Lecture 6 Flashcards
What does Next generation sequencing refer to?
How do they differ from first generation technologies?
Large-scale DNA sequencing technology that allows for querying the entire genome (whole genome), the exons within all known genes (whole exome), or only exons of selected genes (target panel).
Cheaper, faster, considerably smaller sample size needed and higher accuracy. Also, because it is quicker and cheaper, it is possible to sequence more repeats than with Sanger sequencing. More repeats means greater coverage, which leads to a more accurate and reliable sequence, even if individual reads are less accurate for NGS.
What are the four NGS sequencing technologies?
Illumina (Solexa) sequencing
Roche 454 sequencing
Ion Torrent: Proton / PGM sequencing
SOLiD sequencing
Describe the Illumina (Solexa) sequencing technology
Vast number of short reads sequenced in a single stroke (100-150 bp reads are used)
Four basic steps: library preparation, cluster generation, sequencing, data analysis
Describe the Roche 454 sequencing technology
Pioneer NGS platform launched in 2005
Can sequence much longer reads than Illumina (up to 1kb)
Steps: Library preparation Emulsion PCR PTP loading Pyrosequencing reaction
Describe the Ion Torrent: Proton / PGM sequencing technology
Method based on fact that addition of a dNTP to a DNA polymer releases an H+ion.
Steps:
DNA fragmentation (200-400bp)
Emulsion PCR
Sequence on Ion chip
Describe the SOLiD Sequencing technology
Sequencing byOligoLigation andDetection
Run requires ∼5 days and produces 3–4 Gb of sequence data
Average read length of 25–35 bp
Steps:
Library preparation
Emulsion PCR
Polony sequencing
What are two long-read sequencing/ third generation sequencing technologies?
Oxford Nanopore
PacBio - SMRT DNA Sequencing
What are two features of long-read sequencing/ third generation sequencing technologies?
Single molecule sequencing
Real time sequencing
What are the advantages of long-read sequencing/ third generation sequencing technologies?
No need for amplification
- PCR-free
- PCR based
Simplified and less ambiguous genome assembly
Ability to span repetitive regions (eg. transposons, satellites, gene duplications)
Identification of large structural variation (SV) and detection of epigenetic marks
What are 4 applications of Next generation sequencing technologies?
DNA sequencing
-Genome wide variant profiling
RNA-Seq
-Deep sampling of transcriptomes
ChIP-Seq
- Genome-wide mapping of
DNA-protein interactions
Bisulfite-Seq
-Detection of DNA methylation
What options comprise NGS DNA Sequencing?
Whole genome sequencing (WGS)
Whole Exome Sequencing (WES)
NGS targeted sequencing
What is the aim of Whole genome sequencing (WGS)?
What does it involve?
Aim: characterization of the entire genome of an individual
Fragments are generated from the whole genome
Identification of SNPs, CNVs, duplications, inversions, and other forms of structural variations
What is the aim of Whole Exome Sequencing (WES)?
What is the reasoning for this aim?
Aim: sequencing all protein coding regions of genes in genome
Although very small percentage of the human genome encodes for protein, about 1%, exons harbor about 85% of the mutations with large effect on disease development
What is the aim of NGS targeted sequencing?
What does this use?
What does this require?
Aim: sequence specific areas of the genome for in-depth analyses more rapidly and cost-effectively thanWGS.
Uses deep sequencing to detect known and novel variants within your region of interest
Generally requires less sample input and produces a smaller amount of data than WGS
What are the advantages of RNA Sequencing?
Provides a comprehensive view of the transcriptome
Not dependent on prior sequence knowledge
Detection of structural variations such as gene fusions and alternative splicing events
Quantifies discrete read counts aligned to a particular sequence
Increased specificity and sensitivity
Allows differentiation of isoforms
Data analysis more complex
