Lecture 1 "Sequence to Function"

a phenotyp is an individual obervable trait, such as height, eye color, and blood type. the genetic contribution to the phenotype is calles the genotype. some traits detemined by the genotype, while other traits are largely detemined by environmental factors.

• the enirely of DNA in a living cell.

• for practical reasons we differentiate between the nuclear, the mitochindrial, and the plastid genomes.

• DNA is typically organized in chromosomes, either circular (bacteria) or linear (eukaryotes) molecules

• there can be extra chromosomal DNA, e.g. in form of plasmids. in the case of linear chromosomes, we distinguish telomers (chromosome ends) and centromer

1. extract DNA

2. prepare Library

3. make copies

4. sequncing

5. analysis of data

• gene comprises 14,271 putative protein-coding genes

• an estimated 20% function in metabolism

• the network comprises 1080 genes, associated with 2190 reactions and 1068 unique metabolites

• iRC1 080 accounts for the activity of 32% of the estimated genes with metabolic functions

• Acces to the genome sequence of related organisms/species

• catalogues of:

  • the encoded protein-coding and RNA genes

  • transposable elements

  • structural variation

  • genetic diversity within population

  • metabolic pathways > natural compounds

• assesing the metabolic capacities of species

• understanding the link between genotype and phenotype

• reconstruct evolutionary events both on organisms and molecular level

1st generation (1977)

• Sanger method: Sequencing by synthesis

• maxam gilbert method: chemical sequencing

2nd generation (“next generation”; 2005)

• 454 - pyrosequencing

• SOLiD - sequencing by synthesis

• Ion torrent - ion semiconductor

• Pac Bio - Single Molecule Real-Time sequencing, 1000 bp

3rd generation (2015)

• Pac Bio - SMRT, Sequel system, very long reads

• Nanopore - ion current detection, very long reads

• transcription mobilizes information encoded in the genome

• Ribosomal (rRNA)

  • responsible for protein synthesis

  • 60% of a ribosome; up to 95% of total RNA in a cell

• Messenger (mRNA)

  • Translated into protein in ribosome

  • 3-4% of total RNA in a cell

• Micro (miRNA)

  • sort (22 bp) non-coding RNA involved in expression regulation

• Transfer (tRNA)

  • bring sepicific amino acid for protein synthesis

• Others (IncRNA, shRNA, siRNA, snoRNA..)

• build new or improved profile of transcribed regions (“gene models”) of an uncharacterized genome

• rapid access to (protein-coding) genes without bothering with genome assembly and gene predicition

• transcriptome analysis of. community of different species (e.g., gut bacteria, hot springs, soil)

• gain insights on the functioning and activity rather than just who is present

  
  

• quantitative evaluation and comparison of transcript levels, usually between different groups

• vast majority of RNA-Seq is for DGE

• “under which condition do gene express”

  
  

gain insights into regulatory networks controlling gene expression

• synthesis in cycles

  • attached fluorescent marker

• pictures taken after removing terminator

• errors during PCR amplification render copies not 100% identical. Especially errors at an early stage of the PCR can mimic heterozygous positions

• not every pool of millions of sequences will incorporate a base in each cycle. with increasing number of cycles the length heterogenity of the already sequenced fraction will increase and the sequencing will get out of phase

1. size seperation via electrophoresis and detection of fluorescence markers

2. detecting

3. base calling

4. check for quality paramters for Phred Score:

   • peak sequencing - how is it distributed?

   • uncalled/calles ration - clear peaks?

   • peak resolution - how deep is the valley?

• sequencing by synthesis

• terminator free technology

• fluorescent labeled phosphate chain

• uses DNA polymerase, fixed

• read length - 20 kbp

• individual reads have a substantial sequencing error

• optimal for repeat resolution, general genome assembly and saffolding

• library of overlapping inserts

• hairpin adaptors create a circular molecule

• adaptors contain binding site for DNA polymerase

• sequencing results in a long sequencing read

• can genearte multiple subreads from one template

• combine subreads to create circulation consenus reads

• sensed DNA by measuring changes to ion flowbase-pairs at a time

• multiple base-pairs at a time (k-mers)

• characterisitic current signature is comverted to nucleotide sequences

• no theoretical upper limit to sequencing read length, practical limit only in delivering DNA to the pore intact

• Map Illumination reads with bowtie2 and minimap2 for Pacbio and ONT alignment, use samtolls to compare to reference

• Illumina reads align almost perfectly, with a per read median of 99,3% correct. Indels almost never occur.

• PacBio reads which have an median of 89.2% of the read correct. Most frequent error type: insertions (7.45% median) with mismatches only 1.5% median of % read

• ONT reads have per read median of 92.4% correct, with deletions (9%) and mismtaches (4.5%) both at a relatively high mediat per read

• pre-processing