ECON 546 Lecture Notes - Lecture 2: Methyltransferase, Metagenomics, Transcriptome

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568: Alexandre Monpetit L2: 11/1/1
L2: Genome technology and NGS Methods
Stats for Human Genome Project:
1. Approx. 95% of genome sequence is considered “completed” – not finished.
2. Still 526 gaps in assembly, most in centromere, telomere and heterochromatin.
3. According to HUGO there are ~20,000 protein coding genes, but <75% have been validated.
6000 ncRNA, but many thousand more estimated to exist.
What cannot be resolved with one genome sequence?
oThe exact number of genes and their functions
oRoles of non-coding DNA
oThe 3D chromosomal structure and organisation
oThe regulation of gene expression in each cell type
oIdentifying the genes that cause disease in humans or interesting phenotypes in
plants/animals
Variation in the genomic sequence is needed to understand the role of DNA.
Individual genetic variation:
1. Genome sequence variation: Bi-allelic variation (SNPs); insertions/deletions; repeat
polymorphisms; copy number polymorphisms; inversions; translocations
2. Epigeneticsdon’t affect base sequence but affect what surrounds the DNA: DNA
methylation, histone modification, ncRNA regulation
Together have effect on expression:
oGene expression levels (mRNA, ncRNAs); protein expression levels; alternative splicing; RNA
editing; post-translational modifications
oMost common way of measuring the effect of variation is by gene expression levels.
oEnvironment also can affect variation (genome sequence & epigenetic) and expression.
How to study genomic variation?
DNA Microarrays
oCollection of microscopic DNA spots attached to a solid surface, each spot contains a short,
specific, single-stranded DNA sequence.
oPhysically separate DNA fragments and interrogate the presence/absence of a specific
sequence and its relative quantity.
oMany different types:
Mass Array – separation by MS;
Integrated Fluidic Circuit: physical separation in small chambers (Fluidigm)
Probes spotted on chip > 100k probes/array
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568: Alexandre Monpetit L2: 11/1/1
Probe synthesized on chip (Affymetrix, NimbleGen, Agilent) > 1M probes/array
Bead array (Illumina) > 5M probes/array
Last two cover almost entire genome
Applications:
oGenotyping i.e. allele typing, het/homo: discover or validate gene-disease association
oGene expression: extent to which certain genes are turned on or off in cells – use RNA.
oDNA Methylation Analysis
oChIP-on-Chip (chromatin immunoprecipitation) to measure DNA-protein interactions
oCNV (copy number variation)
Commercial arrays covering the genome
Affymetrix: in situ synthesised array
oDetermine which genes exist in a sample by detecting specific pieces of mRNA.
oSingle chip can analyse thousands of genes in one assay – up to 1M probes (~25 bases) per
array.
oCan measure allelic variation in genome (genotyping – emit different wavelengths) or RNA
expression levels – compare levels between control and sample.
Bead arrays: synthesis of oligo done separately, then put together.
oEvery bead is covered with hundreds of thousands of copies of the specific oligonucleotide
that corresponds to one region of the genome
oBeads put on arrays. Each bead has a barcode, telling position of genome.
Advantages of DNA microarrays:
oMultiplexing (>1M) – simultaneously measure multiple analytes in a single run of assay.
oAutomation of analysis
oAllow the survey of the genome (identify variants associated with trait or disease) or of the
transcriptome (identify gene whose expression correlate with a trait) relatively cheaply.
oCan measure simultaneously nucleotide variants (SNPs) and some structural variants (indels,
CNVs)
Limitations:
oNot possible to assay the entire genome
oSignal can be noisy (amplification artefacts/ allelic bias/ cross-hybridisation between probes –
regions of genome are similar).
oInterpretation bias (3’ probes used as surrogate for expression of whole gene)
oSelection bias (only what is known is on the array)
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Document Summary

95% of genome sequence is considered completed not finished: still 526 gaps in assembly, most in centromere, telomere and heterochromatin, according to hugo there are ~20,000 protein coding genes, but <75% have been validated. 6000 ncrna, but many thousand more estimated to exist. Variation in the genomic sequence is needed to understand the role of dna. Individual genetic variation: genome sequence variation: bi-allelic variation (snps); insertions/deletions; repeat polymorphisms; copy number polymorphisms; inversions; translocations, epigenetics don"t affect base sequence but affect what surrounds the dna: dna methylation, histone modification, ncrna regulation. Integrated fluidic circuit: physical separation in small chambers (fluidigm) Probes spotted on chip > 100k probes/array. Probe synthesized on chip (affymetrix, nimblegen, agilent) > 1m probes/array. Bead arrays: synthesis of oligo done separately, then put together: every bead is covered with hundreds of thousands of copies of the specific oligonucleotide that corresponds to one region of the genome, beads put on arrays.

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