Functional Genomics

genome

gene + chromosome

coding gene

gene → expression → protein (function)

chromatin

DNA + histones

heterochromatin

densely packed, stained, transcriptionally inactive

euchromatin

loose, transcriptionally active

flow of genetic information

DNA → pre mRNA → MRNA → protein

phenotype

genotype + environment + life history + epigenetics

Expressed Sequence Tag (EST)

measures expression

repetitive regions

minisatellites, microsatellites, telomeres, SINES, LINES

how much of genome is repetitive regions

50%

SNP

single nucleotide polymorphism (sequence variation)

indels

nucleotide insertion / deletion (structural variation)

CNV

copy number variation (structural variation)

mapping

1) assembly of genetic/physical map

-linkage, recombination frequency

-contig, scaffold

2) mapping expressed gene sequences in genome order

-cDNA clones, EST

3) annotate genes

-sequence search

-open reading frame

variation

4) sequence variation

-SNP

-haplotype

-linkage disequilibrium

5) comparative genomics

-synteny

functional genomics

6)

genetic map

relative order of genetic marker in linkage group; distance between markers expressed in recombination units

physical map

assembly of contigs; distance between landmarks expressed in kB

contigs

continuous stretches of chromosomal DNA

assemble contigs workflow

1) alignment of randomly isolated clones based on restriction fragments length profiles

2) hybridization-based approaches

-chromosome walking

-Sequence Triggered Sites

chromosome walking

screening DNA fragments and overlapping fragments to map & identify desired sequence

cytological map

physical location of genes on chromosomes based on banding

synteny

conservation of gene order

homologs

features derived from common ancestor

orthologs

different species (evolution)

paralogs

same species (gene duplication)

Sanger Sequencing

-PCR chain terminated by dideoxy NTP

-separated by gel electrophoresis

*chain termination method A, G, T, C simultaneously

• fluorescence labelling

• capillary electrophoresis

  • laser detection → base calling

PHRED Sequencing

Q = -10log(10) P(error rate)

shotgun sequencing

random fragmentation of genome (reconstructed)

hierarchical shotgun sequencing

-mapping before sequencing

-BAC to BAC scaffold (physical map) -< supercontig; sequenced contig scaffolding + mate pair cDNA sequencing information (fill gaps)

whole genome shotgun sequencing

-reconstructing using overlaps

-assembly + mate pair from larger fragments

x problem: repetitive sequences, untigs, repeat resolution

untigs

unique contiguous sequence alignments

coverage

avg # nucleotide is represented by base in collection of random raw sequences

de novo sequencing

determination of full genome w/out known reference sequence

resequencing

determination of full genome when reference sequence is known

single end read

multiple matches

paired end read

anchored match

DNA sequencing workflow

1) library prep

2) amplification

• emulsion PCR

• bridge PCR2

3) sequencing chemistry

• ligase reaction

• translocation

4) signal detection

• fluorescence

• electrical signal

454

emulsion PCR + bead deposit + pyrosequence

X problem: homopolymer, precise concentrations

Illumina NGS

1) load library into lane of flow cells

2) bridge amplification

3) sequencing by synthesis

• Reversible Terminator Chemistry

X Problem; difficult to increase length, ^ errors

semiconductor sequencing

release of H+ → pH change → electric signal

Oxford Nanopore

electrical current flows through hole → molecule produces squiggle → base calling→ real time RNA/DNA sequence

Ion Torrent

emulsion PCR → bead deposit → semiconductor pH detection

Novaseq

short read, cluster generation, sequencing by synthesis

DNB

single strand circularization → pattern array

Aviti NGS

avidity, nucleotide identification w/ ligands on dye-labelled polymerase → polymer → nucleotide complexes bound to clonal copies of DNA targets

pair wise alignment

multiple sequence alignment

global alignment

similar sequences, similar length

local alignment

isolate regions in sequences, database searching

hamming distance

# of positions w/ mismatched characters

levenshtein distance

inserting gaps to maximize # of letters

score

match or mismatch penalty - gap penalty

Basic Local Alignment Search Tool (BLAST)

1) List

2) Scan

3) Extend

4) Statistical Significance

segregation

separation of corresponding alleles during reproductive processes

linkage disequilibrium

alleles @ 2+ loci do not segregate, process independently

D

linkage disequilibrium coefficient

Dmax

maximum possible value of D

NGS Library Prep

1) DNA fragmentation/target selection

• sonication (shearing)

• DNAse treatment

2) Adapter ligation

-amplification

-T-overhangs

-forked structure

3) Size selection

-spectrophotometer

-bioanalyzer

-quantitative PCR

-gel electrophoresis

-beads

4) Library quantification

NGS sequencing processing

NGS library prep → NGS → FASTQ Format→ NGS sequencing mapping

genome-wide association study

analyzes the DNA of population to find genetic variations linked to a disease/trait

quantitative trait loci

stretch of DNA that underlies a quantitative trait

phylogenetics

study of evolutionary relationships through groups of organisms

clustering method

distance based

eg. UPGMA, neighbor-grouping

cladistic method

character based methods

eg. maximum parsimony

maximum likelihood

find the tree topology and branch lengths that maximize the probability

exome-seq

sequencing of exons

(+) of exome-seq

more sequence coverage, lower cost, less background noise

(-) of exome-seq

incomplete, limited to coding regions, less SV detection

forward genetics

phenotype → gene ; random mutagenesis

reverse genetics

gene → phenotype ; targeted mutagenesis