MCB104 - Lecture 14 - Genome Annotation and Assembly & Genotypic Variation

What else needed on top of having a full genome sequence?

• Full genome sequence - ONLY STEP ONE

• We need to annotate the sequence to indicate what sequences promote what tasks

• Without annotating, genome sequence is just a series of letters

What are Omics?

• Collective characterization of complete sets of specific type of biological information, typically in high throughput manner

  • Genome: All DNA Sequences

  • Transcriptome: All transcribed RNAs

  • Exome: All Exons

  • Proteome: All Proteins

  • Metabolome: All metabolites

  • Connectome: All neural pathways

• All these methods involve massive amounts of data, requiring various tools to filter out meaningful info

What is Euchromatin?

NOT Heterochromatin

What are Genomes?

• Genome: All DNA Sequences

What are Transcriptome?

All transcribed RNAs

What are Exome?

All Exons

What are Proteome?

All Proteins

What are Metabolome?

All metabolites

What are Connectome?

All neural pathways

How can you identify genes through ORFs?

• An open reading frame is a reading-frame uninterrupted by stop codons

• DNA can be read in 6 reading frames (3 from each strand due to codon length)

• Coding sequences tend to have longer ORFs

How can you identify genes through Phylogenetics?

• Comparing genomes can help track natural selection

• Phylogenetic trees depicting relatedness can be made by comparing DNA sequences among organisms

  • Branch points represent a series of nested common ancestors

  • Number at each branch point is millions of years before the present

What is Genome Conservation?

• When comparing complete genome sequences, there are far less conservation than when only look at coding DNA sequences (CDS)

What is Visualizing Homology?

• A homology map for a 100kb region of the human genome compared to 3 other species shows a range of overlap

  • Zebrafish overlap is largely contained to exons only, increasing overlap proportional to relatedness of species

What are Transcriptomics?

• Sequencing RNA transcripts provides info about what sequences are actually transcribed

• Directly sequencing RNA is difficult

  • Fragile/Unstable

  • Any given RNA sequence is pretty uncommon

  • Tech required is elaborate/expensive

• Solution? CONVERT TO DNA

  • Use retroviral reverse transcriptase to copy RNA into more stable complementary DNA (cDNA)

How can cDNA Library be created?

• Isolate RNA from sample

• Add primers

  • Use oligo-dT primers to bind to poly-A tails

• Add Reverse Transcriptase and dNTPs

• DNA Strand is synthesized

• Denature and use RNase to degrade mRNA

• Add DNA polymerase to synthesize second strand

  • Can self prime: 3’ end forms a hairpin loop

  • If used, need S1 nuclease to cleave loop after

• Now have dsDNA sequences of mRNAs

How can cDNA library be preserved?

• To preserve - clone it into vectors

• Alternatively can be used as is for:

  • Next Gen Sequencing

  • Targeted PCR amplification

  • Many others

• cDNA library includes only some exons

  • Only those transcribed in sample cells

• Genomic library represents all regions of DNA equally, including introns

How does alternative splicing relate to cDNAs?

• Alternative splicing complicates the prediction of proteome

• Comparing cDNAs from different tissues can show where splicing occurs

  • Both “where in primary transcript” and “in which tissues”

What is the Genome Architecture?

• The human genome has about 28,000 genes

• Most DNA sequences are introns and other noncoding DNA:

  • Exome = 1.5% to 2%

  • Remainder is: introns, centromeres, telomeres, transposable elements, etc

  • Variation in genome size mostly due to changes in noncoding DNA rather than gene number or size

What are Repetitive Sequences?

• Most of DNA outside genes is repetitive

  • Particular DNA sequences found many times in genome

  • 2 types: multicopy tandem repeats and transposable elements

  • Repetitive DNA with no known function referred to ask junk DNA

Are Centromere/Telomere junk DNA?

• NO

• Centromeres anchor kinetochores

• Telomeres protect the ends of DNA molecule and protect against replicative degeneration

What are gene-rich regions?

• Chromosomal regions that have many more genes than expected from average gene density over entire genome

• Example in human genome - class III region of major histocompatibility complex

What are gene deserts?

• Regions that have no identifiable genes

• Largest is 5.1 Mb on chromosome 5 with no identified genes

Describe gene-rich/gene deserts establishment with arrangement of genes

• Biological significance of gene-rich regions and gene deserts isn’t well established

  • Both connected someway to gene regulation

What is MHC Region?

