Flashcards in MBB6344 Genomic Science Deck (369)
What are the first generation sequencing technologies?
- Maxam Gilbert (Not used)
- Sanger dideoxy sequencing
What are the second generation sequencing technologies?
- Illumina (only one widely used)
- Ion torrent
What are the third generation sequencing technologies?
- Pacific biosciences
- Oxford Nano pore
When was Sanger sequencing first available?
What is the read length of Sanger sequencing?
Up to ~1kb
How many reads per run does Sanger produce?
One (some machines allow up to 96 at a time)
What is the accuracy of Sanger sequencing?
Highly accurate (>99.9%)
What are the applications of Sanger sequencing?
Good for targeted sequencing e.g. PCR products
When was Illumina sequencing first available?
What is the read length of illumina sequencing?
How many reads per run does illumina produce?
Millions (MiSeq), Billions (HiSeq)
What is the accuracy of illumina sequencing?
Highly accurate (>99.9%)
What are the applications of illumina sequencing?
Draft genome sequencing, resequencing, functional genomics
When was PacBio sequencing first available?
What is the read length of PacBio sequencing?
Up to 50kb
How many reads per run does PacBio produce?
What is the accuracy of PacBio sequencing?
Raw reads ~85% accurate, can be improved to >99.8% with circular consensus sequencing
What are the applications of PacBio sequencing?
Complete genome sequencing, detection of DNA methylation (epigenetics)
When was Oxford nano pore sequencing first available?
What is the read length of Oxford nanopore sequencing?
Can be >2Mb (theoretically unlimited – how intact can DNA remain)
How many reads per run does Oxford nanopore produce?
Up to 1 million (MinION)
What is the accuracy of Oxford nanopore sequencing?
Raw reads now ~95% accurate
What are the applications of Oxford nanopore?
Complete genome sequencing, epigenetics, direct RNA-seq, metagenomics
Outline the main steps in illumina sequencing?
- Sample Preparation
- Bridge amplification
- Image analysis
How is the sample prepared in illumina sequencing?
- Take genomic DNA and break into fragments of around 200-300bp
- Short linker sequences are attached to both ends of the fragments and made single stranded by incubating with sodium hydroxide
- DNA fragments washed across the flow cell and the complementary DNA binds to complementary primers on the surface of the flow cell
What is bridge amplification in illumina sequencing?
- Bridge amplification occurs on the flow cell surface to amplify the single molecule of DNA to produce a cluster of fragments
- Unlabelled nucleotides and DNA polymerase lengthen and join the DNA strands to create bridges of double stranded DNA between the primers on the flow cell surface (look at diagram)
- Double stranded DNA is then broken down into single stranded DNA so that one strand is present on each primer on the cell surface (clonal amplification). This is repeated.
- Produces several million clusters
How does sequencing occur in illumina?
- Primers and fluorescently labelled bases are added to the flow cell. DNA polymerase then binds to the primer and adds the first labelled base to the new DNA strand
- Fluorescence is detected with a camera and recorded
- The next fluorescently labelled terminator can be added. This is repeated until millions of clusters have been sequenced
How is sequencing analysed in illumina?
What determines the number of reads in illumina sequencing?
Each cluster is derived from a single initial molecule and will correspond to a separate read