LT manuscript TODOs

1. Define "promoter types" by examining the frequency and distribution of the 'GRGGC' motif on both + and - strands within the promoter region, and categorize promoters based on the identified "promoter types."

2. Investigate the reasons for observing only a small proportion of peaks in the promoter regions in my analysis (e.g., 203 out of 1938 peaks in NHDF samples).

3. Evaluate the consistency of peaks between replicates for both NHDF and K331A samples.

4. Identify the overlap between peaks and integration sites.

5. Perform enhancer analysis using the additional data for H3K4me1 and H3K27ac marks, obtained from public repositories. /home/jhuang/DATA/Data_Denise_sT_immune_evasion_PUBLISHING/Data_Denise_ChIPSeq_Protocol2/Data_H3K27ac/Raw_Data

   #H3K4me3_H3K27ac__H3K27me3_H3K9me3
   V_8_1_6_p601_d8_D1_H3K4me3.fastq.gz,V_8_1_6_p601_d8_D1_input.fastq.gz,p601_H3K4me3_D1
   V_8_1_5_p601_d8_D2_H3K4me3.fastq.gz,V_8_1_5_p601_d8_D2_input.fastq.gz,p601_H3K4me3_D2
   V_8_1_6_p604_d8_D1_H3K4me3.fastq.gz,V_8_1_6_p604_d8_D1_input.fastq.gz,p604_H3K4me3_D1
   V_8_1_5_p604_d8_D2_H3K4me3.fastq.gz,V_8_1_5_p604_d8_D2_input.fastq.gz,p604_H3K4me3_D2
   V_8_1_6_p601_d8_D1_H3K27me3.fastq.gz,V_8_1_6_p601_d8_D1_input.fastq.gz,p601_H3K27me3_D1
   V_8_1_5_p601_d8_D2_H3K27me3.fastq.gz,V_8_1_5_p601_d8_D2_input.fastq.gz,p601_H3K27me3_D2
   V_8_1_6_p604_d8_D1_H3K27me3.fastq.gz,V_8_1_6_p604_d8_D1_input.fastq.gz,p604_H3K27me3_D1
   V_8_1_5_p604_d8_D2_H3K27me3.fastq.gz,V_8_1_5_p604_d8_D2_input.fastq.gz,p604_H3K27me3_D2
   V_8_1_7_p601_d8_D1_H3K9me3.fastq.gz,V_8_1_6_p601_d8_D1_input.fastq.gz,p601_H3K9me3_D1
   V_8_1_7_p601_d8_D2_H3K9me3.fastq.gz,V_8_1_5_p601_d8_D2_input.fastq.gz,p601_H3K9me3_D2
   V_8_1_7_p604_d8_D1_H3K9me3.fastq.gz,V_8_1_6_p604_d8_D1_input.fastq.gz,p604_H3K9me3_D1
   V_8_1_7_p604_d8_D2_H3K9me3.fastq.gz,V_8_1_5_p604_d8_D2_input.fastq.gz,p604_H3K9me3_D2
   V_8_1_8_p601_d8_D1_H3K27ac.fastq.gz,V_8_1_6_p601_d8_D1_input.fastq.gz,p601_H3K27ac_D1
   V_8_1_8_p601_d8_D2_H3K27ac.fastq.gz,V_8_1_5_p601_d8_D2_input.fastq.gz,p601_H3K27ac_D2
   V_8_1_8_p604_d8_D1_H3K27ac.fastq.gz,V_8_1_6_p604_d8_D1_input.fastq.gz,p604_H3K27ac_D1
   V_8_1_8_p604_d8_D2_H3K27ac.fastq.gz,V_8_1_5_p604_d8_D2_input.fastq.gz,p604_H3K27ac_D2
   
   #H3K27ac_H3K4me1_public
   #https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSM733662
   #https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSM1003526
   NHDF_H3K4me1_r1.fastq.gz
   NHDF_H3K4me1_r2.fastq.gz
   NHDF_H3K27ac_r1.fastq.gz
   NHDF_H3K27ac_r2.fastq.gz
   NHDF_Control_r1.fastq.gz
   NHDF_Control_r2.fastq.gz
   

6. In the PCA plot, adjust the x-axis, y-axis and z-axis to their actual scales, and exclude d3 samples.

7. Identify the significant genes by conducting analyses focused on the specific sequencing data of interest (for example, only input the LT_d8 and control_d8 data for the LT_vs_control analysis), and then compare the lists of significant genes derived from these targeted analyses with those from the comprehensive analysis.

