LEC43: Intro to Genomics

Question 1

what does every human cell contain?

Answer 1

complete genetic code

Question 2

when are chromosomes visualizable?

Answer 2

during mitosis, when chromosomes condense

Question 3

what was first genome screen technology?

Answer 3

karyotyping by G-banding

Question 4

what was reference human genome, its use?

Answer 4

traditional sequencing method: took DNA from individual, arranged into pieces of chromosomes, chopped up, individually sequenced, stitched back together, compared to reference human genome

= sanger sequencing

costly and slow, not method anymore

Question 5

what did human genome project do?

Answer 5

sequence entirety of human genome

took 10 yrs, $1 billion

info acquired allowed cataloging of complete set of human genes

Question 6

what did human genome proj find?

Answer 6

complete set of human genes = similar to number observed in other model organisms like mouse, watercress plant, roundworm

explanation: complexity in mammals due to alternative splicing, permitting increased number of potential proteins

Question 7

how much of our DNA is protein coding genes?

Answer 7

1.5%

Question 8

what is difference between number of genes, gene density, in humans vs plants?

Answer 8

genome size is similar

but gene number is much greater in humans

thus human’s avg gene density is much lower; only 1.5% of our DNA encodes proteins

due to alternative splicing

Question 9

what is result of alternative splicing?

Answer 9

from 1 single gene, exons’ arrangement can be different, get different resulting proteins!

only 1.5% of our DNA is protein coding, though

Question 10

how much of non-gene DNA is conserved?

Answer 10

2-5% of non-gene DNA is conserved through evolution

Question 11

if a piece of DNA is conserved, what does that suggest?

Answer 11

that it’s important

basis for idea that there’s functionality among non-gene portions of our DNA that’ve been conserved through ages/across animals

suggests these regions have important regulatory role in genome function

Question 12

HOXD gene cluster function?

Answer 12

basic body patterning control

example of conserved region of essential proteins that regulate genome function

Question 13

how much of our genome is repeat elements?

what are they relics of?

Answer 13

50%

relics of retrovirsues and ‘genomic parasites’ that invaded our DNA in evolutionary history, i.e. HIV - ‘junk DNA’

Question 14

what causes finger webbing?

Answer 14

mutation in hoxD gene cluster, as HoxD genes encode for basic body patterning

Question 15

segmental duplications?

Answer 15

blocks of DNA 1-500 kb in length that occur at multiple sites in the genome, share a high level of sequence identity

~5% of our DNA

can be intrachromosomal (same chromsome) duplications or interchromosomal (between chromosomes)

Question 16

what role do segmental duplications play in genetic disease?

Answer 16

these large highly idneitical repeats often flank certain regions of the genome that are thus prone to misalignment during meiosis, leading to improper recombination

if any repeats are dosage sensitive, results in genomic deletions and/or duplications that are associated w/ a particular genetic disease

Question 17

examples of recurrent genomic disorders?

Answer 17

velocardiofacial syndrome

angelman/prader-willi syndrome

charcot-marie tooth disease

x-linked hemophilia

all caused by mechanism of recombination between large high-identity repeats

Question 18

recurrent deletion on chromosome 15 causes what/example of what?

Answer 18

causes intellectual disability, dysmorphisms, epilepsy

deletion = most common known genetic cause of epilepsy, present in ~1% of epilepsy patients

example of recurrent genomic disorder caused by aberrant recombination between large high-identity repeats

Question 19

how many bases of difference exist between 2 individuals?

Answer 19

avg ~6 million bases, ~0.1% of genome

means 99.9% shared DNA among humans

Question 20

what are the types of variation in the human genome? from smallest to largest

Answer 20

1) single base-pair changes - point mutatoins/SNVs/SNPs
2) small insertions/deletions & microsatellites
3) mobile elements - retroelement insertions (300 bp - 10kb in size)
4) large-scale genomic variation (>1 kb) - deletions, duplications
5) chromosomal variation - translocatoins, inversions, trisomy

Question 21

most common type of genetic variant?

Answer 21

SNVs, single nucleotide variants or polymorphism or point mutation

occurs 1x every 1,000 bp = 3-5 million SNVs in individual genome

Question 22

where do SNVs usually occur?

Answer 22

most in non-coding regions - may have regulatory effects, but not well understood

however, 10,000 per genome (0.3%) are in coding regions, & cause changes in protein sequence

Question 23

what do SNPs within coding regions cause?

Answer 23

sometimes, no change, since a.as are reduntant

sometimes, changes amino acid, different protein results

Question 24

what do SNPs outside of coding regions cause?

how much of SNPs are outside of genes?

Answer 24

can influence disease by altering gene regulation

i. e. if change a ntd within txn factor binding site code, txn factor may not recognize, may not bind to DNA, no activation occurs, gene may be OFF when should be ON
99. 7% of SNPs are outside of genes

Question 25

what does microarry on SNP chips show?

