Functional Genomics- Ch

Homozygote

An individual with two identical alleles for a particular gene, resulting in uniform expression of that trait. (BB)

Heterozygote

An individual with two different alleles for a particular gene, leading to mixed expression of that trait. (Bb)

Segregation

The separation of homologous chromosomes (aka when paternal and maternal chromosomes pair together) after recombination during meiosis. Or, seperation of corresponding alleles during the reproductive process.

Recombination

Chromatids from each homologous chromosome exchanges segments of alleles, which results in different gene combinations. Results in genetic diversity.

Chromosomes pair up with their homologous partners (same genes), and recombination shuffles the alleles between them.

Alelles

An allele is a different version (or variant) of the same gene, and each version can lead to a different trait or form of a trait.

Ex: B and b are on/from the same gene (like coding for eye color), but different alleles (B for brown eyes and b for blue eyes).

Linkage

• Refers to the physical proximity of genes on the same chromosome.

• Genes that are close together tend to be inherited together because there’s less chance of recombination separating them.

• Ex: If Gene A and Gene B are close on chromosome 1, a crossover is less likely to occur between them. So they “travel together” during meiosis = linked.

Linkage Disequilibrium

• Refers to non-random association of alleles at two or more loci in a population.

• Aka …some combinations of alleles appear together more often (or less often) than expected by chance even though they are far apart.

Ex: Locus 1 has alleles A and a. Locus 2 has alleles B and b.

Even if these two loci are far apart on the chromosome, they might be in LD if you find that:

• AB combination appears more frequently than Ab or aB, even though they are physically far apart.

Genetic Map

A diagram that shows the arrangement of genes and their relative distances on a chromosome, based on the frequency of recombination events. b

• Higher % = alleles are further apart, higher likelihood of crossing over

• Lower % = alleles are closer together, lower likelihood of crossing over

centiMorgan (cM): 1cM = recombination frequency of 0.01

“closely linked” = they are more likely to be inherited together (aka linkage disequilibrium) because it is more than expected

Physical Maps (Mapping Genomes)

An assembly of long, continuous pieces of DNA (contigs), and on these contigs, scientists mark known DNA sequences (landmarks) and measure the physical distance between them in kilobases.

• Provide information about the arrangement and location of genes on chromosomes to help study genomes.

Methods of Assembly Contig: Hybridization Techniques

• chromosome walking

• sequence-tagged sites (STS): single occurance in the genome

Chromosome Walking

It’s a method to find and connect overlapping DNA fragments to gradually move along a chromosome, starting from a known DNA sequence. Start from a known DNA sequence (probe), connect it to another fragment that contains the same DNA sequence (overlapping sequence), sequence the unknown parts, create another DNA probe at the end of the sequence, repeat.

Sequence tagged sites

An STS is a short, unique DNA sequence that occurs only once in the genome. Because it's unique, if a clone contains an STS, scientists know exactly where that clone came from in the genome. STSs mark precise locations on the DNA. STSs help scientists match up and organize DNA fragments by anchoring them to a known position in the genome.

Cytological Map

A cytological map is a chromosome map created by analyzing the physical structure of chromosomes using microscopy techniques. Provide a way to visualize and locate specific regions on chromosomes based on banding patterns that appear when chromosomes are stained.

Synteny

The conservation of blocks of genes or genetic sequences on chromosomes between different species. In simpler terms, it means that genes are found in the same relative positions on the chromosomes of different organisms.

Homologs

Genes with very similar sequences, evolved from a common ancestral DNA sequences
1) Orthologs

2) Paralogs

Orthologs

homologs in different species (evolution)

• Genes in different species that evolved from a common ancestral gene by speciation.

• Usually retain the same or similar function.

Paralogs

homologs within the same species (gene duplication)

• Genes that are related by duplication within the same genome.

• Often evolve new functions, even if they’re related.

