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  <head>
    <title>102-02 Molecular, genetic, omics, single-cell, and computational methods</title>
    <ownerName>Integrated Medical Foundations</ownerName>
  </head>
  <body>
    <outline text="Molecular, genetic, omics, and computational methods">
      <outline text="Specimen versus representation">
        <outline text="Extraction, amplification, labelling, filtering transform">
          <outline text="Each step can lose or enrich material"/>
          <outline text="Controls must follow the whole chain"/>
        </outline>
        <outline text="Extraction: lyse, inactivate nucleases, recover">
          <outline text="Yield, purity ratio, integrity differ"/>
          <outline text="RNA changes during ischaemia and handling"/>
          <outline text="Blanks catch contamination; spike-ins track recovery"/>
          <outline text="Kit differences can resemble biology"/>
        </outline>
      </outline>
      <outline text="PCR and quantification">
        <outline text="Denaturation, primer annealing, extension"/>
        <outline text="End-point PCR poorly quantitative after saturation"/>
        <outline text="Quantitative PCR uses threshold-crossing cycle">
          <outline text="Valid only with sound efficiency and baseline"/>
        </outline>
        <outline text="Reverse transcription varies with enzyme and priming"/>
        <outline text="Reference genes must be stable in the experiment"/>
        <outline text="Digital PCR counts positive partitions">
          <outline text="Probability model estimates molecule number"/>
        </outline>
      </outline>
      <outline text="Sequencing">
        <outline text="Sequencing by synthesis on clonal templates"/>
        <outline text="Short reads accurate but ambiguous in repeats"/>
        <outline text="Long reads span structural variants and isoforms"/>
        <outline text="Coverage improves low-frequency variant detection">
          <outline text="Cannot repair systematic mapping or amplification bias"/>
        </outline>
        <outline text="Workflow from library preparation to annotation">
          <outline text="Index misassignment moves reads between samples"/>
          <outline text="Duplicate reads may reflect amplification"/>
          <outline text="Reference bias misses novel sequence"/>
          <outline text="Orthogonal confirmation when consequences are high"/>
        </outline>
      </outline>
      <outline text="Detecting variants and associations">
        <outline text="Sanger for small regions and confirmation">
          <outline text="Consensus signal misses low-frequency variants"/>
        </outline>
        <outline text="Chromosome analysis: large abnormalities, dividing cells"/>
        <outline text="Fluorescence in situ hybridisation">
          <outline text="Selected loci in cells or tissue"/>
        </outline>
        <outline text="Copy-number methods may miss balanced rearrangements"/>
        <outline text="Method follows variant type and question"/>
        <outline text="Genome-wide association tests common variants">
          <outline text="Linkage disequilibrium: marker may only tag"/>
          <outline text="Control structure, relatedness, multiple testing"/>
          <outline text="Fine mapping narrows; experiments test mechanism"/>
        </outline>
        <outline text="Polygenic scores weaken in underrepresented groups"/>
      </outline>
      <outline text="Genome editing">
        <outline text="Guide directs nuclease cutting">
          <outline text="Repair gives indels or incorporates template"/>
        </outline>
        <outline text="Base and prime editors avoid double-strand break"/>
        <outline text="Measure off-target, mosaicism, rearrangement, p53"/>
        <outline text="Rescue or independent guides confirm the target"/>
      </outline>
      <outline text="Transcripts, proteins, metabolites">
        <outline text="RNA-seq counts depend on length, depth, mapping"/>
        <outline text="Bulk change: within-cell or cell-mixture shift"/>
        <outline text="Transcript does not equal protein or activity">
          <outline text="Translation, degradation, modification intervene"/>
        </outline>
        <outline text="Proteomics digests to peptides for tandem spectra">
          <outline text="Isoforms need distinguishing peptides"/>
        </outline>
        <outline text="Metabolites shift fast with diet, time, handling">
          <outline text="Many untargeted features stay unidentified"/>
          <outline text="Identify by mass, retention, fragments, standards"/>
        </outline>
      </outline>
      <outline text="Epigenomic and single-cell views">
        <outline text="Bisulfite conversion distinguishes modified cytosine"/>
        <outline text="Chromatin immunoprecipitation needs specific antibody"/>
        <outline text="Accessibility enzymes enter open chromatin"/>
        <outline text="Single-cell barcodes sample transcriptomes sparsely">
          <outline text="Capture, dissociation, viability bias cells seen"/>
          <outline text="Ambient RNA, doublets, dropout"/>
          <outline text="Clusters are analytic and need validation"/>
        </outline>
      </outline>
      <outline text="Spatial and multi-omic integration">
        <outline text="Spatial methods preserve location">
          <outline text="Resolution, sensitivity, segmentation trade off"/>
          <outline text="Deconvolution depends on references and models"/>
          <outline text="Proximity is not proof of communication"/>
        </outline>
        <outline text="Multi-omics multiplies dimensions and missingness">
          <outline text="Correlated features let many models fit"/>
        </outline>
        <outline text="Pathway enrichment generates hypotheses">
          <outline text="Selecting and testing on same data is circular"/>
        </outline>
      </outline>
      <outline text="Batch effects and pipelines">
        <outline text="Date, lot, instrument, operator differ systematically"/>
        <outline text="Batch confounded with group is uncorrectable"/>
        <outline text="Randomise across batches with bridge controls"/>
        <outline text="Correction can remove biology or fake similarity"/>
        <outline text="Pipelines are part of the instrument">
          <outline text="Versions, references, seeds change output"/>
        </outline>
        <outline text="Prespecify QC; rebuild from raw inputs"/>
      </outline>
      <outline text="From data to causal inference">
        <outline text="Bottleneck shifts to causal interpretation"/>
        <outline text="Signatures can classify without mechanism"/>
        <outline text="Enrichment may reflect composition or stress"/>
        <outline text="Discover, validate, localise, perturb, link phenotype"/>
        <outline text="Omics does not repeal controls and replication"/>
      </outline>
    </outline>
  </body>
</opml>