Answer 25

useable to genotype millions of SNPs in a single experiment

can find identity of a base pair at an SNP

floursescently labeled DNA is hybridized to an array of probes immobilized on a glass slide that bind either to normal or variant DNA

Question 26

how does array CGH work?

Answer 26

label a control and patient DNA w/ flourscent dyes

cohybridize them together onto a slide that contians DNA corresponding to different parts of the genome

flourescently labeled DNA hybridizes to the slide

scan it, get image

YELLOW indicates no gain or loss or duplication on the array

however if see color of flourescence of sample, know there is duplication in, for ex., patient’s DNA, at that position

Question 27

what does array CGH enable?

Answer 27

detection of copy number changes that’re too small to be seen by karyotyping

Question 28

what do different chip/microarray tests give info about?

Answer 28

sattistical testing for assocaition between diseases of interset and SNPs at specific chromosomal locations

DNA copy number across genome

detection of sub-microscopic gains or losses of material for rare conditions and common conditions alike

duplications of genomic regions that’re associated w/ protection from disease

Question 29

how can array-based technology be used to inform diagnosis and treatment of cancer?

Answer 29

take cancerous tumor DNA and match to control DNA from same person’s blood or non-tumorous site

compare the 2 to see what happened in tumor

see where chromosomes have extra copies, see deletion of known tumor suppressor

see massive amplification of EGFR gene region, growth-promoting gene and see deletion of tumor suppressor genes: so can develop drugs to inhibit this gene where see amplification b/c clearly key event in tumorogenesis

Question 30

what are tandem repeats?

Answer 30

serial repetition of 2 bases (acacacacac…)

inherently unstable highly repetitive sequences

are rich source of variation in genome b/c polymerase working on DNA at repeat site will add or delete copies of repeats

highly variable regoins btwn individuals

Question 31

what are triple repeat expansions associated w?

Answer 31

neurological diseases

Question 32

what is cause of fragile x?

Answer 32

CGG motif repeat has 5-50 copies in healthy individuals; in ill individuals, can be up to 50-200 copies; in patient w/ fragile X, hundreds/thousands of repeats

this switches off nearby gene, causes disease

causes breakage of chromosome, making DNA polymerase unable to replicate

causes mental retardation w/ distinct dysmorphic features, accompanied by a ‘fragie site’ on X chromosome (= original name)

Question 33

what can a large tandem repeat contain?

Answer 33

entire genes

may be true for genes present in multiple copies, e.g. salivary amylase

Question 34

are genomic duplication regions ever protective from disease?

Answer 34

yes

eg. cheokine CCL3L1, inflammatory signaling molecule

it’s binding partner of CCR5, major receptor molecule for HIV cell entry

more copies of CCL3L1 gene is inversely correlated w/ susceptibility to HIV infection

Question 35

is complete personal genome sequencing expensive?

Answer 35

no! quick and cheap now

Question 36

what is focus of next generation sequencing?

Answer 36

whereas old sanger sequencing focused on 1 gene at a time,

next gen sequencing permits analysis of massively parallel sequencing- more data simultaneously

Question 37

describe process of next generation genome sequencing

Answer 37

1) extract genomic DNA
2) shear DNA into small 200-500 ntd pieces
3) ligate adaptors to ends of fragments
4) enrich and amplify library by PCR
5) sequence on microscopic scale, from adaptor w/ platform

wash through w/ bases that floursece differently; each cluster of DNA will flouresce

measure that flourescence or electrochemical energy, detemrine which base was added durign each step of DNA synthesis rxn

Question 38

describe whole genome shotgun sequencing

Answer 38

can stictch back together fragments of DNA by mapping onto reference human genome

due to random nature of sequences, depth of coverage at any 1 place in genome is variable

reads also contain errors (1%)

therefore need **high redundancy **to generate high-quality gap-free sequence (20x-20x)

Question 39

what is imperfect about whole genome shotgun sequencing?

Answer 39

random errors in sequencing occur

thus cannot know if heterozygous SNV or sequencing error or random error has occurred when a base is mismatched

so SNV calling in genome sequencing is a probabilistic exercise

Question 40

what are barriers to personalized genomics being the be-all/end-all of medical treatment today?

Answer 40

cost is falling rapidly ($1500-2k now)

BUT knowledge of how to interpret consequences of majority of genetic variation is limited

geneticists only know phenotypes caused by mutations in ~3200 of ~25,000 human genes (13%)

each human has ~3 million SNVs, 1200 CNVs - what are effects of these on individual disease risk?

even for the ~10,000 that change a.a. sequence of proteins, currently we can interpret effects of a minority of these, + these are 0.3% of each person’s variation