Illumina Sequencing

–Sequencing and synthesis are the same thing

-Primer binding uses base pairing

DNA Sequencing

  • The process whereby the identity and order of nucleotides in a DNA molecule are determined
  • Early sequencing timeline:
  • Protein sequencing came first then other methods sequecing of the beta chain
  • RNA was the first nucleic acid sequence, it was a tRNA molecule (for alanine) and this was the work of Holley et all (1968)
  • 1977, Fred Sanger invented, “Sanger Sequencing”

DNA replication in a cell

  • DNA seq is DNA replication in a test tube modified for tech/science purposes

The essential parts for our purposes:

  • Complementary Base Pairing
  • Directionality
  • The idea of TEMPLATE
  • The role of the 3’OH group in phosphodiester bond formation and thus the structure of nucleotide

Sanger Sequencing relies on synthetic dideoxynucleotides (ddNTPs)

  • Also called chain terminator nucleotides

Template DNA

-Normal Nucleotides (A,T,C,G)

-Fake Nucleotides (A,T,C,G)

-An enzyme to build DNA

Illumina Sequencing

  • Illumina sequencing is also called Solexa sequencing or illumina dye sequencing
  • Next generation technology (3rd generation)
  • Used to sequence whole genomes, and metagenomes

How we will approach illumina sequencing:

1.Learn the concept of “sequencing by synthesis” which relies on reversible dye-terminators which tell you what nucleotide has been incorporated into a growing strand

2.Consider LIBRARY preparation and what the fragments of DNA in a library means

3.Learn how to make “clonal clusters”

4. Learn the steps in sequencing the clonal clusters

Sequencing by synthesis: the perspective of a single DNA fragment being sequenced in one direction

DNA polymerase attached the next nucleotide into position

Laser energy causes the emission of florescent light, which is detected and used to identify the nucleotide incorporated

The nucleotide has its “blocker” removed, regenerate the 3OH

Where sequencing by synthesis fits into the larger process

  • prepare the sample, make a library”
  • Attach the little tony pieces of DNA to a slide which places the DNA to be sequenced on a solid surface
  • Amplify them into a bunch of tiny clusters, each cluster contains many molecules of the same tiny piece of DNA attached to the slide (clonal clusters)
  • Sequence by synthesis, one strand (forward reads) indicates (labeling pieces) and the other strand (reverse reads) suing reversible dye terminators and READ the sequence by detecting little tiny flashes of light
  • Link the flashes of light with the position on the slide so that you know what the sequence of each cluster’s DNA is

 

2/20/18

Paired end sequencing

“read”- length of sequence

(250 bp long)

Pale blue is what is sent in for sequencing (DNA of interest) with adapter bits on each side

Adapter sequences serve general functions:

  • Attach library fragments to the flow cell (physical location where sequencing occurs)
  • Contain “bar codes” (index sequence) which label the sample
  • Contain Sequencing primer binding sites

-Outer sequences are for attaching- attaches molecule to the surface of the flow cell

-Broke base pairs and floated across the cell- so if base pairing can happen it will.

-Predictable known size, pay money for library preparation

-Wash of original molecules, molecule in the other orientation, continue on with bridge amplification (PCR)- Bridges are made and amplified

-The goal is to make more

-Always a chance for error

-Orientations will be from both original strains, doesn’t work well with sequencing, wash off one of the two fragments, keep clonal cluster

-Adapter sequences are unique, binding and primer sites are common

-Read 1 first sequenced- two ends of the same fragment (going to know that they are pairs)

 

 

 

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