Functional Genomics Midterm

genome

gene + chromosome

Giemsa Stainin (G banding)

DNA staining at AT rich egions

chromatin

DNA + histone complex (structual information)

parts of chromosome

p-arm, q-arm, centromere

heterochromatin

densely packed, stained, transcriptionally inactive

euchromatin

loose, transcriptionally active

genomics

genome (all genes) -(gene expression→ phenotype (all phenotypes)

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%

nucleosome

DNA + Histone (2A, 2B, 3, 4) x 2

homozygote

indiv that has 2+ identical alleles at some locus

heterozygote

indiv that has two different alleles at some locus

linkage

absence or reduction of independent assortment of parental gens, which are usually transmitted together because they lie on the same chromosome

recombination rate

frequency with which homologous chromosomes exchange genetic material during meiosis

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

sequence-tagged sites (STSs)

single occurence in genome

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

fragment

small piece of genomic DNA (100bp or so)

single end read

technique in which sequence is reported from only one end of fragment; multiple matches

paired end read

technique in which sequence is reported from both ends of a fragmen; anchored match

coverage

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

read length

the # of bases reported from a single experiment on single fragment

assembly

the inference of the complete sequence of a region from the data on indiv fragments from the region by piecing together overlaps

contig

a partial assembly of data from overlapping fragments into a contiguous region of sequence

de novo sequencing

determination of full genome w/out known reference sequence

resequencing

determination of full genome when reference sequence is known

DNA sequencing workflow

1) library prep

2) amplification

• emulsion PCR

• bridge PCR

3) sequencing chemistry

• ligase reaction

• translocation

4) signal detection

• fluorescence

• electrical signal

Illumina/Solexa NGS

1) load library into lane of flow cells

2) bridge amplification (solid-phase)

3) sequencing by synthesis

• Reversible Terminator Chemistry

4) color detection

X Problem; difficult to increase length, ^ errors

Oxford Nanopore

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

Pacific Bioscience

single-molecule → real-time sequencing → electrical signal

longer read (3-15kb) accuracy (95%) 30min

pyrosequencing

nucleotide incorporation → ATP → light (fluoresence) + excess nucleotides (recycled)

emulsion PCR

DNA amplification in water-in-oil emulsion droplets

emulsion oil + PCR mix + bead + library DNA

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

Haplotyping

local combinations of genetic polymorphisms that tend to be co-inherited

linkage disequilibrium

alleles @ 2+ loci do not segregate, process independently

D

linkage disequilibrium coefficient

Dmax

maximum possible value of D

D = Dmax, D’ = 0

completely linked

D = 0, D’ = 1

completely independent

H

expected average heterozygosity per nucleotide

genome-wide association study

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

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

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

(+): better coverage, less data, less cost, less bg noise

(-): incomplete, limited to CD mutations, difficult to detect structural variations, intrinsic variations caused by probe hybridization

forward genetics

phenotype → gene ; random mutagenesis

reverse genetics

gene → phenotype ; targeted mutagenesis

types of genomic variations

structural, sequence

structural variations

chromosome number, segmental duplications, copy number variations, translocations, inversion

sequence variations

sequence repeats, transposable elements, short deletions and insertions, tandem repeats, nucleotide insertion and deletions, single nucleotide polymorphisms, mutations

genetic variations

germline variation, somatic variation

germline variation (Blood, normal tissue)

GWAS, Targeted genetic study

somatic variation (Tumor tissue)

cancer genomics

Loss of heterozygosity (LOH)

marker to locate tumor suppressor gens

whole genome sequencing (WGS by NGS) for variants

DNA fragmentation → Library preparation → NGS -FASTQ→ mapping -BAM→ variant cling

high coverage needed

RNAi screening

high throughput method to study gene function through loss of gen function

gene knockout

mutating germ-line DN to create null allele

loss of function

RNA induced to selectively degrade RNA transcripts of specific gene (RNA interference & morpholino antisense oligos)

gain of function

transfection of exogenous gene copy to overexpress (dominant negatives)

dominant-negative

mutant gene product interferes with normal gene product

3 major mutagen types

gamma rays, chemicals, insertions

gamma rays

strong mutations, disrupt multiple genes, laborious cloning

chemicals

full spectrum of mutations, random distribution, mutation detection difficult

insertions

mutation is tagged, reversible, nonrandom distribution

ChIP Sequencing - Chromatin Immunoprecipitation Sequence

for DNA-protein interactions

1) crosslink cells w/ formaldehyde

2) shear chromatin w/ sonicator

3) enrich target using antibody (IP)

4) reverse cross-link, purify DNA, amplify w/ PCR

5) identify sequences, genome-wide association map