Knut Erik Aslaksen & Sollid, Ludvig Magne (2019). On the immune response to barley in celiac disease: Biased and public T-cell receptor usage to a barley unique and immunodominant gluten epitope. European Journal of Immunology. ISSN 0014-2980. p. 1–14. doi: 10.1002/eji.201948253. Full text in Research Archive
• Eggesbø, Linn Margrethe; Risnes, Louise Fremgaard; Neumann, Ralf Stefan; Lundin, Knut Erik Aslaksen; Christophersen, Asbjørn & Sollid, Ludvig Magne (2019). Single-cell TCR sequencing of gut intraepithelial γδ T cells reveals a vast and diverse repertoire in celiac disease. Mucosal Immunology. ISSN 1933-0219. p. 1–9. doi: 10.1038/s41385-019-0222-9. Full text in Research Archive
• Bartolomé Casado, Raquel; Landsverk, Ole J. B.; Chauhan, Sudhir Kumar; Richter, Lisa Maria; Phung, Danh & Greiff, Victor [Show all 18 contributors for this article] (2019). Resident memory CD8 T cells persist for years in human small intestine. Journal of Experimental Medicine (JEM). ISSN 0022-1007. 216(10), p. 2412–2426. doi: 10.1084/jem.20190414. Full text in Research Archive
• Iversen, Rasmus; Roy, Bishnudeo; Stamnæs, Jorunn; Høydahl, Lene Støkken; Hnida, Kathrin & Neumann, Ralf Stefan [Show all 9 contributors for this article] (2019). Efficient T cell–B cell collaboration guides autoantibody epitope bias and onset of celiac disease. Proceedings of the National Academy of Sciences of the United States of America. ISSN 0027-8424. 116(30), p. 15134–15139. doi: 10.1073/pnas.1901561116. Full text in Research Archive
• Dahal-Koirala, Shiva; Ciacchi, Laura; Petersen, Jan; Risnes, Louise Fremgaard; Neumann, Ralf Stefan & Christophersen, Asbjørn [Show all 11 contributors for this article] (2018). Discriminative T-cell receptor recognition of highly homologous HLA-DQ2-bound gluten epitopes . Journal of Biological Chemistry. ISSN 0021-9258. 294(3), p. 941–952. doi: 10.1074/jbc.RA118.005736. Full text in Research Archive
• Bråte, Jon; Neumann, Ralf Stefan; Fromm, Bastian; Haraldsen, Arthur Alexander Blørstad; Tarver, James E. & Suga, Hiroshi [Show all 11 contributors for this article] (2018). Unicellular origin of the animal microRNA machinery. Current Biology. ISSN 0960-9822. 28(20), p. 3288–3295.e5. doi: 10.1016/j.cub.2018.08.018. Full text in Research Archive
• Risnes, Louise Fremgaard; Christophersen, Asbjørn; Dahal-Koirala, Shiva; Neumann, Ralf Stefan; Sandve, Geir Kjetil & Sarna, Vikas Kumar [Show all 9 contributors for this article] (2018). Disease-driving CD4+ T cell clonotypes persist for decades in celiac disease. Journal of Clinical Investigation. ISSN 0021-9738. 128(6), p. 2642–2650. doi: 10.1172/JCI98819. Full text in Research Archive
• Gunnarsen, Kristin Støen; Høydahl, Lene Støkken; Neumann, Ralf Stefan; Bjerregaard-Andersen, Kaare; Nilssen, Nicolay Rustad & Sollid, Ludvig Magne [Show all 8 contributors for this article] (2018). Soluble T-cell receptor design influences functional yield in an E. coli chaperone-assisted expression system. PLOS ONE. ISSN 1932-6203. 13:e0195868(4), p. 1–15. doi: 10.1371/journal.pone.0195868. Full text in Research Archive Show summary

  There is a quest for production of soluble protein of high quality for the study of T-cell receptors (TCRs), but expression often results in low yields of functional molecules. In this study, we used an E. coli chaperone-assisted periplasmic production system and compared expression of 4 different soluble TCR formats: single-chain TCR (scTCR), two different disulfide-linked TCR (dsTCR) formats, and chimeric Fab (cFab). A stabilized version of scTCR was also included. Additionally, we evaluated the influence of host (XL1-Blue or RosettaBlueTM) and the effect of IPTG induction on expression profiles. A celiac disease patient-derived TCR with specificity for gluten was used, and we achieved detectable expression for all formats and variants. We found that expression in RosettaBlueTM without IPTG induction resulted in the highest periplasmic yields. Moreover, after large-scale expression and protein purification, only the scTCR format was obtained in high yields. Importantly, stability engineering of the scTCR was a prerequisite for obtaining reliable biophysical characterization of the TCR-pMHC interaction. The scTCR format is readily compatible with high-throughput screening approaches that may enable both development of reagents allowing for defined peptide MHC (pMHC) characterization and discovery of potential novel therapeutic leads.