Class III regions of the human major histocompatibility (MHC) complex

• Contains 60 genes within a 700 kb region

• Most gene-rich region of human genome

What is Genomic Evolution

• Exons often encode protein domains: sequence of amino acids that fold into functional units

• Shuffling, addition, or deletion of exons during evolution can create new domain architectures

  • Domain architecture: number, kind and order of protein domains

What is Domain Analysis?

• Function of a new protein can be deduced if it contains a domain known to play a role in other proteins

• Shown below: Homeobox Domain (found in Hox genes)

Can exon shuffling create new genes?

• YES

• After exon shuffling, protein products have novel domain architectures

• Moving entire exons is more forgiving than moving parts of exons

  • Exons that have a specific function are more likely to stick when moved

What are Gene Families?

• Groups of genes closely related in sequence and function

• Evolve via Duplication and Divergence

  • Duplicated DNA sequence products start out identical

  • Eventually diverge via accumulation of mutations

• EX; Globin, Hox, Small Monomeric GTP (Rho, Ras, etc)

What is Homologous Genes?

Any evolutionary related sequences

What is Orthologous Genes?

Arose form same gene in common ancestor, usually retain same function

What is Paralogous Genes?

Arise by duplication, often refers to members of gene family

What are Psuedogenes?

• Sequences that look like, but don’t function as genes

• Rapidly accumulate mutations

• Common features:

  • Missing promotor/start codon

  • Early frameshift/nonsense mutations

What are de novo genes?

• Genes without homologs

• Young genes that evolved recently from ancestral intergenic sequences

What are synthetic blocks?

Homologous blocks of chromosomal sequence

What are chromosomal rearrangements:

The cutting and reassembling of chromosomal blocks accompanying evolutionary divergence

How does Chromosomal Rearrangements relate to Mouse/Human genomes

• Mouse and human genomes diverged 85 mya, but can be compared via chromosomes to visualize similarities

Is the number of genes in the genome the number of proteins the genome can generate?

NO, IT IS NOT

What does combinatorics and complexity relate?

• Combinatorial amplification allow a comparatively small number of genes to produce a large variety of proteins

  • Remember product rule

• Can occur at different levels

  • DNA level: The DNA itself is rearranged into different combinations

  • RNA level: Alternative Splicing

  • Protein level: Multimeric proteins and post translational modifications

What is VDJ Recombination?

• Best studied DNA-level combinatorial amplification

• T-cell receptors have Variable (V), Diversity (D), Joining (J) and Constant (C) segments

• DNA rearrangement in T cell precursors combines V, D, and J segments into an exon

  • Done by deleting intervening sections

• Result is about 1000 different combinations

• Only occurs once per T-cell precursor (all other are gone, DNA itself is edited, happens only ONCE)

What is an example of Combinatorial Amplification: Alternative Splicing

• EX: Neurexin Genes

  • 2 alternative promoters; 5 sites for alternative splicing

  • 3 different neurexin genes

  • Can generate >2000 different mRNAs

What is Bioinformatics?

• The science of using computational methods to decipher biological meaning of information contained in organismal systems

What is GenBank (Bioinformatics)

• Database established by NIH in 1982

• Online repository of sequence data

What is RefSeq (Bioinformatics)

• Single, complete, annotated version of species’ genome

• Agreed upon standard for comparison

• Maintained by NCBI

What is Basic Local Alignment Search Tool (BLAST)

• Aligns query sequences with sequences in a database and finds areas of homology

• Has variants for nucleotide and amino acid sequence searches

  • Amino acid search often includes information on how similar 2 amino acids are to improve alignment

    • Similar to conservative vs. nonconservative subtitutions

    • i.e: Leucine vs. Isoeucine Aspartic Acid vs. glutamic acid

What is Hemoglobin?

• Hemoglobin carries oxygen in blood

• Adult hemoglobin consists of 4 polypeptide chains

  • 2 alpha (a) globins

  • 2 beta (b) globins

• Each polypeptide chains surrounds a heme group that binds and releases oxygen

How is hemoglobin during development?

• Embryonic (Z and e) and fetal (y) hemoglobins bind more tightly to oxygen to facilitate transfer of oxygen from mother to embryo or fetus

• Adult hemoglobin binds oxygen less tightly to allow delivery of oxygen to organs

What is the Hemoglobin Cluster?

• Globin Genes are in 2 clusters: (a and B)

  • Face same direction and are in order they are expressed during development

• Clusters are controlled by locus control regions (LCR)

  • LCR are long range cis-regulatory elements with many enhancer sites

• One model for function is that LCR forms loops to specific sites and as development progresses, the available sites change

What is the hereditary persistance of fetal hemoglobin?

