DL 5: Genomics, Bioinformatics, and Functional Annotation

Genomics

The study of the DNA sequence

Recombinant DNA

Technology that enables cloning and manipulation of genes; foundational for genomics.

cDNA Cloning

Uses reverse transcriptase to synthesize complementary DNA (cDNA) from mRNA templates.

Reverse Transcriptase

Enzyme that converts single-stranded RNA into double-stranded DNA (cDNA).

Poly-T Primer

Oligonucleotide primer that binds to the poly-A tail of eukaryotic mRNA for cDNA synthesis.

cDNA Library

Collection of cDNA molecules representing all mRNAs expressed in a cell or tissue.

gDNA Library

Collection of genomic DNA fragments

Coverage

The average number of times each base is sequenced in a dataset; determines sequencing depth.

Library Coverage Calculation

Calculated by multiplying the number of clones by average insert size and dividing by genome size.

Unequal cDNA Representation

mRNAs vary in abundance

Sanger Sequencing

Uses dideoxynucleotides (ddNTPs) to terminate DNA synthesis and determine base order.

Whole-Genome Shotgun (WGS) Sequencing

Randomly shears DNA

Contigs

Overlapping DNA fragments assembled into continuous stretches of sequence.

Coverage in WGS

Typically 10–12× genome length to ensure complete assembly.

Repetitive Sequences

Make de novo genome assembly difficult due to ambiguous alignments.

Illumina Sequencing

Next-generation sequencing method that sequences millions of short fragments in parallel.

Flow Cell

Surface where Illumina sequencing occurs

Read Length Comparison

Sanger: ~900 bp; Illumina: ~150 bp paired-end.

Throughput Comparison

Sanger produces hundreds of reads per run; Illumina produces billions.

Cost Comparison

Sanger >$1M per gigabase

De Novo Assembly

Building genomes from scratch without a reference sequence; challenging for repetitive genomes.

Assembly by Reference

Aligning reads to a known reference genome to identify SNPs and small indels.

Human Genome Sequencing

Achieved through both clone-by-clone and WGS approaches; completed in 2003.

Genome Coverage Today

Human genome sequencing can reach ~20× coverage for <$1

Metagenomics

Sequencing mixed DNA from environmental samples to study uncultured microbial communities.

Metagenome

The collective genome of microbial populations from a given environment.

Bioinformatics

The computational analysis and annotation of biological data such as DNA sequences.

Genome Annotation

Identifying genes

Santa Cruz Genome Browser

Online tool for viewing

Gene Prediction

Computational identification of coding regions using ab initio (in silico) or experimental methods.

Ab Initio Gene Prediction

Statistical models predict gene structure directly from DNA sequence; fast but may be inaccurate.

Functional Annotation

Experimental verification of predicted genes via RNAseq and other molecular methods.

RNAseq

Sequencing-based method to measure gene expression and identify exons and splice variants.

RNAseq Output

High read density at exons; splice junction reads define intron–exon boundaries.

Functional Gene Annotation

Integrates RNAseq

Comparative Genomics

Uses cross-species sequence comparison to identify conserved functional regions.

Evolutionary Signatures

Conserved sequences indicate functional importance (e.g.

Phylogenetic Conservation

Higher conservation across species suggests essential function.

Conservation Example

Exons conserved between humans and rhesus monkeys more than with chickens or dogs.

Comparative Gene Expression

Measures expression across tissues or timepoints to infer gene function.

Gene Expression Profiling

Uses arrays or RNAseq to quantify expression levels across samples.

Heatmap

Graphical representation of gene expression patterns; clusters genes with similar expression.

GTEx Database

Provides tissue-specific gene expression data across human tissues.

ChIP-seq

Chromatin Immunoprecipitation Sequencing; identifies protein-DNA binding sites such as enhancers and promoters.

DNAse I Hypersensitivity Sequencing

Identifies open chromatin regions accessible to regulatory proteins.

Open vs Closed Chromatin

Open regions are transcriptionally active; closed chromatin wrapped around histones is inactive.

Enhancers

Regulatory DNA elements marked by histone modification (e.g.

1000 Genomes Project

Large-scale effort to catalog common human genetic variation by re-sequencing 1

OMIM

Online Mendelian Inheritance in Man

Repeat Masker

Computational tool identifying repetitive elements such as transposable elements in the genome.

HEXA Gene

Mutations in HEXA cause Tay–Sachs disease

Impact of Genomics

Technologies like sequencing and bioinformatics have revolutionized our understanding of human biology and disease.

Functional Genomics

Combines genomics and experimental methods to determine gene function.

The Upshot

Genomic and bioinformatic tools now allow disease analysis and discovery from a laptop or smartphone.