Intro to Genomics

Genomic

study of genome in their entirety, used for system biology, utilize global and genome wide analysis of physiological roles of gene products

Genomics objective

to learn how combination of experimental and computational methods used to sequence genomes and identify functional elements

Forward genetics approach

start with phenotype and then find gene

Reverse genetics approach

mutate genes to cause phenotypic change

Bioinformatics

analysis of entire genome, number and types of gene and gene products, location/number/type of binding sites on DNA and RNA, what control production at correct time and place

Comparative genomics

consider genomes of closely and distantly related species for evolutionary insight

Functional genomics

use various methods like reverse genetics, understanding gene and protein function in biological processes

Human Genome project

took 13 years and $100M to ‘finish’ sequence (99.99% accurate)

Genomic Revolution

1 million fold increase in throughput, price decrease to under 100$ for human genome

Logic obtaining genome sequence

DNA broken up into million of overlapping segments, computationaly find overlap among small segments (Contigs), continue overlapping until segments are linked

First generation technology

Dideoxynucleotide DNA sequencing (Sanger), automated DNA sequencing

Sanger dideoxynucleotide sequencing

can sequence 800-100 bases at time, labour intensive and costly, chain terminating

mechanism of sanger sequencing

4 seperate reactions - one each base as dDNA, molecules separated by size - Gel electrophoresis (bottom 5’ to top 3’), used radioactive labels to read autoradiograms, complementary to template

Automated DNA sequencing

supplanted manual dideoxy sequencing, automated and computational - assemble at rate 10 000 to 20 000 bp per hour, dNTP labelled with fluorescent dyes (unique wavelengths), analyze 4 reaction at once, High throughput(8-96 reactions), capillary based

Whole-genome shotgun (WGS) sequencing

general strategy for obtaining and assembling sequence of genome, random DNA fragment assemble based on matching sequences

Contigs

sequential assembly of short DNA sequences, single continuous assembly of reads

Traditional WGS sequencing

construction genomic libraries using restriction enzymes, cloning into bacterial cell, partially sequenced using primers based sequence of adjacent vector, sequecne assembled into consensus sequence covering whole genome

Next generation WGS Illumina sequencing

cell free reactions (no vectors), millions of individual DNA fragemnts isolated and sequenced in parallel, extremely small reaction volumes

Illuina sequencing steps

Library preparation, Cluster generation - PCR, Sequencing by Synthesis, Data analysis

Illumina Library preparation

genomic DNA fragmented into smaller uniform fragments, Adaptors (short knwon sequences) added to both ends

Cluster generation - Illumina

flow cell glass slide coated with oligonucleotides complementarty to adapter sequences, ssDNA fragments bound to flow cell via adaptors, bridge formation + Bridge amplication (by PCR), one end dissociates allowing other round (several cycles), forms clusters (contains 1000ds fragments of same DNA)

Sequencing by Synthesis - Illumina

sequence of each cluster, dNTP labelled with different fluorescent dye, emits different wavelength based on which base added, signal integrated to generate complementary sequence of template

Data analysis - Illumina

tagged nt emits specific wavelength, DNA sequence determined by synthesis, generates complementary sequence

Whole genome sequence assembly

random DNA fragments assembled based matching sequences, contigs linked into whole genome, repetitive elements collapsed into single contig - challenge for genome assembly

Paired end reads

pairs of sequence read from opposite ends of insert in same clone, one end of insert is part of one contig and other end is part of second contig, insert spans gap between two contigs - correct order and orientation of contigs

Whole genome shotgun sequencing

scaffolds(supercontigs), sequence overlaps buold contigs, paired end reads span gaps and order/orient contigs into larger units - scaffolds

Exome

complete set of exons (protein-coding regions) within a genome

Transcriptome

complete set of RNA transcripts, dynamic - active transcription

Targeted Gene panel

sequence selected known genes, disease search

Epigenome

epigentic markers

advantages of paired end reads

high throughput, produced by circularization - without genomic library construction

reads

small DNA sequences, not yet overlapped, essential for constructing sequence

Scaffolds

Supercontigs, assembly of contigs, use pair end reads to order and orient contigs

NGS features

high-throughput, whole genome, cell free, complex data processing, sequence billions of reads, used cancer genomic/ disease studies/ microbial analysis

Sanger sequencing features

low throughput, single gene/gaps, use vectors(plasmids) and bacterial transformation, high accurary, simple data, use mutation screening/ plasmid and small gene sequencing