• Rare condition of continued fetal globin expression

  • Deletion of omega and beta genes (should be lethal)

  • y genes continue to be expressed

  • Results in near normal level of health

• LCR cannot switch to adult conformation as looping sites were deleted

What are globin related genetic disorders?

• Hemolytic anemias

  • Changes amino acid sequence of alpha- or beta- globin chain

  • Causes destruction of red blood cells

  • Ex: Sickle Cell Anemia

• Thalassemias

  • Mutations reduce or eliminate production of ½ globin polypeptides

  • Range of phenotypes

How are Thalassemias associated with alpha-globin deletions

• Severity of thalassemia correlates with copy number

• Requires at least 3 copies of alpha-globin to have normal blood

  • alpha 1 and 2 (a1 and a2) are relatively interchangeable

What is B-thalassemia?

• Can occur via deletions of various B-globins however

• Severe B-thalassemia occurs when LCR is deleted

  • Silences all B-globin genes even if all other regulatory sequences are preserved

Is there a “THE” human genome?

No,

• The genome sequences of only 3 people reveal over 5.6 million DNA polymorphisms - sequence differences

Do most polymorphisms have a phenotype?

• Codons make up less than 2% of human genome

• Many mutations in codons don’t change amino acid

• Many deleterious mutations disappear from population through natural selection

What are categories of variation?

• Single nucleotide polymorphisms (SNPs)

  • • One base pair changes, the most common genetic variant

• Deletion-insertion polymorphisms (DIPs or InDels)

  • Short insertions or deletions of a single or a few base pairs

  • In protein-encoding regions, DIP variants are frameshift mutations, unless a multiple of 3

• Simple sequence repeats (SSRs or microsatellites)

  • 1 to 10 base sequence repeated typically ~5-50 times in tandem, can rarely be >100

  • Most common repeating units are one-, two-, or three-base sequences

• Copy number variants (CNVs)

  • Large blocks of genetic material up to 1 Mb in length that are variable in copy number in
    the genome

  • Most important mechanism producing CNVs is unequal crossing-over in meiosis I

What are single nucleotides polymorphisms (SNPs)

• Single nucleotide polymorphisms (SNPs)

  • • One base pair changes, the most common genetic variant

What are Deletion-insertion polymorphisms (DIPs or InDels)?

• Short insertions or deletions of a single or a few base pairs

• In protein-encoding regions, DIP variants are frameshift mutations, unless a multiple of 3

What are Simple sequence repeats (SSRs or microsatellites)?

• 1 to 10 base sequence repeated typically ~5-50 times in tandem, can rarely be >100

• Most common repeating units are one-, two-, or three-base sequences

What are Copy number variants (CNVs)?

• Large blocks of genetic material up to 1 Mb in length that are variable in copy number in
  the genome

• Most important mechanism producing CNVs is unequal crossing-over in meiosis I

What are the frequencies of Variations?

What is the Origin of Variations?

• To determine when a variation occured, must compare genomes

• Comparison of a human and chimpanzee genomes reveals the changes that has occured since divergence of these species

• The single base change at loci 2 is polymorphic in humans

  • C is ancestral, present in ancestral organisms

  • T is derived, changed nucleotide

What are CNVs?

• CNVs are tandem sequence repeats more than 10bp long

• Misalignment during meiosis leads to unequal crossing over

• Not common event, so most CNVs are inherited rather than being a new mutation

• Example: Humans are fewer than 1000 olfactory receptor genes are different loci

  • At each locus, copy number varies

What is an overview of PCR?

• Polymerase chain reaction (PCR)

  • Method of making many copies of target region of DNA

  • First developed in 1985

  • Fast and extremely efficient: can amplify DNA from single cell

• Hinges on thermostable DNA polymerase

  • Originally Taq

• Exponential increase in targeted DNA

What are the steps of PCR?

CYCLE 1

1) Denature strands

2) Base-pairing of primers

3) Polymerization of primers along templates

CYCLE 2

1) Denature strands

2) Base-pairing of primers

3) Polymerization of primers along templates

How does Genotyping via Sequencing work?

• PCR amplify a targeted sequence

• Use same primers or nested one to sequence allele

  • Usually Sanger Sequencing variant

• EX: Sickle Cell anemia is caused by SNP in Hbbeta gene (Hbb)

  • Genotyping can idetify carriers and homozygous individuals

How does Genotyping by PCR product size work?

• Amplify target sequence

• Run PCR product on gel

• If alleles have different length then PCR products with those alleles will run at different speeds

• Can distinguish between homo and heterozygotes

• VERY EASY TO DO YAYYYYY

What is PCR Product Size Variations?