   #Error during "macs2 callpeak"
   (rnaseq) jhuang@hamm:~/DATA/Data_Denise_LT_DNA_Binding/ChIPseq_histone_hg38/H3K27ac_H3K4me1_public$ nextflow run NGI-ChIPseq/main.nf --reads '/mnt/h1/jhuang/DATA/Data_Denise_LT_DNA_Binding/ChIPseq_histone_hg38/H3K27ac_H3K4me1_public/Raw_Data/*.fastq.gz' --genome hg38 --macsconfig macs.config --saveReference --saveAlignedIntermediates --singleEnd --blacklist_filtering -profile standard --project NHDF_enhancer_analysis_hg38 -resume
   

   new

   | Cell line | T antigens | Nr of peaks | | ----------- | ----------- | ----------- | | 293 | Early region | 2219 | | 293 | LTtr | ? | | PFSK1 | Early region | 7795 | | PFSK1 | LTtr | ? | | nHDF | LT | 1938 | | nHDF | LTtr | 381 | | WaGa | sT+LTtr (endogenous) | ? |

   old

   | Cell line | T antigens | Nr of peaks | | ----------- | ----------- | ----------- | | 293 | Early region | 2219 | | 293 | LTtr | ? | | PFSK1 | Early region | 7795 | | nHDF | LT | 1938 | | nHDF | LTtr | ? | | nHDF | LTtr | 381 | | WaGa | sT+LTtr (endogenous) | ? |

   

   

8. A brief overview of each cell line and their relevance to MCPyV:

   • HEK293:

     • Origin: Human embryonic kidney cells, but with characteristics of immature neuronal cells.
     • MCPyV: These cells are often used in virology research due to their high transfection efficiency. They are not naturally permissive to MCPyV infection, but they can be used to study the virus by introducing viral genes through transfection.

   • PFSK-1:

     • Origin: Derived from a primary cerebral tumor of a 22-month-old child, these cells have neuronal properties.
     • MCPyV: Not typically associated with MCPyV research, as these cells are of neural origin. MCC typically does not arise from neural tissue.

   • nHDF (Normal Human Dermal Fibroblasts):

     • Origin: These are primary cells derived from normal human dermal skin.
     • MCPyV: While not derived from Merkel cells, fibroblasts like nHDF can be used in studies to understand the impact of MCPyV on skin cells or to provide a non-cancerous control in MCPyV studies.

   • WaGa:

     • Origin: A cell line derived from human Merkel cell carcinoma.
     • MCPyV: WaGa cells are MCPyV-positive, meaning they contain the virus, and are used in research to understand the biology of MCPyV-associated MCC.

   • MKL-1:

     • Origin: Also a cell line derived from human Merkel cell carcinoma.
     • MCPyV: MKL-1 cells are MCPyV-positive and have been used extensively in studies to understand MCPyV’s role in Merkel cell carcinoma pathogenesis.

   Each of these cell lines offers a different context for studying the biology of MCPyV, its infection mechanism, the cellular response to the virus, and the development of MCC. The HEK293 line is often used for molecular cloning and gene expression studies due to its ease of transfection and high growth rate. In contrast, WaGa and MKL-1 cells, being derived from MCC, provide a more disease-relevant system to study the virus' oncogenic mechanisms.

   In virology and cancer research, choosing the appropriate cell line is crucial, as it can significantly affect the relevance and applicability of the findings. The HEK293 cells would provide a basic understanding of the virus-host interactions at the molecular level, whereas WaGa and MKL-1 cells would be used to study the virus in the context of its natural disease state. PFSK-1 and nHDF might serve as non-MCC comparators or be used in MCPyV research for more specialized purposes.

9. Sample code explanation

Markdown | Less | Pretty --- | --- | --- Still | renders | nicely 1 | 2 | 3

| Item | Price | # In stock | |--------------|-----------|------------| | Juicy Apples | 1.99 | 7 | | Bananas | 1.89 | 5234 |

    | Sample Code | Sample Name |
    | ----------- | ----------- |
    | untreated_d0_DonorI | untreated_Day0 |
    | untreated_d0_DonorII | untreated_Day0 |
    | p601_d3_DonorII | mCherry-control_Day3 | 
    | p604_d3_DonorII | sT_Day3 |
    | p601_d8_DonorII | mCherry-control_Day8 |
    | p604_d8_DonorII | sT_Day8 |
    | p601_d3_DonorI | mCherry-control_Day3 |
    | p604_d3_DonorI | sT_Day3 |
    | p601_d8_DonorI | mCherry-control_Day8 |
    | p604_d8_DonorI | sT_Day8 |
    | p600_d3_DonorII | GFP-control_Day3 |
    | p605_d3_DonorII | LTtr_Day3 |
    | p600_d8_DonorII | GFP-control_Day8 |
    | p605_d8_DonorII | LTtr_Day8 |
    | p600_d3_DonorI | GFP-control_Day3 |
    | p605_d3_DonorI | LTtr_Day3 |
    | p600_d8_DonorI | GFP-control_Day8 |
    | p605_d8_DonorI | LTtr_Day8 |
    | p602_d8_DonorII | LT_Day8 |
    | p602_d8_DonorI | LT_Day8 |
    | p600and601_d12_DonorI | GFP+mCherry-control_Day12 |
    | p604and605_d12_DonorI | sT+LTtr_Day12 |
    | p600and601_d9_DonorII | GFP+mCherry-control_Day9 |
    | p604and605_d9_DonorII | sT+LTtr_Day9 |
    | p602_d3_DonorI | LT_Day3 |
    | p602_d3_DonorII | LT_Day3 |
    | p602and604_d3_DonorI | sT+LT_Day3 |
    | p602and604_d3_DonorII | sT+LT_Day3 |

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