8 - Genomes

Diff between genomics and genetics

Genomics

• larger scale and complexity of data/datasets

• closely related to computer/technology and bioinformatics

CoT Analysis/Reassociation kinetics

• Estimate the complexity and repetitive content of DNA → see how qucikly they anneal

• extract DNA → hear DNA and fragment to single stranmd → wait and let cool to measure reassociation → plot ssDNA vs log C0T

• x-axis - log10cot, y-axis % single-strand DNA

• more “bumps” and smaler dropoffs = less repetiitive DNA, bigger dropoff w? less bumps = more repetitive DNA

Genetic Map

• based on recombination frequency

• measure relative positions of genes + affected by recombination hotspots

• made by loofing at crossing over

• “How often trians switch tracks between stations”

Physical Mapping (STS, contig, BAC, fingerprinting, FPC, YAC, two ways to close gaps (library and PCR))

• ordered collection of clones from genomic library → find order and physical distance between DNA base pairs by DNA markers

• Involves:

  • BAC - Bacterial Artificial Chromosome, clonging vector used with physical maps

  • STS - Sequence tagged site, marker, only hybridize one locaiton in genome

  • contig - set of overlapping DNA clones

  • Fingerprint - unique pattern of restriction fragments used to orient and order clone

  • FPC - fingerprinted contigs, contigs made with fingerprinting

  • YAC - Yeast artifical chromsome, used to potentially fill in gaps + some seq poisonous in BAC (and vice-versa)

  • Filling in gaps for physical map

    • 1) long-range PCR

    • find clones in genomic libraries hybriziing to ends of contigs

Physical Map - X-value and coverage

• denotes how much sequence has been duplicated

• ex: 3X coverage means each base covered about 3 times

• Coverage = 1-e^-x

• e^-x = how much not covered

Reference genome and why it isn’t representative

• complete sequence of an organisms chromosome

• artificial because

  • involves many different people’s genomes

  • haploid

Sanger era (golden path, finishing, scaffold, shotgun seq) + issues of sanger

• Human genome mainly used Sanger sequencing

• picked the “golden path” with given physical map

• Bac clone sequence with shotgun seq - random chosen sub-clones of BAC)

• seq aligned were grouped into contigs → single consensus seq

• finishing - filling in the gaps of contigs, often with PCR

• scaffolds - connecting contigs logically but not by using contiguous sequence

• Issues

  • 1) needed to create physical map

  • 2) subcloning BACs to sequence them

Short-read era (adv+dis of shortread, N50

• used PCR instead of cloning DNA

• SOLiD, Illumina, pyrosequencing

• Advatange → cheap, high coverage

• Disadvatange → short read (100-150 bp) anmd genome has may repeats, make assembly difficult

• N50 → measure quality and contiguity of genome assembly

  • add up total contig sizes

  • add up contigs until hits half of total size

  • last number to reach 50% threshold is the N50

Long-read era

• PacBio and Oxford

• huge read with high error rates → long reads better for genome assembly

Genoem facts

• largest genome → amoeba, 670Gb

• Pufferfish have more genes than humans but 1/10th gneome size

• S. cerevisae only few huynder iuntrons in 6.3k gnees, anothe rfungus C, neofromans has 5.3 introns per gene

• C. elegnas have trnas and cis pslicing

• most common repeat in pine is 5% genoem, most common repeat in corn in 75% of genome

• intron of animal genomes are huge, but not plants or fungi

• some highly conservedseq in mammmal don;t code fvor protein

Tandem Repats - micro/mini satelite, replication slippage

• occur next to each other

• microsatelite - coupkle of nt long

• ministatelite - 10-60 bp long

• long tandem repeats - duplications or inversions

• satelite grow and shirnk via replication slippage  - DNA pol slips causing indel

Intersperesed repeats - gene conversion

• distributed all over genome

• can copy to new locations in the genome and disrupt normal genes

• “junk dna”

• combat gene conversion

• interspersed repeats find way into introns and make genes look dissimilar, preventing homologous conversion and gene conversion

RNA transposons (LTR, non-LTR (LINE and SINE))

• also known as retrotransposons, are common in eukaryotic genomes, 42% of human genome

• get name from going through RNA intermediate, from RNA →: DNA

• RNA transposon types

  • 1) LTR (long terminal repeats)

    • have long terminal repeats and encode enzymes allowing self replication and integration in genome

    • couplkt hundred nucleotides and enzymes are 5 kb, look similar to retrovirus

  • 2) Non-LTR

    • also called LINEs (long interspersed nuclear elements), 21% of genome

    • LINE1 - 6kb lonmg, RT activity

    • many LINE truncated because RT falls off before ocmpleting transcript -:> explains nonfunctionality

    • another type → SINE, 100-700 nt long

    • human genome, SINE = Alu elements → 300 nt and 1 million present anout 10% genoem

    • SINE originate from small RNA, transcribed by RNA p[ol III

DNA transposons - Helitrons and politrons

• dont use RNA intermediate

• two types

  • Helitrons - eukaryotic DNA transposon, make copies via rolling circle)

  • Politrons - 15-20 kb lonf an related to viruses,

Psuedogene - retrosuedogenes.proicessed, non-processed, unitary, psuedo-psuedogene

• look like broken protein coding genes, 4 majopr classes

• 1) Process psuedogenes (retropsuedogene)

  • mRNBA rev transcribed to DNA and inserted into geneome

  • contian a poly-A tail and have no intron

  • often form highly expressed genes

  • can create a higher BLASTX score than regular DNA

• 2)Non-processed

  • DNA psuedo genes

  • reuslt of incomplete duplication

  • retian OG sequence and look like real genes

• 3) Unitary

  • gene nonfuinctional without dupliocation

  • ex: GULOm, why we need Vitamin C

• 4) Psuedo-Psuedogenes

  • genes seem broken with nonsense mutation but may actually funmction

  • stop codons read through

Variation - SNP, SNV, SV, CNV

• humans vary 1bp/1000bp (human/chimp = 1/100)

• replication errors 1/100,000bp (30,000 errors per haploid gneome)

• SNPs (single nucleotide polymorphisms)\

  • present in1% or more of population

  • often used as markers for GWAS

  • SNV → also simlar but cna be rare

  • calssified about where they occur in genome (ex: non0-coding, coding, indel)

• SV (structural variant)

  • gneetic differences that make larger chagnes to genoeme

  • ex: indel, inversion, translocationm

  • CNV (copy miumber variant) →: region of chromosome where ther is differnece of repeasts

    • some diseases incluse Fragile X, Dup15q

  • SV harder to asses than SNPs

Human gneoem Trivia

• Size - 3 billion bp

  • LKongest chromosome: 1 - 249Mbp, 2- 242bp, 3- 198 bp

  • Shortest Chromosome: 21: 47 Mbp, 22 - 51 Mbp, Y - 57 Mbp

• Genes

  • 20KL protein-coding genes

  • 1% of genome corresponmds to protien-coding DNA

  • unknown number of RNA genes

• Repeats - >50% repetitive geneome

  • 13% SINE (11% Alu)\

  • 20% LINE ( 17% LINE1)

  • 8% LTR transposons

  • 3% DNA elements

  • 3% SSR

  • 3% Duplications

• variation - 1bp/1000bp

Metagenomics - 16S rDNA, rarefaction curve, OTU

• Environmental sequencing

• look at 16S rDNA, part of small ribosomal subunit

• rarefaction curve → unique OTU (operational taxnomical units/unique seq) y-axis vs. # of sequences x-axis

  • tells us hwo manyh differnet species present