19.02.14 NGS technologies Flashcards
What is library preparation?
All NGS platforms need to prepare DNA/RNA libraries. To do this, you need to fragment the DNA/RNA and ligate adaptors and indices. If not doing WGS (so exome, panels or single genes) also need an enrichment step to capture the region of interest.
Enrichment methods
- Multiple kits available - which ones depends on sample type (FFPE, genomic DNA), technology you’re using, panel size, TAT and cost.
- Two main types - amplicon (PCR) and hybridisation-based methods.
Enrichment method - Amplicon (PCR based)
- Use long range PCR to amplify large genomic regions containing multiple exons
- Kits include Truseq/Nextera XT (illumina)
- The produce sequencing libraries from these fragments, before ligating with adaptor oligonucleotides for sequencing
Advantages - cheaper, need less starting material and faster
- Good for small regions of interest (single gene/small panel)
- Suitable for FFPE
Disadvantages - Can get preferential amplification
- Amplification can introduce artefacts
- Hard to multiplex primers to look at multiple regions
- Read depth can’t be used for CNV analysis
Enrichment method - Hybridisation
- Rely on solution-based capture of target regions
- Common kits are agilents SureSelect and Illuminas Trusight
- Method involves fragmentation of genomic DNA, tagging of fragments with sequence-ready adaptors and barcodes, and then capture of the libraries using DNA/RNA-based oligonucleotides
- The oligos anneal to specific genomic regions to result in a range of captured fragments that are representative of the entire region of interest
Advantages - More uniform coverage
- Better coverage of GC rich regions
- Read depth can be used for CNV calling
- Good for large panels/exomes
- Suitable for FFPE material
Disadvantages - Need more starting material
- More expensive
- Longer preparation time
Truseq/Nextera XT (illumina) - Amplicon (PCR) enrichment method
- Can amplify fragments over 300bp in size
- Transposomes cleave dsDNA breaks in genomic DNA at random
- They then ligate adaptor oligonucleotide to the 5’ end of the fragment
- Can use different transposons to get fragments with different sequence tags
- The do a limited cycle PCR reaction to add indexes and full adaptor sequences ready for sequencing
Agilent SureSelect - Hybridisation enrichment method
- Start with gDNA and shear it into small fragments
- Prepare library with sequencer specific adaptors and sample specific barcodes
- They are then hybridised with biotinylated RNA library baits
- Select targeted regions using magnetic streptavidin coated magnetic beads
Wash beads and digest the RNA - Amplify and load on sequencer
NGS platforms
- Second generation platforms have an amplification step prior to sequencing
- Third generation platforms don’t have this
- Examples of second generation platforms include 454 by Roche, SOLiD by life technologies, Ion torrent by ThermoFisher and MiSeq/HiSeq/NextSeq by Illumina
- Most platforms use sequencing by synthesis, except for SOLiD - which is a cyclical process which occurs in a flow cell, process involves nucleotide addition, washing and signal detection.
NGS platform - Illumina workflow (MiSeq, HiSeq, Nextseq)
1) Library preparation
- DNA is fragmented, enzymatic trimming, and adenylation of the fragment ends and ligation of specific adaptors
- Flow cell is flat glass with eight microfluidic channels, each covered in adaptor sequences complementary to the library adaptors
- Precisely diluted solution of library fragments is amplified in situ on the flow cell surface by the use of bridge amplification (creates clusters)
- The clusters of DNA fragments are amplified in a process called cluster generation, resulting in millions of copies of single-stranded DNA.
- Chemical step to release fragment ends that have the same adaptor
- This is then primed with a complementary synthetic DNA (primer) to provide free 3’-OH groups (which are extneded in next seq reaction)
- Sequencing by synthesis (SBS) - chemically modified nucleotides bind to the DNA template strand through natural complementarity
- Each nucleotide contains a fluorescent tag and a reversible terminator that blocks incorporation of the next base (terminator at 3’-OH position of the ribose sugar)
- The fluorescent signal indicates which nucleotide has been added, and the terminator is cleaved so the next base can bind
- After reading the forward DNA strand, the reads are washed away, and the process repeats for the reverse strand. This method is called paired-end sequencing
NGS platform - ThermoFisher - Ion Torrent
- Principle is based on release of a hydrogen ion as a by-product of the incorporation of a nucleotide into a strand of DNA by a polymerase
- Uses a chip with a high-density array of micro-machined wells - each well holding a different DNA template
- Below well is an ion-sensitive layer, and below that a proprietary ion sensor
- Library molecules are clonally amplified by emulsion PCR
- Each vesicles contains one ion sphere, one library molecule and all PCR reagents
- One PCR primer is bound to the sphere and the other is in solution
- Individual library molecules are amplified to millions of identical copies, all bound to sphere
- individual beads/spheres are then sequenced
- Ligation of fluorescently labelled hybridisation probes to deduce sequence 2 bases at a time
- As it uses two base encoding, it provides inherent error correction
