APOC3 ODN-Seq Project BN24-9851

Project Overview

Description

• Objective: Assay ODN-Seq for 8 APOC3 guides
• Jira Ticket: https://lilly-jira.atlassian.net/browse/GTEN-249
• Purpose: Nomination of 8 guides from ODN-Seq
• Benchling: https://lilly.benchling.com/lilly/f/lib_dLCNmSei-apoc3-off-target/etr_3aChcz3F-apoc3-odn-seq-top-8-sgrna-screen-hepg2/edit

Directory Structure on Lilly Lift and Shift Environment

   
├── raw data 
    └── $lrld/BN24-9851_APOC3_Top8ODN/raw/
├── samplesheet/
    └── $lrld/BN24-9851_APOC3_Top8ODN/BN24-9851_nf.csv   
├── odnseq pipeline
    └── [workflow pull https://github.com/EliLillyCo/nf-lly-odnseq]
  ├── [results in $lrld/BN24-9851_APOC3_Top8ODN/nf_results]
    └── aligned_bams
        └── $lrld/BN24-9851_APOC3_Top8ODN/nf_results/aligned_bams/sorted_bam_bai
    └── bowtie2
    └── multiqc 
        └──/$lrld/BN24-9851_APOC3_Top8ODN/nf_results/multiqc/broad_peak/multiqc_report.html
    └── preprocessing
    └── project_data 
    └── results_offtarget
       └── peaks and double stranded breaks results from Macs2 and in-house per sample
    └── stats 
    └── summarization
├── combined in-house and Macs2 peaks and double stranded breaks per sample
    └── [workflow pull https://github.com/EliLillyCo/ODNseq_2ndary_analysis]
    └── [results in $lrld/BN24-9851_APOC3_Top8ODN/2ndary_analysis/cameron_macs_combOTsumm]
├── final results/
    └── [workflow pull https://github.com/sgupta-lilly/SiteSeqSecondaryAnalysis.git branch ODNSecondaryAnalysis ]
    └── [ results in $lrld/BN24-9851_APOC3_Top8ODN/nf_results/stats/result_secondary_pipeline]
    └── published reports 
        └── $lrld/BN24-9851_APOC3_Top8ODN/nf_results/stats/SiteSeqSecondaryAnalysis
$lrld = "/lrlhps/genomics/prod/lgm/dna_editing/"

Samplesheet

datatable(
  head(metadata),
  caption = htmltools::tags$caption(
    style = 'caption-side: bottom; text-align: center;',
    'Samplesheet: ', htmltools::em('BN24-9851_nf.csv')
  )
)

Odn-seq workflow

Step1 : nf-lly-odnseq for primary workflow

cd /lrlhps/genomics/prod/lgm/dna_editing/BN24-9851_APOC3_Top8ODN/
!git clone https://github.com/EliLillyCo/nf-lly-odnseq
!nextflow run nf-lly-odnseq -profile omicsprd

Review qc with Multiqc

Step2: Jupyter notebook to combine results of the two peak callers

cd /lrlhps/genomics/prod/lgm/dna_editing/BN24-9851_APOC3_Top8ODN/2ndary_analysis/
!git clone https://github.com/EliLillyCo/ODNseq_2ndary_analysis
jupyter nbconvert --to notebook --execute --inplace ODNseq_OT_combo_sum.ipynb

All unfiltered off-target sites from the two peak callers

datatable(
  head(all_result),
  caption = htmltools::tags$caption(
    style = 'caption-side: bottom; text-align: center;',
    'Unfiltered OT sites: ', htmltools::em('Unfiltered OT sites')
  )
)

Step3: Final off-target sites with ranks and annotations

Setup

Guides in the study 1. 17rev 2. 29rev 3. 41rev 4. 52rev 5. 53rev 6. 67forw 7. 68forw 8. 6rev

$lrld = "/lrlhps/genomics/prod/lgm/dna_editing/"
mkdir $lrld/BN24-9851_APOC3_Top8ODN/BN24-9851_APOC3_Top8ODN/nf_results/stats/result_secondary_pipeline
cd $lrld/BN24-9851_APOC3_Top8ODN/BN24-9851_APOC3_Top8ODN/nf_results/stats/result_secondary_pipeline
!git clone https://github.com/sgupta-lilly/SiteSeqSecondaryAnalysis/tree/ODNSecondaryAnalysis
!wget https://ftp.ebi.ac.uk/pub/databases/gencode/Gencode_human/release_46/gencode.v46.annotation.gtf.gz
gunzip gencode.v46.annotation.gtf.gz

Step3a: Run secondary_analysis.py

secondary_workflow {
samplesheet = "$lrld/BN24-9851_APOC3_Top8ODN/BN24-9851_nf.csv" 
aligned_bams = "$lrld/BN24-9851_APOC3_Top8ODN/nf_results/aligned_bams/sorted_bam_bai/" 
combined_results = "$lrld/BN24-9851_APOC3_Top8ODN/2ndary_analysis/cameron_macs_combOTsum/"

!python3 $lrld/BN24-9851_APOC3_Top8ODN/nf_results/stats/SiteSeqSecondaryAnalysis/secondary_analysis.py samplesheet aligned_bams combined_results

Step3b: Run SiteSeqGuideReport.rmd for each guide

input = $guide
output = $guide+'.html'
!Rscript -e "rmarkdown::render('/lrlhps/genomics/prod/lgm/dna_editing/BN24-9851_APOC3_Top8ODN/nf_results/stats/SiteSeqSecondaryAnalysis/SiteSeqGuideReport.rmd',  params = list(guidename =input, results_folder='/lrlhps/genomics/prod/lgm/dna_editing/BN24-9851_APOC3_Top8ODN/nf_results/', samplesheet='/lrlhps/genomics/prod/lgm/dna_editing/BN24-9851_APOC3_Top8ODN/BN24-9851_nf.csv', result_secondary_pipeline='/lrlhps/genomics/prod/lgm/dna_editing/BN24-9851_APOC3_Top8ODN/nf_results/stats/result_secondary_pipeline/'), output_file = output))"

Step3c: Run annotation.py for each guide

input = $guide +'.OT_rank_out' 
!python3 annotation.py 17rev.OT_rank_out cancer_gene_census.csv

}

Review the results

guide 17rev

guide 29rev

guide 41rev

guide 52rev

guide 53rev

guide 67forw

guide 68forw

guide 6rev

datatable(
  head(dat),
  caption = htmltools::tags$caption(
    style = 'caption-side: bottom; text-align: center;',
    'Summary of Sites: ', htmltools::em('https://lilly-jira.atlassian.net/browse/GTEN-249')
  )
)