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22 Cards in this Set
- Front
- Back
- 3rd side (hint)
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Why Sequence Genomes |
Identify Organism Public health/ epidemiology Identify virulence factors Discover new enzymes Discover antibiotic resistance mechanisms Discover new antibiotics Study of evolution in near real time |
7 things |
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Key steps of whole genome sequencing (WGS) |
1) Isolate genomic DNA 2) Fragment DNA (shotgun approach) 3) Prepare genomic library- add adapters or clone 4) Sequence 5) QC- read trimming and quality filtering 6) De Novo assembly/ reference mapping/ SNP calling 7) Contig Reordering 8) Annotation and comparison |
8 steps |
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Give four examples of Sequencing History |
1- Maxam Gilbert 1976 2- Sanger later 70’s 3- Next Gen early 2000’s 4- 3rd gen (now) |
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What are the steps of sanger chain termination? |
Step 1) Primer added 2) Reaction ingredients added 3) Primer extension chain termination and product recovery 4) Electrophoresis, imaging and data analysis. |
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Name the reaction ingredients in sanger chain termination |
DNA polymerase dATP dCTP dTTP Small amounts of ddNTPs with fluorochromes ( each of these have a different fluorescent dye) |
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What is the purpose of fluorescent labels |
Fluorescent dyes emit at different wavelengths. |
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The cost of sequencing has went up over the last 15 years- True or False |
FALSE The cost of sequencing has went down over the last 15 years. It has become more widely available. |
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Give three Key properties of Ion Torrent. |
Sequencing chemistry- Ion semiconducter sequencing Amplification approach- Emulsion PCR Mb per run- 100Mb-1Gb + rising Time per run- 2hrs Read Length- >200bp +rising Cost per run- 500 USD Cost per instrument- 50,000 USD |
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Give three key properties of 454 sequencing |
Sequencing chemistry- pyrosequencing sequencing Amplification approach- emulsion pcr Mb per run- 100Mb Time per run- 7hrs Length- 400bp Cost per run- 8,438 USD Cost per instrument- 500,000 USD |
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Give three key points of Illumina sequencing |
Sequencing chemistry-Polymerase based sequence approach. sequencing Amplification approach- Bridge amplification Mb per run- 600GB Time per run- 9Days Length- 2x 100bp Cost per run- 20,000 USD Cost per instrument- 600,000USD |
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Give three key points of SOLiD |
Sequencing chemistry- Ligation- based sequencing sequencing Amplification approach- Emulsion PCR
Mb per run- 3000Mb Time per run- 5 days Length- 35- 50bp Cost per run- 17,447 USD Cost per instrument- 591,000 USD |
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Illumina Sequencing Chemistry Step 1. |
Step 1- Library preparation Genomic DNA is fragmented -shotgun approach Adapters are ligated into DNA fragments to immobilise DNA onto flow cell. |
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Illumina Sequencing Chemistry Step 2. |
Step 2- cluster generation Flow cell is coated in complementary adapters DNA library is diluted so that fragments are evenly distributed across flow cell Bridge PCR amplifies each fragment at that location forming cluster |
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Illumina Steps |
1) Prepare genomic DNA sample 2) Attach DNA to surface 3) Bridge Amplification 4) Fragments become double stranded 5) denature the double stranded molecules 6) complete amplification |
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Illumina Step 3 |
Add unlabelled nucleotides and enzyme to initiate solid-phase bridge amplification Fluorescent bases are inserted and the flow cell image is recorded in real time Colour of cluster indicates base being incorporated at that moment Fluorescent chain termination is reversible |
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Illumina Step 4 |
The enzyme incorporates nucleotides to build double-stranded bridges on the solid-phase substrate. |
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Illumina Step 5 |
Denaturation leaves single-stranded templates anchored to the substrate |
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Illumina step 6 |
Several million dense clusters of double-stranded DNA are generated in each channel of the flow cell |
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Paired end reads |
Once sequencer has done one round it repeats the process but from the other end. Very useful for analysing rearrangements and insertions etc |
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Ion Torrent- EXPLAIN |
Post light sequencing by proton |
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PacBio SMRT sequencing |
PacBio immobilises the polymerase |
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PHRED score- EXPLAIN |
PHRED is a quality score for the probability of incorrect base call |
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