Reconstruction of paired TCR sequences from Single cell RNA-seq (PMID: 26950746)

Paper: T cell fate and clonality inference from single-cell transcriptomes. Nature Methods. (2016)
Organism: Mus Musculus
Cell types: CD4 T-cells
Sample number: 272
Library preparation: Smart-seq

  1. 329 single cells from 5 C1(small) chips (68.5% efficiency)
  2. Mapping is performed against genome and ERCC sequence with GSNAP followed by gene count by HGSeq
  3. Combinatorial recombine files are created from ImMunoGeneTics information system
  4. Junctional diversity during V(D)J recombination is not generated, but ambiguous ‘N’ is introduced, 7 Ns for TCR-beta and 1 N for TCR-alpha
  5. 20Ns nucleotides are added at the 5′ end of the V sequences to permit alignment of reads that include the leader sequence
  6. TCR-alpha/-beta constant region are downloaded from ENSEMBL and append to the 3′ end of each combined sequence
  7. Reads from each cell are aligned to each combinational recombine independently with Bowtie2 (-no-unal -k 1-np 0-rdg 1,1-rfg 1,1)
  8. Aligned reads to appropriate recombine are used as input to the Trinity RNA-seq
  9. Contigs from Trinity are used as input to IgBLAST
  10. For determination of productive/nonproductive, full-length TCR mRNA sequences are generated, variation outside junctional region are ignored, check reading frame
  11. Expression level of TCR genes are quantified by appending full-length recombinant sequences to entire mouse transcriptome for the pseudoalignment-based Kallisto algorithm
  12. Cells with <2000 detected genes or >10% mitchondrial genome are descarded
  13. TCR sequence diversity of mouse is estimated as 5×10^21 combinations
  14. TraCeR – reconstruction of T cell receptor sequences from single-cell RNA-seq data.
  15. Align reads to all possible combinations of V and J segments
  16. Detect at least 1 productive alpha-chain with 74-96%, beta-chain with 70-93%
  17. 2 alpha-chain are detected from 42% of cells, 2 beta-chain from 22%
  18. 2 alpha-chain are detected with at least 1 productive from 19% of cells, 2 beta-chain from 6%
  19. No TCR sequences are detected from embryonic stem cells
  20. TraCeR was applied to published Th17 related paper, detecting expanded clonotypes in EAE model
  21. Cell groups that share TCR-beta sequences but have different TCR-alpha sequences are detected
  22. ICA for 14,899 informative genes
  23. Cells derived from the same progenitor can be seen in activated Th1 effector/effector+memory
Posted in Bioinfomatics, Immunology, Reference, Single cell and tagged .

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