Parse-seq Pipeline

Input Data

  • TrailMaker Outputs:
    • anndata.h5ad : Cell by Gene Matrix ( cell_id=”bc_well”, gene_id=pref+”gene_name”+suffix)
    • cluster_assignment.csv : Cell + Cluster Table
    • meta file : Cell, Sample w/o Group (uh..)
  • CNF references for back calculation: 
    data/All_Cells_NMF_Average_Gene_Spectra.txt
data/Malignant_Cell_NMF_Average_Gene_Spectra.txt
data/Myeloid_NMF_Average_Gene_Spectra.txt
data/T_Cell_NMF_Average_Gene_Spectra.txt
  • Marker set :
    • @Ty : Cluster-base DGE
    • @Edward : Using Reference Annotation Markers

Pipeline

parse-prepro <anndata.h5ad> <meta file> <group_sample file> => <anndata1.h5ad>
parse-backcal <anndata.h5ad> <spectra> ... =>  [<_spectra.csv> ... ]
parse-integrate [<_spectra.csv> ...] => <final_spectra.csv>|<final_spectra.xsls>
parse-scanpy <anndata.h5ad> # scanpy processes ( qc, filter, embedding, umap, visualization )

UMAPs

TrailMaker UMAP TrailMaker by Sample
image image
Cell-Type @Ty per Cluster
umap_cell-type
Cell-Type @Edward per Marker
image

Testing Hypothesis

Input

  • Count frequency per Sample (tm) in Group(uh)
  • Effects of Conditions to test

Model

  • Fit a mixed-effects model for each cell type
  • Random intercept for group accounts for subject variability
  • Perform pairwise comparisons for each cell type (t-statistics on Frequency)

Results : Celltype Frequency Altered (pvalue < 0.05)

Screen Shot 2025-01-16 at 8 37 39 PM test_condition_celltype

                        contrast   estimate         SE      p.value                           ftype
1        priimary_manual - vt104  0.2528811 0.07964959 4.833048e-02                          Cancer
2                  dmso - fhd286  0.4008129 0.08471378 9.695398e-05                         Myeloid
3           fhd286 - ihk44_100nM -0.5085499 0.10896785 1.323738e-04                         Myeloid
4             fhd286 - ihk44_5nM -0.4850313 0.10896785 3.639328e-04                         Myeloid
5       fhd286 - priimary_manual -0.3585190 0.08596997 1.257893e-03                         Myeloid
6      fhd286 - priimary_octomac -0.3827086 0.08596997 3.630806e-04                         Myeloid
7           fhd286 - priimary_s2 -0.4387606 0.08596997 1.472604e-05                         Myeloid
8                 fhd286 - vt104 -0.5296484 0.08471378 1.817826e-08                         Myeloid
9                  dmso - fhd286 -0.3431723 0.06735977 1.544115e-05 Myeloid_Immune_treated_with_FHD
10                fhd286 - ihk44  0.3432714 0.08250026 1.309383e-03 Myeloid_Immune_treated_with_FHD
11          fhd286 - ihk44_100nM  0.3433299 0.08250026 1.305444e-03 Myeloid_Immune_treated_with_FHD
12           fhd286 - ihk44_20nM  0.3433299 0.08250026 1.305444e-03 Myeloid_Immune_treated_with_FHD
13            fhd286 - ihk44_5nM  0.3433299 0.08250026 1.305444e-03 Myeloid_Immune_treated_with_FHD
14      fhd286 - priimary_manual  0.3433043 0.05325524 5.148475e-09 Myeloid_Immune_treated_with_FHD
15     fhd286 - priimary_octomac  0.3431691 0.05325524 5.235334e-09 Myeloid_Immune_treated_with_FHD
16          fhd286 - priimary_s2  0.3433043 0.05325524 5.148475e-09 Myeloid_Immune_treated_with_FHD
17                fhd286 - vt104  0.3433007 0.06735977 1.528751e-05 Myeloid_Immune_treated_with_FHD
18      ihk44_20nM - priimary_s2 -0.0682645 0.02144191 4.705988e-02                           Oligo
19             dmso - ihk44_20nM -0.2783620 0.04746508 2.018153e-07                        Pericyte
20       dmso - priimary_octomac  0.1219240 0.03329123 9.430297e-03                        Pericyte
21           fhd286 - ihk44_20nM -0.3710949 0.04746508 2.978728e-13                        Pericyte
22            ihk44 - ihk44_20nM -0.3560539 0.05772919 3.112213e-08                        Pericyte
23      ihk44_100nM - ihk44_20nM -0.3081091 0.05328732 3.305075e-07                        Pericyte
24        ihk44_20nM - ihk44_5nM  0.2809911 0.05328732 5.956525e-06                        Pericyte
25  ihk44_20nM - priimary_manual  0.3806557 0.04257911 6.872281e-14                        Pericyte
26 ihk44_20nM - priimary_octomac  0.4002860 0.04257911 9.403589e-14                        Pericyte
27      ihk44_20nM - priimary_s2  0.3738004 0.04257911 7.671641e-14                        Pericyte
28            ihk44_20nM - vt104  0.3203449 0.04746508 6.692835e-10                        Pericyte

