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Summary All presentations of next generation sequencing
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Summary of all presentations of NGS
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NGSIllumina
Sanger sequencing -> low throughput
Illumine -> parallel sequencing of 1000sequences at the time
Short read sequencing
1. sample preparation
DNA sample of interest is extracted and purified
Transposome fragments are added and tag DNA with adapters
Additional motifs are added: primer binding sites, indices and regions that are
complementary to oligonucleotides on flow cell
2. cluster generation
-> fragment molecule amplification
-> flow cell consists of a glass slide with lanes (each lane is coated with 2 types of oligos)
-> type 1 oligo is complementary to the adapter region of one of the DNA fragment strands
-> hybridization with type 1 oligo
-> polymerase makes the complement of the molecule
-> ds molecule is denatured -> original strand is washed away
-> a ds bridge is formed -> polymerase makes a complementary strand
-> bridge gets denatured -> 2ss copies of the molecule (forward and reverse strand)
-> this process is repeated multiple times
-> reverse strands are cleaved and washed away -> this leaves 1 forward strand with 3’ end
1
, 3. sequencing
-> once the clusters have been generated -> sequence process can start
-> primers are added with fluorescent labled nucleotides
-> every base gets incorporated -> releases a fluorescent signal which is specific for that nucleotide
-> generating many reads simultaneously
-> the indices get sequenced by the same process
-> the reverse strand gets sequenced -> this strand needs to be regenerated at first then wash the
forward strand away
4. data analysis
-> after obtaining forward, reverse reads and both indices -> sequences are aligned to the reference
genome to produce 1 continuous sequence
Advantages Disadvantages
Cost effective Limited in resolving complex regions
High speed, high throughput Hard to detect inversions and translocations
High accuracy No real time data access
Broad range of applications Short read lengths -> may be a problem with
repetitive sequences
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