• Gunnarsen, Kristin Støen; Høydahl, Lene Støkken; Risnes, Louise Fremgaard; Dahal-Koirala, Shiva; Neumann, Ralf Stefan & Bergseng, Elin [Show all 15 contributors for this article] (2017). A TCRα framework–centered codon shapes a biased T cell repertoire through direct MHC and CDR3β interactions. JCI Insight. ISSN 2379-3708. 2(17). doi: 10.1172/jci.insight.95193. Full text in Research Archive
• Roy, Bishnudeo; Neumann, Ralf Stefan; Snir, Omri; Iversen, Rasmus; Sandve, Geir Kjetil & Lundin, Knut Erik Aslaksen [Show all 7 contributors for this article] (2017). High-Throughput Single-Cell Analysis of B Cell Receptor Usage among Autoantigen-Specific Plasma Cells in Celiac Disease. Journal of Immunology. ISSN 0022-1767. 199((2)), p. 782–791. doi: 10.4049/jimmunol.1700169. Full text in Research Archive
• Bråte, Jon; Adamski, Marcin; Neumann, Ralf Stefan; Shalchian-Tabrizi, Kamran & Adamska, Maja (2015). Regulatory RNA at the root of animals: dynamic expression of developmental lincRNAs in the calcisponge Sycon ciliatum. Proceedings of the Royal Society of London. Biological Sciences. ISSN 0962-8452. 282(1821), p. 1–9. doi: 10.1098/rspb.2015.1746. Full text in Research Archive Show summary

  Long non-coding RNAs (lncRNAs) play important regulatory roles during animal development, and it has been hypothesized that an RNA-based gene regulation was important for the evolution of developmental complexity in animals. However, most studies of lncRNA gene regulation have been performed using model animal species, and very little is known about this type of gene regulation in non-bilaterians. We have therefore analysed RNA-Seq data derived from a comprehensive set of embryogenesis stages in the calcareous sponge Sycon ciliatum and identified hundreds of developmentally expressed intergenic lncRNAs (lincRNAs) in this species. In situ hybridization of selected lincRNAs revealed dynamic spatial and temporal expression during embryonic development. More than 600 lincRNAs constitute integral parts of differentially expressed gene modules, which also contain known developmental regulatory genes, e.g. transcription factors and signalling molecules. This study provides insights into the non-coding gene repertoire of one of the earliest evolved animal lineages, and suggests that RNA-based gene regulation was likely present in the last common ancestor of animals.

• Kumar, Surendra; Krabberød, Anders Kristian; Neumann, Ralf Stefan; Michalickova, Katerina; Zhao, Sen & Zhang, Xiaoli [Show all 7 contributors for this article] (2015). BIR pipeline for preparation of phylogenomic data. Evolutionary Bioinformatics. ISSN 1176-9343. 11, p. 79–83. doi: 10.4137/EBO.S10189.
• Neumann, Ralf Stefan; Kumar, Surendra; Haverkamp, Thomas Hendricus Augustus & Shalchian-Tabrizi, Kamran (2014). BLASTGrabber: A bioinformatic tool for visualization, analysis and sequence selection of massive BLAST data. BMC Bioinformatics. ISSN 1471-2105. 15(128). doi: 10.1186/1471-2105-15-128. Full text in Research Archive
• Neumann, Ralf Stefan; Kumar, Surendra & Shalchian-Tabrizi, Kamran (2014). BLAST output visualization in the new sequencing era. Briefings in Bioinformatics. ISSN 1467-5463. 15(4), p. 484–503. doi: 10.1093/bib/bbt009.

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• Eggesbø, Linn Margrethe; Risnes, Louise Fremgaard; Neumann, Ralf Stefan; Lundin, Knut E. A.; Christophersen, Asbjørn Otto & Sollid, Ludvig Magne (2021). Single-cell TCR repertoire analysis reveals highly polyclonal composition of human intraepithelial CD8⁺ αβ T lymphocytes in untreated celiac disease. European Journal of Immunology. ISSN 0014-2980. 51(6), p. 1542–1545. doi: 10.1002/eji.202048974.
• Haraldsen, Arthur Alexander Blørstad; Bråte, Jon; Neumann, Ralf Stefan; Fromm, Bastian; Grini, Paul Eivind & Shalchian-Tabrizi, Kamran (2016). Pre-metazoan origin of animal microRNAs.
• Bjorbækmo, Marit Markussen; Neumann, Ralf Stefan; Haraldsen, Arthur Alexander Blørstad & Shalchian-Tabrizi, Kamran (2015). Expanding our knowledge about parasites using PATHOS-DB (The PArasiTe–HOSt-DataBase).
• Bjorbækmo, Marit Markussen; Neumann, Ralf Stefan; Haraldsen, Arthur Alexander Blørstad & Shalchian-Tabrizi, Kamran (2015). HIDDEN PROTIST PARASITES: HOW CAN THEY BE DETECTED?
• Høydahl, Lene Støkken; Neumann, Ralf Stefan; Qiao, Shuo Wang; Sollid, Ludvig Magne; Sandlie, Inger & Løset, Geir Åge (2011). Development of a High Affinity TCR Reagent by Phage Display to Study Antigen Presentation in Celiac Disease. Scandinavian Journal of Immunology. ISSN 0300-9475. 73(4), p. 372–372.

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