Program Frequency Altered (p-value < 0.001)

Screen Shot 2025-01-16 at 8 42 14 PM

test_condition_program

                         contrast    estimate         SE      p.value        ftype
1              dmso - ihk44_100nM -0.63396927 0.10984349 3.516713e-07    Microglia
2            fhd286 - ihk44_100nM -0.50758919 0.10984349 1.648621e-04    Microglia
3   ihk44_100nM - priimary_manual  0.44564786 0.10002695 3.571141e-04    Microglia
4  ihk44_100nM - priimary_octomac  0.49253049 0.10002695 3.722986e-05    Microglia
5       ihk44_100nM - priimary_s2  0.45430884 0.10002695 2.393522e-04    Microglia
6             ihk44_100nM - vt104  0.62106581 0.10984349 7.005935e-07    Microglia
7  ihk44_100nM - priimary_octomac -0.18541568 0.04356689 8.699461e-04   Macrophage
8       ihk44_100nM - priimary_s2 -0.18987889 0.04356689 5.535857e-04   Macrophage
9         dmso - priimary_octomac -0.07755659 0.01798885 6.820141e-04          cDC
10             dmso - priimary_s2 -0.07855443 0.01798885 5.329064e-04          cDC
11            priimary_s2 - vt104  0.07645102 0.01798885 8.928575e-04          cDC
12                  dmso - fhd286  0.30934498 0.06996251 4.163705e-04 SystematicI.
13             dmso - ihk44_100nM  0.56732735 0.09011186 1.373849e-08 SystematicI.
14         dmso - priimary_manual  0.58414517 0.07165515 1.360023e-13 SystematicI.
15        dmso - priimary_octomac  0.57494486 0.07165515 1.778577e-13 SystematicI.
16             dmso - priimary_s2  0.56579688 0.07165515 2.726708e-13 SystematicI.
17                 fhd286 - vt104 -0.36215579 0.06996251 1.001944e-05 SystematicI.
18            ihk44_100nM - vt104 -0.62013817 0.09011186 2.657734e-10 SystematicI.
19   ihk44_20nM - priimary_manual  0.35440933 0.08287418 7.951362e-04 SystematicI.
20        priimary_manual - vt104 -0.63695599 0.07165515 7.205347e-14 SystematicI.
21       priimary_octomac - vt104 -0.62775568 0.07165515 7.982504e-14 SystematicI.
22            priimary_s2 - vt104 -0.61860769 0.07165515 8.637535e-14 SystematicI.
23                  dmso - fhd286 -0.07525473 0.01736212 6.158151e-04          IFN
24            fhd286 - ihk44_20nM  0.09258370 0.02184240 9.373499e-04          IFN
25             fhd286 - ihk44_5nM  0.09228030 0.02184240 9.954395e-04          IFN
26       fhd286 - priimary_manual  0.08115221 0.01522594 4.371028e-06          IFN
27      fhd286 - priimary_octomac  0.08278375 0.01522594 2.414737e-06          IFN
28           fhd286 - priimary_s2  0.08896314 0.01522594 2.299101e-07          IFN
29        ihk44_5nM - priimary_s2  0.11566498 0.02681330 6.749821e-04         G1_S

Summary

  • Implemented Pipeline from the TrailMaker Input Matrix and clusters
  • Test Hypotheses using the annotated CellTypes and Programs