Next Generation Sequencing Flashcards
What was the human genome project?
Human Genome Project (1990 - 2003)
3 billion base pairs long
All done with traditional Sanger Sequencing
Unravelled the first Human Genome Sequence to drive genetics research
3 billion dollars cost
We can now achieve this amount of sequencing in as little time as one day!
What is PCR?
Polymerase Chain Reaction (PCR)
Fundamental principle for any DNA sequencing application
PCR is used to amplify a specific region of DNA; primers flank the region you want to amplify.
Each cycle doubles the amount of DNA copies of your target sequence
Amplify enough DNA molecules so that we have sufficient material to sequence or for other DNA applications
What is Sanger sequencing and some of its features?
Invented by Fred Sanger in 1977 Cycle Sequencing Based on PCR Modified nucleotides Chain Terminators Nucleotide specific colour tag
A small proportion of the free nucleotides are modified this way to allow every base in the sequence to be read
One reaction = one sequence Up to 800 bp per reaction Accurate (99.99%), Slow and low-throughput Used predominantly until the late 2000s Costly ££££
What are the four steps of next-generation sequencing?
Four step process
1. DNA library Construction 2. Cluster Generation 3. Sequencing-by-synthesis 4. Data analysis
What is DNA library construction?
A DNA library is a collection of random DNA fragments of a specific sample to be used for further study; in our case next generation sequencing
The DNA can come from just about anywhere, but in human genetic research generally it’s derived from patients blood.
What are the steps for DNA library construction for next-gen sequencing?
Step 1: DNA library construction:
- In the wet lab – first we need to prepare the DNA sample for sequencing
- Essentially the DNA is chopped into small fragments (typically 300bp ). This is called shearing
- This can be achieved chemically, enzymatically or physically (sonication)
Step 2:
1. We have to repair the end of the sheared DNA fragments called end repair
2.Adenine (A) nucleotide overhangs are added to end of fragments
3.Adapters with Thymine (T) overhangs can be ligated to the DNA fragments with the adenine nucleotides
The end result is the DNA library of literally billions of small, stable random fragments representative of our original DNA sample
Step 3:
Adapters contain the essential components to allow the library fragments to be sequenced
Sequencing Primer binding sites
P5 and P7 anchors for attachment of library fragments to the flow cell
Describe the cluster generations step of next-gen sequencing.
Step 2: cluster generation
- Hybridise DNA library fragments to the flow cell - Hybridization to the flow cell is a Random process - But we can’t measure individual single molecules of our DNA library –too small - We need to amplify the fragments to a bigger size that we can measure - Perform bridge amplification to generate clusters - Many billions of clusters originating from single DNA library molecules - Clusters are now big enough to be visualised - Flow cell is now ready to be loaded on to the sequencing platform to perform the sequencing
Describe the sequencing-by-synthesis step of next-gen sequencing.
Step 3: sequencing-by-synthesis:
Modified 4 bases (ATCG) with:
- Chain terminators
- Different fluorescent colour dye
Sequence each single nucleotide 1 cycle at a time in a controlled manner
Single nucleotide incorporation (DNA polymerase)
Flow cell wash
Image the 4 bases (digital photograph)
Cleave terminator chemical group and dye with enzyme
Repeat (n) times for full length sequence
Camera sequentially images all 4 bases on the surface of the flow cell each cycle
Each cycle image is converted to a nucleotide base call (ACGT)
Cycle number anywhere between 50 – 600 nucleotide base pairs
How do we analyse NGS data?
Short read sequences from the sequencing machine need to be re-assembled like a jigsaw
Mapping locations of our sequence reads on the reference genome sequence
To generate a consensus sequence of our original DNA sample library
In comparing this consensus sequence against the human genome reference and looking for the genetic variants
Dedicated software and bioinformatics tools will achieve this
How do we perform whole exome sequencing?
- Capture target regions of interest with baits which are complementary to the target regions of interests
- We incubate and perform hybridisation reaction
- We capture the targeted regions using streptavidin coated magnetic beads
- We now have an enriched library and we can sequence
- Potential to capture several Mb genomic regions of interest
- Exome would be 50Mb in size
What are the applications for exome sequencing?
ollecting disease affected individuals and their families
Use of NGS in disease gene identification
Perform exome sequencing
Compare variant profiles of affected individuals
Try to identify the variant or mutation shared buy the affected individuals
What is RNA sequencing?
RNA-seq experiments use the total RNA (or mRNA) from a collection of cells or tissue
RNA is first converted to cDNA prior to library construction
NGS of RNA samples determine which genes are actively expressed.
Single experiment can capture the expression levels of thousands of genes
The number of sequencing reads produced from each gene can be used as a measure of gene abundance
Quantification of the expression levels
Calculation of the differences in gene expression of all genes in the experimental conditions
With appropriate analysis, RNA-seq can be used to discover distinct isoforms of genes are differentially regulated and expressed