Sanger Sequencing

1. Denature DNA- Split strands

2. Bind primer (small section of DNA)

3. DNA polymerase lengthens strand according to the template strand

4. DNA Polymerase randomly places dideoxy nucleotide, which terminate chain

5. Chain terminated

Process repeated above until sample contains fragments of all different lengths (all terminated by differently clored flourescently labeled dideoxynucleotides)

6. Fragments separated by size, and the color of dideoxy nucleotdes can tell us the template stand nucleotide sequence

Dideoxy Nucleotide

Missing an OH group, which is what allows the next nucleotide group to bind, randomly placed by DNA Polymerase, dyed according to the base last placed.

Cyclic Sequencing

Cyclic sequencing refers to a DNA sequencing method where the process happens in repeated cycles, each cycle revealing one base (A, T, C, or G) of the DNA sequence at a time. (concept behind many NGS)

Capillary electrophoresis (done after sanger sequencing)

1. loaded into capillary tube filled with gel

2. electric current is applied

   • fragments migrate through the gel/ capillary tube

   • smaller fragments move faster through the gel and exit the capillary tube faster

3. Laser and detector at the exit of the tube

   • Laser: excitrd the dye

   • Detector: reads the color of the dye

Base Calling

Software records the order of the colors and translates it into a DNA sequence

• Each peak is a detected base

Chromatogram: this is what the color peaks are called

Phred Scores

(q) Number assigned to each base in a DNA sequence to represent how confident the software is that the correct abse was identified

Phred Score formula

Phred Score (Q) = -10 log10(P)

p= probability that the base call is incorrect

q= quality score

Interpretation of Phred Score

Q20 score or higher is considered high quality base calling

Good: sharp clear peaks (Q20 and higher)

Bad: messy/overlapping peaks (lower than Q20)

Bad: no phred score could be calculated, sequencer could not determine which base was present (N designated for base)

Heirarchal Sequencing

1. Break the entire genome (~3 billion bp) into large chunks (150bp) using BACs

2. Build a map: align and organize these large clones (chunks) into a scaffold using…

   • chromosome walking

   • finger printing

3. Subdivide each BAC clone into smaller pieces (3kb)

4. Sequence smaller fragments (Sanger Sequencing)

5. Assemble fragments into contigs, then super-contigs using overlapping regions

6. Fill gaps using cDNA or mate pair reads

BAC

Bacterial Artificial Chromosome, used to clone large DNA fragments.

• Tiny, circular piece of DNA (plasmid)

• Use to clone large pieces of DNA

• Cut up BACs to get smaller fragments for sequencing.

Fingerprinting

1. enzymes slice DNA at specific recognition sites to create fragments of known specific lengths

2. Measure fragment size

3. Size of the fragment is the “fingerprint”

Fragment size/pattern tells you which DNA seqeunces go together by comparing their sizes and patterns

You want to line up the BACs before you sequence them

cDNA

complementary DNA

• Can onyl help fill gaps inside genes

• Can help locate genes → since you already know what that gene sequence looks like

Mate Pairs

Come from ends of large DNA fragments — before it is cut up into smaller pieces

• So you know which contigs/ sequence on the contigs are on both ends of the long DNA fragment (as well as how many bp are between them) and if they are near each other

Whole Genome Shotgun Sequencing

1. Break entire genome into large DNA chunks

2. Skip initial scaffolding step! Go straight to…

3. Cut up large DNA chunks into smaller chunks

4. Sequence smaller chunks

5. Assemble all reads into contigs, unitigs, and eventually scaffolds

6. Use mate pairs to help assemble unitigs into contigs/scaffolding

Problems with whole genome sequencing

Repetitve sequences because there is no intiial scaffold/map to go off of

You can’t tell which unitigs go on which end of repeats (ATATAT…)

Mate pairs will tell you which unitig will go on each end of the repeat sequence, as well as how many bp are supposed to be between each unitig

Unitig

made up of multiple smaller fragments

• perfectly ovrelapping, no ambiguity (no gaps, etc.)

• 100% confident

Contigs

may include multiple contigs, contains gaps etc. made up of multiple small DNA chunks

chrUn

unlocalized contigs → These are sequences from a genome that haven't been assigned a specific location on a chromosome yet.

