Real skills tested on publication datasets, taking you from raw FASTQ reads to a biological narrative.
Navigate NCBI GEO and SRA to download raw reads with fasterq-dump. Execute FastQC, assess Phred scores, inspect GC bias, and compile MultiQC dashboards.
Perform poly-G and adapter trimming via fastp, execute unspliced Bowtie2 alignment, apply SAMtools MAPQ ≥ 30 filters, mark PCR duplicates, and filter ENCODE blacklists.
Model local Poisson background from input controls with MACS3 (narrow & broad peaks). Build consensus binding matrices and identify statistically differential peaks in R via DiffBind + DESeq2.
Annotate peak genomic distributions with ChIPseeker, discover enriched GO Biological Processes and KEGG pathways via clusterProfiler, and identify TF binding motifs with HOMER.
Compute genome-wide normalized BigWig coverage tracks, plot clustered TSS signal heatmaps and composite profiles using deepTools, and visually inspect differential binding at Huntington's Disease target loci (BDNF, DARPP-32) in IGV.
Structured into 4 progressive phases spanning 10 hands-on modules:
Biophysical principles of chromatin immunoprecipitation, antibody validation, cross-linking dynamics, active vs repressive histone marks (H3K4me3, H3K27ac, H3K27me3), input/mock controls, and designing a rigorous case study in Huntington's Disease striatal neurodegeneration.
Fetching raw ChIP-seq sequencing data from NCBI GEO and SRA using fasterq-dump, evaluating base quality scores with FastQC, trimming adapters and poly-G tails with fastp, and compiling interactive MultiQC dashboards to identify low-complexity or degraded libraries.
Configuring Bowtie2 for un-spliced epigenomic alignment against GRCh38, understanding end-to-end vs local alignment modes, multi-threading, SAM to BAM conversion, sorting, indexing, and filtering low-quality or multi-mapping reads with SAMtools (MAPQ >= 30).
Differentiating optical from PCR duplicates with Sambamba / Picard, masking high-signal anomalous regions using ENCODE v2/v3 Unified Blacklists with bedtools, and computing Normalized Strand Cross-correlation (NSC) and Relative Strand Cross-correlation (RSC) with phantompeakqualtools.
Deep dive into Model-based Analysis of ChIP-Seq (MACS3). Estimating fragment size (d) via sonication shifts, calculating dynamic local Poisson lambda from matched input controls, selecting narrowPeak vs broadPeak modes, setting q-value thresholds, and calculating the Fraction of Reads in Peaks (FRiP).
Configuring DiffBind sample sheets, building a consensus peak matrix across biological replicates, affinity score normalization (TMM / RLE), contrast design (Huntington's Disease vs Healthy Controls), and executing negative binomial GLMs via DESeq2 to detect hyper- and hypo-methylated promoter regions.
Mapping differential peak coordinates to genomic features (promoter, 5' UTR, 3' UTR, exons, introns, distal intergenic) using ChIPseeker with TxDb.Hsapiens.UCSC.hg38.knownGene, plotting TSS distance distributions, and performing Gene Ontology and KEGG pathway over-representation analysis with clusterProfiler.
Uncovering transcription factor binding motifs within differential H3K4me3 peak summits. Running HOMER findMotifsGenome.pl with GC-content matched background, identifying known and de novo motifs (SP1, CREB, REST/NRSF), and mapping transcriptional repressor occupancy in Huntington's Disease.
Generating genome-wide coverage tracks in BigWig format with deepTools bamCoverage using 1x genome normalization (RPKM/BPM), calculating coverage matrices around Transcription Start Sites (TSS +/- 3kb), and generating publication-ready composite profile plots and clustered heatmaps with plotHeatmap.
Loading multi-track BigWig files, narrowPeak calls, and gene models into IGV for visual verification; exploring critical Huntington's Disease loci including BDNF, HTT, and DARPP-32/PPP1R1B; synthesizing the entire multi-day analytical journey into a compelling, publication-ready biological narrative.
Huntington's Disease ($HD$) provides one of the clearest demonstrations of chromatin pathology in human medicine. Mutant huntingtin protein contains an expanded polyglutamine tract that binds directly to transcriptional co-activators and histone acetyltransferases ($CBP/p300$), while disrupting histone methyltransferase complexes that deposit the active promoter mark H3K4me3.
Post-mortem striatum & prefrontal cortex from Huntington's patients vs. age-matched neurotypical controls (NCBI GEO GSE68952).
Verified ENCODE Quality Standards