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Genomics > Next Gen sequencing > Flashcards

Next Gen sequencing Flashcards

1
Q

PCR

A
  • Fundamental principle for any DNA sequencing application
  • PCR is used to amplify a specific region of DNA; primers flank the region you want to amplify.
  • Each cycle doubles the amount of DNA copies of your target sequence
  • Amplify enough DNA molecules so that we have sufficient material to sequence or for other DNA applications
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2
Q

Sanger sequencing

A
  • Invented by Fred Sanger in 1977
  • Cycle Sequencing
  • Based on PCR
  • Modified nucleotides
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3
Q

Next Generation of DNA sequencing (NGS1)

A
  • Technological advances since the end of the human genome project
  • Decrease in the cost of DNA sequencing
  • Since the end of 2007, the cost has dropped at a rate faster than that of Moore’s law
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4
Q

NGS 2

A
  • Development of new NGS methods began 13 years ago with 454 pyrosequencing
  • DNA sequencing throughput jumped 10 orders of magnitude
  • Solexa sequencing-by-synthesis (SBS) developed end of 2005
  • Sequencing market to this day is now dominated by Illumina SBS sequencing
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5
Q

NGS process

A

• Four step process

  1. DNA library Construction
  2. Cluster Generation
  3. Sequencing-by-synthesis
  4. Data analysis
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6
Q

Step 1 DNA library construction

A
  • In the wet lab – first we need to prepare the DNA sample for sequencing
  • Essentially the DNA is chopped into small fragments (typically 300bp ). This is called shearing
  • This can be achieved chemically, enzymatically or physically (sonication)
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7
Q

What is a DNA library?

A

A DNA library is a collection of random DNA fragments of a specific sample to be used for further study; in our case next generation sequencing
The DNA can come from just about anywhere, but in human genetic research generally it’s derived from patients blood.

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8
Q

Part 2 of DNA library

A
  • We have to repair the end of the sheared DNA fragments
  • Adenine (A) nucleotide overhangs are added to end of fragments
  • Adapters with Thymine (T) overhangs can be ligated to the DNA fragments
  • The end result is the DNA library of literally billions of small, stable random fragments representative of our original DNA sample
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9
Q

Part 3 of DNA librarys

A
  • Adapters contain the essential components to allow the library fragments to be sequenced
  • Sequencing Primer binding sites
  • P5 and P7 anchors for attachment of library fragments to the flow cell
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10
Q

Step 2 - Cluster Generation 1

A
  • Hybridise DNA library fragments to the flowcell
  • Hybridization to the flowcell is a Random process
  • But we can’t measure individual single molecules of our DNA library –too small
  • We need to amplify the fragments to a bigger size that we can measure
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11
Q

Part 2 - Cluster Generation 2

A
  • Perform bridge amplification to generate clusters
  • Many billions of clusters originating from single DNA library molecules
  • Clusters are now big enough to be visualised
  • Flow cell is now ready to be loaded on to the sequencing platform to perform the sequencing
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12
Q

Step 3 - Sequencing by synthesis

A

• Modified 4 bases (ATCG) with:
Chain terminators
Different fluorescent colour dye
• Sequence each single nucleotide 1 cycle at a time in a controlled manner

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13
Q

Part 2 - Sequencing by synthesis

A
  • Single nucleotide incorporation (DNA polymerase)
  • Flowcell wash
  • Image the 4 bases (digital photograph)
  • Cleave terminator chemical group and dye with enzyme
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14
Q

Part 3 - Sequencing by synthesis

A
  • Camera sequentially images all 4 bases on the surface of the flowcell each cycle
  • Each cycle image is converted to a nucleotide base call (ACGT)
  • Cycle number anywhere between 50 – 600 nucleotide base pairs
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15
Q

Part 4 - Sequencing by synthesis

A

Machine DNA base calls

Millions of short-read sequences representing our original DNA library

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16
Q

Analysis of NGS data

A
  • Short read sequences from the sequencing machine need to be re-assembled like a jigsaw
  • Mapping locations of our sequence reads on the reference genome sequence
  • To generate a consensus sequence of our original DNA sample library
  • In comparing this consensus sequence against the human genome reference and look for the genetic variants
  • Dedicated software and bioinformatics tools will achieve this
17
Q

NGS v Sanger sequencing

A
  • NGS (left) produces a digital readout. Sanger (right) produces an analogue readout
  • Sanger is one sequence read
  • NGS is a consensus sequence of many reads
18
Q

Whole-exome sequencing part 1

A
  • There are ~21,000 genes in the human genome
  • Often, we are only interested in the gene protein-coding exons or ‘exome’ represents 1-2% of the genome
  • Some ~80% pathogenic mutations are protein-coding
  • More efficient to only sequence the bits we are interested in, rather than the entire genome
  • Costs £1,000 for a genome, but only £200-£300 for an exome
19
Q

Whole-exome sequencing part 2

A
  • Target enrichment
  • Capture target regions of interest with baits
  • Potential to capture several Mb genomic regions of interest
  • Exome would be 50Mb in size
20
Q

Application of exome Sequencing

A
  • Collecting disease affected individuals and their families
  • Use of NGS in disease gene identification
  • Perform exome sequencing
  • Compare variant profiles of affected individuals
  • Try to identify the variant or mutation shared buy the affected individuals
21
Q

RNA sequence 1

A
  • NGS not just for studying DNA.. RNA-seq experiments use the total RNA (or mRNA) from a collection of cells or tissue
  • RNA is first converted to cDNA prior to library construction
  • NGS of RNA samples determine which genes are actively expressed.
  • Single experiment can capture the expression levels of thousands of genes
22
Q

RNA sequence 2

A
  • The number of sequencing reads produced from each gene can be used as a measure of gene abundance
  • Quantification of the expression levels
  • Calculation of the differences in gene expression of all genes in the experimental conditions
  • With appropriate analysis, RNA-seq can be used to discover distinct isoforms of genes are differentially regulated and expressed

Genomics (19 decks)

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