Next generation sequencing Flashcards
(13 cards)
What is the basic principle of NGS?
The genomic strand is fragmented, and the bases in each fragment are identified by emitted signals
What are the advantages of NGS over sanger sequencing?
- Increase in sample size
- Increase in sequencing speed
- Reduced cost
- Increased accuracy
What are the steps of NGS?
- Isolate nucleic acids
- Library preparation
- Sequencing
- Data analysis
What happens in the ‘isolate nucleic acids’ step of NGS?
- DNA or RNA
- Confirms yield, purity and quality of nucleic acid
What happens in the ‘library preparation’ step of NGS?
- Nucleic acids fragmented into shorter sequences and adapters attached to the sequences to ensure compatibility with the sequencing platform.
- Can include PCR amplification
What happens in the ‘sequencing’ step of NGS?
- The prepared libraries
- Several platforms (illuminated, 454 etc…)
What happens in the ‘data analysis’ step of NGS?
- Bioinformatics:
- Read processing - base calling, QC, removal of adaptors.
- Sequence analysis - alignment, assembly, annotation, gene counts, differential expression
What does Assembly mean in terms of NGS?
A generic term to describe methods to generate a consensus sequence from large amounts of DNA/RNA sequencing data
What is De novo genome assembly?
The process of generating a new genome sequence from NGS data
What is De novo transcriptome assembly?
- More complicated than genome assembly, because:
– data more heterogenous in coverage
– because of the possibility of alternative splicing events
– one output of the process is a measure of transcript abundance
What does alignment/mapping mean in NGS?
A generic term to describe methods to find the approximate origin of a sequence, in an existing genome or transcriptome
What does annotation mean in NGS?
The process of marking specific features in a DNA, RNA or protein sequence with descriptive information about structure or function
