onek1k_scd

Phase 2 analysis for OneK1K data in hg38 and new cell type annotation

Create Seurat objects

• input: /directflow/SCCGGroupShare/projects/data/experimental_data/CLEAN/OneK1K_scRNA/OneK1K_scRNA_V3
• output: results/2021-10-26_raw_seurat_objects
• script: raw_seurat_object.R

This version creates a Seurat object for each pool and calculates the percentage of mitochondrial expression. Results are saved in separate .RDS objects. Pool number is appended to each barcode to avoid duplicates when pools are combined.

Classify cells pre-QC

• input: results/2021-10-26_raw_seurat_objects
• output: results/2021-10-26_pre-qc_annotation
• script: pre-qc_annotate.qsub.sh
  • https://github.com/sc-eQTLgen-consortium/WG2-pipeline-classification/tree/c30cd57cca5c0bf17e587b8abb53ce5380f0a709

Aggregate metadata pre-QC

• input: results/2021-10-26_pre-qc_annotation
• output: results/2021-10-26_pre-qc_metadata_aggregation
• script: pre-qc_metadata_aggregation.R

Perform QC

• input: results/2021-10-26_pre-qc_metadata_aggregation
• output: results/2021-10-28_qc_filter_barcodes
• script: QC.R

Filter barcodes

• input:
  • results/2021-10-28_qc_filter_barcodes
  • results/2021-10-26_pre-qc_annotation
• output: results/2021-10-28_cleaned_barcodes
• script: clean_barcodes.R

Cell type classification

• input: results/2021-10-28_cleaned_barcodes
• output: results/2021-10-28_cell_type_annotation
• script: cell_type_annotation.sh

Combine pools

• input: results/2021-10-28_cell_type_annotation
• output: results/2021-10-30_combine_pools
• script: combine_pools.sh

Align pools

• input: results/2021-10-30_combine_pools
• output: results/2021-10-30_aligned_data
• script: align_embeddings.R

Add metadata

• input: results/2021-10-30_aligned_data
• output: results/2021-11-10_add_metadata
• script: add_sex_age.R

This step also adds ancestry information

Convert Seurat to Scanpy

• input: results/2021-11-10_add_metadata
• output: results/2021-11-10_h5ad
• script: convert_seurat2scanpy.R

Stores raw counts, azimuth reductions, and harmony embeddings

Apply SCTransform v1

• input: results/2021-11-10_add_metadata
• output: results/2021-11-30_SCT_v1
• script: sct_v1.R

Apply SCTransform v2

• input: results/2021-11-10_add_metadata
• output: results/2021-11-30_SCT_v2
• script: sct_v1.R

Apply scale factor normalization

• input: results/2021-11-10_add_metadata
• output: results/2021-12-02_norm
• script: norm.R

Extract metadata

• input: results/2021-11-10_add_metadata
• output: results/2022-02-22_final_metadata
• script: extract_metadata.R