• Size variations can be detected by gel electrophoresis

• Ex: Huntington disease locus

  • Normal allele has <36 C A G repeats

  • Disease-causing alleles have 36 or more CAG repeats; alleles with 42 or more repeats are completely penetrant

What are Restriction Fragment Length Polymorphisms (RFLPs)

• Amplify target region with PCR

• Digest with restriction enzyme

• If variant disrupts/creates a restriction site, can distinguish between them

How can Fetal and Embryonic cells be genotyped using PCR

• Prenatal genetic diagnosis

  • Genotyping fetal cells isolated by aminocentesis: fetal cells in amniotic fluid are extracted using a needle

• Preimplanatation embryo diagnosis

  • Utilizes in vitro fertilization and PCR

  • Genotype embryos before placing in womb

What are Hybridization Probes?

• Hybridization of short (<40 bases0 oligonucleotides to sample (target) DNAs (allele-specific hybridization)

  • If no mismatch between probe and target, hybrid will be stable at high temperature

  • If mismatch between probe and target, hybrid will NOT be stable at high temperature

What are Microarrays?

• Allele-specific oligonucleotides ( A S Os) are attached to a solid support (like silicon chip)

  • 2 oligonucleotides are shown here, but many can be put on one array

How does microarray for genomic DNA work?

• Preparation of genomic DNA for microarray

  • Fragmented

  • Adapter attached

  • Amplified by PCR denatured to make single stranded

  • Fluorescent dye coupled to end of single stranded DNA

How can a microarray be read?

• Fluorescent output is proportional to number of copies of each allele

  • Can sometimes distinguish between hets and homozygous

• Up to 4 million loci can be genotyped simultaneously for approximately $100

How does Positional Cloning work?

• Positional Cloning

  • Object is to identify disease causing genes by genetic linkage to polymorphic loci

• Strategy

  • Same as linkage analysis using 2 phenotypes, except one gene tracked by phenotype, the other by DNA genotype

  • Use microarrays to simultaneously analyze millions of 2 point crosses, each one a test for linkage between a disease locus and DNA marker

What are steps of positional cloning?

• Region of interest narrowed by finding closely linked DNA markers

• Candidate genes are located in region of interest

• Sequence and expression of candidate genes are determined in normal and diseased individuals

What is an example of using positional cloning?

• NEUROFIBROMATOSIS

  • Autosomal, dominantly inherited

• Causes proliferation of nerve tissue forming tumerous bumps

• Positional cloning example determines whether or not SNP is linked to neurofibromatosis gene

• Children in GEN III are in effect the result of testcross

  • RF = 0.125 (N too small to be confident though)

What are some limitations of positional cloning?

• Configuration of alleles are not always known

• Not all mating are informative

• Difficult to obtain pedigree data in humans

• Identified SNPs aren’t necessarily causative

How are large pedigrees used for positional cloning?

• Mapping of Huntington disease

  • Detection of linkage between DNA marker G8 and HD locus

  • Segregation of G8 DNA marker (4 alleles - ABCD) in a large Venezuelan pedigree affected with HD

What is Lod Score?

• Lod Score (Log of the odds) is used to determine if data is sufficient to conclude with confidence that a disease gene and a marker are linked

What is Lod Score compared to RF? Relationship?

• Relationship between Log Score and assumed RF

  • Lod of 3 means log1000 so 1000x more likely to be linked than not

• Can add Lod scores from different pedigrees together to increase confidence

  • RED: Lod score from Neurofibromatosis pedigree

  • BLUE: sum of Lod scores from 3 such pedigrees

Describe Locus heterogeneity:

• Allelic heterogeneity: disease caused by different mutations in same gene

  • Compound heterozygotes (trans-heterozygotes) - individuals with different mutant alleles of the same gene

  • Individuals with certain alleles may respond to drug treatment, while others do not

• Locus heterogeneity: disease caused by mutation in one or two more different genes

Describe an example with completely sequenced genomes

• MILLER SYNDROME - First sample with completely sequenced genome

• Pedigree:

  • Identical regions: brother and sister share same alleles

  • Nonidentical: siblings share NO alleles

  • Haploidentical maternal: same allele from mother, different from father

  • Haploidentical paternal: same allele from father, different from mother

• Geneticists studying disease in affected children could focus on identical regions

• Identified compound heterozygote in DHOD

How can Gene location be narrowed down?

• Identifying causative alleles via filtering

What are evolutionary conserved amino acids?

• Nic’s XIAP gene had a missense mutation that changed a single amino acid that was completely conserved among humans, frogs, flies, and other species

• Assumption that nonconversative variations in conserved regions are more likely to be causative