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    Checkpoint 4 PP TX

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    Checkpoint 4 PP TX

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    marwaan.nabil1
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    Checkpoint 4 Pp TX

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    6 views29 pages

    Checkpoint 4 PP TX

    Uploaded by

    marwaan.nabil1

    Copyright:

    © All Rights Reserved
    Download as PPTX, PDF, TXT or read online from Scribd
    Download as pptx, pdf, or txt
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    Preprocessing of Genotyping & scRna seq data

    Checkpoint (4) 14/12/2023


    Director: Pr. Lluis Quintana-Murci Co-supervisor: Dr. Maxime Rotival
    Human Evolutionary Genetics Unit
    Marwan Sharawy
    Sars Genes expression
    Previous Progress

    Performed on 88 libraries (176 indiviual) Performed on integrated dataset

    Align raw data to referenceAmbient RNA & Process datasets Merging & batch Automatic celltype Downstream
    & sample demultiplexing Doublets removal (QC, Scran Normalization, HVG...etc) correction datasets annotation analysis

    Starsolo Cellbender Scanpy & Scran SCVI CellID Scanpy & GseaPY..etc
    Demuxlet Doublet Detection

    Reference used for all this Pipline : Human + IAV PR8 + Sars COV2 GE

    Build our own reference using new strain and redo EVERYTHING
    Build reference

    • Download FASTA files for new reference strains.


    • Manually build GTF files as there are no strain-specific GTF files available in databases.
    Previous Progress

    Performed on 88 libraries (176 indiviual) Performed on integrated datast

    Align raw data to referenceAmbient RNA & Process datasets Merging & batch Automatic celltype Downstream
    & sample demultiplexing Doublets removal (QC, Scran Normalization, HVG...etc) correction datasets annotation analysis
    Cellbender
    Starsolo Scanpy & Scran SCVI CellID Scanpy & GseaPY..etc
    Doublet Detection
    Demuxlet Scrublet

    • After building the reference, rerun the entire pipline

    Adjustments:

    • Doublets: KNN + DD led to the leakage of some doublets.

    • In the new pipeline, utilize two different doublet detection tools to


    address missed doublets. Scrublet+DD .

    • Remove anything detected as a doublet by either DD or Scrublet.


    Previous Progress

    Performed on 88 libraries (176 indiviual) Performed on integrated datast

    Align raw data to referenceAmbient RNA & Process datasets Merging & batch Automatic celltype Downstream
    & sample demultiplexing Doublets removal (QC, Scran Normalization, HVG...etc) correction datasets annotation analysis
    Cellbender
    Starsolo Scanpy & Scran SCVI CellID Scanpy & GseaPY..etc
    Doublet Detection
    Demuxlet Scrublet

    • Cell ID: Instead of randomly subsampling the Yann dataset, for use as a
    reference for cell ID.

    • Filtering out only Europeans

    • Increase the number of generated gene signatures per cell type from 500
    to 1000.
    Further QC
    Further QC
    Further QC

    • Visualizing count of cells per cell type for cell that have less than 1500 total count read
    Post QC UMAP
    Annotation

    • Split by condition for finner annotation • Perform clustering


    Anntotaion
    Anntotaion
    Per cluster composition
    Manunal anntotaion
    • Automatic labeling : Very Good but not perfect
    • case example : T.Reg cells
    Manunal anntotaion
    • case example : Mait cells
    SARS genome
    SARS reads

    SARS

    Coverage

    Junctions

    • Most reads come from the end of the genome (3' prime end, "poly A").

    • Are we sequencing the viral genome?


    SARS reads

    • If we are sequencing the genome, why only monocytes?

    • Could it be an infection? Specifically, is phagocytosis by monocytes of the Omicron variant a possibility? Or could this be an
    effect of vaccination or prior exposure?
    SARS reads

    "Spliced reads suggest that viral genes are being processed, indicating successful invasion by the virus."
    Clustering

    ('OT + kNN + Leiden')


    • OT defines distances to compare high-dimensional data represented as probability distributions.

    OT is not scalable for 1 m cell


    To Do

    • further manual cell annotations

    • run compositional analysis (differences in cell composition)

    • GSEA between conditions per celltype

    • Understand what i have .......spend some time with the literature for innate immunity,viral response,
    diffrent immune cell functions and interactions etc..

    • Prepere for labmeeting 8th of janurary

    • compute bulk expression for eQTL


    benchmark of scvi/ pca /topo
    metry /OT
    Raw _control

    10 KNN , using raw data


    PCA

    10 KNN , 30 pca
    Mwogli / Optimal transport

    10 KNN , OT 30 latent_dim
    Mwogli / Optimal transport

    10 KNN , OT 30 latent_dim
    Topometry
    obtain properly weighted eigenbases to represent the underlying data manifold
    the eigencomponents are the 'latent space' (a.k.a. the dimensionality reduced
    spaced), similar to the latent space learned by autoencoders like scVI or the
    principal components learned by PCA.

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