Continuous spectrum of differentiation of HSCs in Zebrafish (PMID: 26804912)

Paper: Single-Cell RNA-Sequencing Reveals a Continuous Spectrum of Differentiation in Hematopoietic Cells. Cell Reports. (2016)
Organism: Zebrafish
Cell types: HSCs (Kidney)
Sample number: 672
Library preparation: Smart-seq2
DATA: E-MTAB-3947
Analysis:

  1. cd41-eGFP zebrafish is used for tracking (CD41:marker of HSCs and the megakaryocytic equivalent in fish)
  2. map to reference genome combined with eGFP and ERCC / STARv2.3 / RSeQC / Sailfish (-l IU), use TPM for analysis
  3. Samples with <50,000 paired reads and <1,000 expressed genes are excluded (for follow-up exclude <200,000PE and <150genes)
  4. ICA is used to identify the latent factors (select 4 out of 10 components, i)progression among EGFPlow, ii)EGFPlow to high iii) progression among EGFPhigh, iv)identify 3outliner cells)
  5. use tSNE to visualize the data (perplexity parameter = 75)
  6. Ward clustering on 4 ICA factors
  7. Pseudotime(3 stages) is based on which cluster cells belong to and which factor explains the variability of the cells of cluster
  8. Another pseudotime is based on application of Bayesian Gaussian process latent variable model with a one-dimensional latent variables. BayesianGPLVM implementation is used with radial basis function kernel on the log(TPM)
  9. Apply the mixtures of hierachiacal Gaussian processes model to identified pseudotime-dependent gene groups
  10. Gene enrichment analysis: gProfiler
  11. Marker gene discovery: random forest for each cluster vs rest of cells using Gini feature importance score. ExtraTreesClassifier with n_estimator = 100,000
  12. Several cell-type markers are found, ccr9a, tcea3, e2f8, top2a etc.
  13. Expression pattern from ohnologs(gene duplication) are also evaluated and divided in to i)expressed exclusive in indivisual, ii)only one of ohnologs is expressed, iii)equally expressed. Nopatterns of ohnolog use over pseudotime are observed
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