ChIP-Sequencing

This presentation is brief introduction to ChIP sequencing from sample preparation to data analysis.

1. ChIP -Sequencing Course: Next Generation Sequencing Analysis

2. Introduction 6/3/2023 10:42 PM 2 Introduction Sample Preparation Data Analysis Considerations References Figure 1: DNA Organization. Adapted from Henrik's Lab. " ChIP seq - Chromatin Immunoprecipitation sequencing”, YouTube, 12 May 2021. A method used to identify genomic regions bound by specific proteins or protein modifications, providing insights into gene regulation and chromatin structure.

3. Sample Preparation 6/3/2023 10:42 PM 3 Chemical treatment (Formaldehyde) TF, Modified Histone, RNA pol Introduction Sample Preparation Considerations Data Analysis References Figure 2: Sample preparation for ChIP -Seq Adapted from Henrik's Lab. " ChIP seq - Chromatin Immunoprecipitation sequencing”, YouTube, 12 May 2021. .

4. 6/3/2023 10:42 PM 4 Sample Preparation 100-300 bp Cell Disruption and DNA fragmentation Introduction Sample Preparation Considerations Data Analysis References Figure 2 (contd..): Sample preparation for ChIP -Seq Adapted from Henrik's Lab. " ChIP seq - Chromatin Immunoprecipitation sequencing”, YouTube, 12 May 2021 . .

5. Target Enrichment 6/3/2023 10:42 PM 5 Immunoprecipitation Introduction Sample Preparation Considerations Dara Analysis References Figure 2 (contd..): Sample preparation for ChIP -Seq Adapted from Henrik's Lab. " ChIP seq - Chromatin Immunoprecipitation sequencing”, YouTube, 12 May 2021.

6. Sequencing 6/3/2023 10:42 PM 6 Cross-linked reversal and Library preparation Sequencing of target DNA fragment NovaSeq 6000 System Introduction Sample Preparation Data Analysis Considerations References Figure 2: Sample preparation for ChIP -Seq Adapted from Henrik's Lab. " ChIP seq - Chromatin Immunoprecipitation sequencing”, YouTube, 12 May 2021.

7. Experimental Design Considerations Antibody selection Chromatin fragmentation Cross-linking conditions Sufficient amount of starting material - 2 x 10 6  cells per immunoprecipitation. Control libraries Reducing artifacts - normalization Biological replicates ≥ 3. 6/3/2023 10:42 PM 7 Introduction Sample Preparation Data Analysis Considerations References

8. Sequencing Considerations Parameters Values Read Length 50-150 bp Sequencing Mode SE, PE Sequencing Depth 20-40 M total read depth (for TF) ≥ 40 M for Histone marks 6/3/2023 10:42 PM 8 Table1 : S equencing considerations for ChIP -Seq Introduction Sample Preparation Data Analysis Considerations References

9. Sequencing Considerations 6/3/2023 10:42 PM 9 Figure 3: No. of peaks called vs. sequencing depth Adapted from Landt et al., " ChIP -seq guidelines and practices of the ENCODE and modENCODE consortia," Genome Research, 22(9), 1813-1831, 2012. Introduction Sample Preparation Data Analysis Considerations References

10. Data Analysis 6/3/2023 10:42 PM 10 Introduction Sample Preparation Data Analysis Considerations References Figure 4: ChIP -Seq data analysis pipeline Quality control Read mapping Peak calling Data visualization Functional analysis Motif analysis Differential analysis Integration with other data types Reproducibility

11. 1. Preprocessing Quality Control (QC) Read trimming and filtering PCR duplicate removal Important Quality Matrices Per base sequence quality GC content Over represented sequences 6/3/2023 10:42 PM 11 FastQC , MultiQC Introduction Sample Preparation Data Analysis Considerations References

12. 2. Alignment 6/3/2023 10:42 PM 12 Preprocessed reads are mapped to the reference genome using tools like BWA or SAMtools Input = FASTQ Output = SAM, BAM BWA, Bowtie, STAR, NovoAlign Figure 5: Alignment results from BWA Adapted from Zymo Research, https://github.com/Zymo-Research/service-pipeline-documentation , Accessed May 6, 2023. Introduction Sample Preparation Data Analysis Considerations References

13. 3. Peak Calling 6/3/2023 10:42 PM 13 Identification of enriched loci in the genome. Output = BED Format MACS, SICER, Bayes Peak Figure 6: Peaks calling summary statistics using MACS2 Adapted from Zymo Research, https://github.com/Zymo-Research/service-pipeline-documentation , Accessed May 6, 2023. Introduction Sample Preparation Data Analysis Considerations References

14. 4. Visualization 6/3/2023 10:42 PM 14 Figure 7: Peaks visualization by DROMPAplus Adapted from Nokato et al., "Methods for ChIP -seq analysis: A practical workflow and advanced applications," Journal of Biochemistry, 159(4), 335-345, 2016, doi : 10.1093/ jb /mvv124. Introduction Sample Preparation Data Analysis Considerations References

15. 4. Visualization 6/3/2023 10:42 PM 15 Figure 8: Peaks visualization by DROMPAplus Adapted from Nokato et al., "Methods for ChIP -seq analysis: A practical workflow and advanced applications," Journal of Biochemistry, 159(4), 335-345, 2016, doi : 10.1093/ jb /mvv124. Introduction Sample Preparation Data Analysis Considerations References

16. 4. Visualization 6/3/2023 10:42 PM 16 Peaks can be viewed directly in genome browser e.g. UCSC Genome Browser ChIPseeker , IGV Figure 9: Peaks visualization by UCSC Genome Browser Adapted from Zymo Research, https://github.com/Zymo-Research/service-pipeline-documentation , Accessed May 6, 2023. Introduction Sample Preparation Data Analysis Considerations References

17. 5. Peak Annotation 6/3/2023 10:42 PM 17 ReMap , MGA, RSAT, rGADEM Figure 10: Peaks annotation by HOMER Adapted from Zymo Research, https://github.com/Zymo-Research/service-pipeline-documentation , Accessed May 6, 2023. Introduction Sample Preparation Data Analysis Considerations References

18. 5. Peak Annotation 6/3/2023 10:42 PM 18 Figure 11: Peaks annotation by HOMER Adapted from Zymo Research, https://github.com/Zymo-Research/service-pipeline-documentation , Accessed May 6, 2023. Introduction Sample Preparation Data Analysis Considerations References

19. References Nakato , R., Shirahige , K., & Takahata , S. (2021). Methods for ChIP -seq analysis: A practical workflow and advanced applications. Genes to Cells, 26(6), 371-382. doi : 10.1111/gtc.12863. Landt , S.G., Marinov , G.K., Kundaje , A. et al. (2012). ChIP -seq guidelines and practices of the ENCODE and modENCODE consortia. Genome Res. 22(9), 1813-1831. doi : 10.1101/gr.136184.111. Zymo Research. (n.d.). Service Pipeline Documentation. GitHub. https://github.com/Zymo-Research/service-pipeline-documentation 6/3/2023 10:42 PM 19 Introduction Sample Preparation Data Analysis Considerations References