Because of repeat sequences and lack of information

Chr1 xxxx random

Unplaced contig names consist of the chromosome number, followed by the NCBI accession number, followed by "random“

• Chr# = chromosome #

• NCBI number = unique identifier for the contig in the GenBank database

• Random = indicates the contig is not placed at a specific location

GC contents in human genome

generally low GC content, but some regions are highly GC rich (CpG island)

CpG Islands

CpG islands are regions with a high frequency of CG dinucleotides, often near gene promoters.

Their methylation status influences gene expression:

• Unmethylated CpGs (hypomethylated) → chromatin open → genes ON

• Methylated CpGs (hypermethylated) → chromatin closed → genes OFF

Segmental Duplication

Segmental duplications are long stretches of DNA that are nearly identical copies of each other — with greater than or equal to 90–95% sequence identity.

• Intrachromosomal

• Interchromosomal

Intrachromosomal Duplication

• Both copies are on the same chromosome.

• Tend to be less similar (less % identity).

• Can be longer.

Interchromosomal Duplication

• Tend to be more similar (higher % identity).

• Usually shorter

• One copy is on one chromosome, the other on a different chromosome

Fragment

a small piecce of genomic DNA - typically several hundred bp in length - subject to an individual partial sequence determination, or read

Single-end read

technique in which sequence is reported from only one end of a fragment

Pair-end read

technique in which sequence is reported from both ends of a fragment (with a number of undetermined bases between the reads that is known only approximately)

Read length

the number of bases repoorted from a single experiment ona single fragment

Assembly

the inderence of the complete sequence of a region from the data on individual fragments from the region, by piecing together overlaps

De novo sequencing

determination of a full-genome sequence without using a known reference sequence from an individual of the species to avoid the assembly step

Resequencing

determination of the sequence of an individual of a species for which a reference genome sequence is known. The assemble process is replaced by mapping the fragments onto the reference genome.

DNA sequencing by NGS

1. start with extracted DNA

2. DNA is broken into small pieces using: sonication, dnase (enzymes)

3. Short artifiical DNA sequences (adapters) are added to bothh ends of each DNA fragment.

   • These help the fragments bind to the sequencing platform and get read

4. PCR Amplification: fragments are amplified (copied) to increase the amount of DNA

5. Sequencing library: a collection of amplfiied DNA fragmentes with adaptors, ready to be sequenced.

6. NGS sequencing platform → put in libaray into NGS machine

Amplification methods

1. emulsion pcr

2. Bridge Amplification

3. NO AMPLIFICATION (single molecule)

Emulsion PCR

1. start with DNA that has been ligated

2. Attqach DNA to beads - each fragment gets attached to a small bead of water

3. Form an emulsion

   • Bead +PCR reagent are mixed with oil to create an emulsion

   • each droplet acts like tiny test tube

4. PCR amplification inside droplets

   • PCR performed

   • within each bead, DNA is copied many times and coats bead

Bridge Amplification

1. Prepare DNA with adapter

   • fragments have special adaptor sequences ligated to both ends

   • complementary to oligos stuck on the surface of the illumina flow cell

2. Bind DNA to flow cell

   • each DNA fragment sticks to the surface via base pairing with attached oligos which is now anchored at one end

3. Bridge Formation

   • Free end bends over like “bridge” and binds to second oligo on surface

4. DNA Polymerase codes second strand

5. Denature both strands → now two complementary strands

6. Repeat

DNA Polymerase Reaction

1. DNA polymerase reads the template strand

2. Adds complemetary strand

3. When it adds a base to the new strand…

   • the base is added to the strand

   • A byproduct (phosphate/ H+) released → that release is what gets detected in sequencing

Pyrosequencing, reversble termination, chain termination

SBS (Sequencing by synthesis) method

pyrosequencing, reversible termination

Pyrosequencing

1. when nucleotide is being added by DNA polymerase, pyrophosphate (PPi) is released

2. Enzymes convert PPi into ATP, which produces light

   • How much light → how many bases were placed

   • One nucleotide added at a time → light tels you if it was added or not

Detection through light (monocolor)

Reversible Termination

1. same as chain termination EXCEPT:

   • chain termiantion reversible through chemically removing dideoxynucleotide, allows for continuation of synthesis

   • after every base, machine snaps picture of fluorescent label

Detection through fluorescence (4 colors)

Alternate Sequencing methods

ligation, translocation

Translocation (channel)

• DNA strand is threaded thorugh nanopore (tiny biological or synthetic pore embedded in a membrane

• As each base passes through the pore, it disrupts an electric current in a base specific way

• Sequencer measures the current changes to identify bases in real time

Electric detection

Ligation

• uses fluorescently labeled probes (oligonucletodies)

• Each broke contains known bases and a dye tag

• Binds to matching sequence, and DNA ligase attaches it

• Fluorescence detected → sequence decoded based on color pattern

• Multiple rounds of probing give full sequence

Illumina

Bridge Amplification → Reversible Termination → Fluorescence (image/color)

Pacific Bioscience

Single molecule (no amplification) → pyrosequencing → Fluorescence

Oxford Nanopore

single molecule (no amplification) → nanopore → H+ (pH)

H+ (pH) detection

• nucleotide added (ATCG)

• H+ is released, changes pH of solution

• pH change detected

• Tells you how many of each nucleotide is added

Sequence Alignment

The process of arranging DNA or protein sequences to identify regions of similarity that may indicate functional, structural, or evolutionary relationships.

• Think of it like comparing two strings of letters to see how closely they match

Common Ancestor: ACGCTGA ←→ ACTGT

Sequence Alignment 1

ACGCTGA
A - - CTGT
Sequence Alignment 2

ACGCTGA

ACTGT - -

You want to maximize the amount of matches you have with minimal gaps or mismatches.

Identity

nucleotide sequences are identical, likely to have similar functions or origins

Substitution

One base swapped for another.

Insertion

extra base added

deletion

base removed

Pairwise Sequence Alignment

• Aligns two sequences and compares them to find matches/mismatches.

• Match = same letter in both sequences.

• Mismatch = different letters.

Optimal Alignment Involves

• Scoring: How similar are the sequences? Use:

  • Distance (like Hamming or Edit distance).

  • Score (based on matches, mismatches, gaps).

• Dynamic Programming: Algorithm that finds the best alignment.

Global Alignment

Aligns entire sequences end to end (useful if sequences are similar).

High sequence similarity, homolog, same functiom

Local Alignment

Finds regions of highest similarity (useful if sequences only share some regions).

Conserved region of sequence > functional domain / element

Hamming distance/ edit distance

Hamming distance: use when there are no caps and count the number of mismatches (or if there are gaps needed, still line them up with no gaps)

Edit distance: how many editing operations there are to transform x to y (how many changes you need to use in order for the sequences to match)

How to measure sequence similarity: score

Score = (match or mismatch penalty) - gap penalty
Score values:

Match =3

Mismatch = -1

Gap = -2

Local Alignment

finds the best matching region(s) between two sequences (better if sequences vary in size or contain different domains).

Global Alignment

aligns sequences from start to finish (useful when they’re similar in length)

Haplotypes

group of genes (DNA regions) in the chromosome that are inherited (segregated) together from a single parent during recombination

• A collection of specific alleles (SNPs) in a cluster of tightly linked genes on a chromosome — likely to be continually passed down unchanged (low rate of mutation)

SNP

An SNP is a single base-pair change in the DNA sequence at a specific position in the genome.

• SNPs differ between people (one person might have A at that specific location, while another person might have G at that specific location)

• However, SNPs in biological families are passed down unchanged

Polymorphism

Tagged SNPs

Specific representative SNPs within a haplotype block (that include other SNPs) that act like “markers” or shortcuts to identify the whole region.

• help in tracking inheritance of traits and diseases.

Three major types of mutagen

gamma rays- strong mutations, disrupt multiple genes

Chemical- full spectrum of mutations, random distribution, mutation detection difficult

Insertion- nonrandom distribution

Structural variations

Large-scale alterations in the genome, including deletions, duplications, inversions, and translocations of DNA segments. They can affect gene function and contribute to genetic diversity and disease.