14- Next Generation Sequencing Flashcards
describe the basis of the term ‘next generation sequencing’ with respect to sanger sequencing
sanger sequencing is a low-throughput method involving sequential chain termination of DNA synthesis using ddNTPs. fragments are out through capillary gel electrophoresis and sequenced separately, one at a time
NGS is a high-throughput, massively parallel method that allows for the simultaneous sequencing of millions of DNA fragments
NGS is massively parallel, high-throughput, faster and more cost-effective in the face of larger genomic studies
NGS produces a digital readout, Sanger seq. produces an analogue readout
NGS generates a consensus sequence of many short-reads with overlapping fragments, Sanger seq. produces one long sequence read
name the four stages of the Illumina NGS process
library construction
cluster generation
sequencing by synthesis
data analysis
describe the stage of library preparation for NGS
DNA sample is prepared for sequencing through shearing - broken into fragments of approx. 300bp via restriction enzymes or sonification
shearing is a damaging process - end repair must occur. an enzyme adds complementary nucleotide bases to the single stranded ends, making them double-stranded blunt ends
adenosine overhangs are added - allows for the ligation of an adapter with T overhangs
adapter has a primer binding site and P5 &P7 anchor sites for the library fragment to anchor to the flow cell
describe the stage of cluster generation for NGS
DNA library fragments are flooded along the flow cell and hybridise at random point – however they’re too small to be visualised
library fragments undergo bridge amplification to generate clusters big enough to be visualised
PCR is done directly to the flow cell = template strand is melted and one molecule is amplified into two… exponential doubling until an amplified clonal cluster of one DNA library fragment is formed big enough to be visualised
flow cell loaded on to sequencing platform - sequencing reaction performed on the surface of the flow cell
describe the stage of sequencing-by-synthesis for NGS
involves the 4 nucleotide bases, modified with chain terminators and different fluorescent colour dyes
flow cell with library fragments is flooded with the modified nucleotides
one nucleotide incorporated per cycle using DNA polymerase
flow cell washed - gets rid of unincorporated nucleotides before imaging
colour signal emitted from fluorescence = can determine what base is at what position by the nucleotide bound to it
reversible chain terminator on the modified nucleotides is cleaved using an enzyme - another cycle can begin
sequence is read one base at a time, all the images of a cluster are sequentially imaged and converted to nucleotide base call by software
describe the stage of data analysis with NGS
NGS produces short-read sequences
can piece the short-read sequences together and compare it to a a reference genome to identify mapping locations on our sequence
genetic variants can be identified when the produced NGS sequence deviated from the consensus sequence
why the interest in whole exome sequencing?
exome considers the protein-coding exons of the genome - make up 1-2% of the genome
majority (80%) of pathogenic mutations are protein coding
more efficient to sequence the parts of the genome we’re interested in - sequencing the exome takes less time than the whole genome
how do we perform WES?
perform target enrichment using baits
RNA baits are synthesised as complementary sequences to the target exon
RNA baits mixed with library DNA sample - hybridise with complementary exon sequences
baits have biotin tags - streptavidin is used to purify the baits by removing biotin
RNA is digested by an enzyme leaving an enriched library of just the target exon
define whole exome sequencing/WES
technique that sequences all the protein-coding regions in a genome
what are the three methods for WES data analysis?
sequence read alignment = aligning the reads to the reference genome
variant calling = identifying variants from sequence read alignment comparisons
variant annotation - identifying the functional significance and genetic impact of a variant
what is the use of WES?
can identify a disease-causing gene within a family - compare variant profiles of affected individuals depending on mode of inheritance
what are the principles of NGS applications with RNA-sequencing
RNA sequencing analyses RNA sequences in cells and tissues
- explore gene expression of many genes within a tissue, find out what specific genes are doing within a tissue
- measure gene abundance - more readouts means more genes are being expressed
- compare gene expression levels before and after treatment
- discover isoforms of genes, their expression and regulation
