single molecule DNA sequencing

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mutations can occur through:

1. errors introduced during DNA replication

2. errors introduced during repair of DNA damage

3. errors introduced by mutagens

different forms of genetic variation

SNPs: single nucleotide polymorphisms, can be silent, missense or nonsense

indels: insertions or deletions of short DNA sequences

CNVs: copy number variations, applying to any DNA length

inversions: reversing the orientation of a segment of a chromosome

translocations: a section of one chromosome is moved and joined to another chromosome

if we could sequence genomes of individual cells, we could…

• estimate the mutation rate in healthy tissues and tumours

• understand evolutionary processes leading to any individual tumour

• understand how specific tumours have acquired transformation and resistance

• provide patients with cocktails of drugs for which the tumour has no current resistance

• monitor development of resistance to keep the drug regime appropriate

problems with single cell sequencing

1. only one cell must be isolated from a mass of tissue

2. only a tiny amount of DNA (just two copies of each gene of interest)

3. amplifying DNA to allow sequencing introduces errors

4. DNA sequencing also introduces errors

what can’t be used to isolate a single cell

• traditional tissue homogenisers break open the cells and mix up their DNA

• classical dissociation enzymes damage cells and lead to mixing of their DNA

how a single cell can be isolated

1. flow cytometry of whole cells

2. flow cytometry if isolated nuclei

3. serial dilution

4. microfluidics

5. laser capture micro-dissection

serial dilution

• simplest method of isolating single cells

• works well for cells already in suspension but can’t be used for cells still within a block of tissue

flow cytometry and sorting

• (FACS): fluorescence activated cell sorting, cells are labelled using fluorescently tagged ABs

• a FACS machine then separates cells, which pass a laser one a time intro collection streams using an electromagnet to push water stream over each collection pot

microfluidics

• movement of tiny volumes of liquid across channels of a specialised silicon chip

• microfluidic platforms can be used to separate cells according to their different physical properties

• only works with cells in suspension

laser capture micro dissection

• a slice of tissue just one cell thick is prepared using a microtome

• tissue is observed with a microscope and a laser is fired at tissue to burn away the tissue around an individual cell of interest

• isolated cell is then removed with fine tweezers

how a whole genome from a single cell is amplified

• degenerate oligonucleotide primed PCR (DOP-PCR)

• isothermal amplification (MDA)

• hybrid methods (PicoPLEX)

degenerate oligonucleotide primer PCR (DOP-PCR)

• PCR methods such as degenerate oligonucleotide primed PCR use random priming followed by PCR amplification which preferentially amplifies specific sites in the genome

• results in low physical coverage but better uniformity of amplification

• uses thermostable polymerases, which are more error prone than traditional polymerases

isothermal amplification

• multiple displacement amplification (MDA)

• isothermal exponential amplification using polymerase at 37 degrees and high processivity and strand displacement activity

• these methods can cover most of the genome but have less uniformity

• introduces less error because it doesn’t use error prone thermostable polymerases

hybrid methods

• initial isothermal pre amplification in which common sequences are added, followed by PCR amplification using those sequences

• intermediate coverage and uniformity when compared to pure PCR and isothermal methods

• starts with isothermal polymerase, then switches to thermostable enzyme

how to ensure the variants are real and not artefacts of amplification

• thousand mutations are introduced during single cell WGA in a 3 Gb human genome

• somatic variant calling requires coverage of a variant allele at a rate that exceeds the sum of the amplification and sequencing error rates

• one way is to sequence 2 or 3 nearby cells

• if mutation is present in all of them, almost certainly a genetic mutation

• should also compare results with bulk sample

why direct sequencing from one DNA strand isn’t possible

• fluorescent signal from single nucleotides is too weak for conventional flow cells to detect

• addition of a single nucleotide will not generate enough pH signal to be detected by ion torrent or light emission from pyrosequencing

• all of these conventional HTS methods require an amplification stem: their main limitation

advantages of single molecule sequencing over other HTS methods

• can avoid amplification step (saves time and cost)

• can use tiny amounts of sample and reagents

• no longer looking at average sequence of sample

• can separate individual DNA sequences from a complex sample of mixed DNA so enables identification of numerous individuals from one sample

Oxford Nanopore sequencing

• sample containing DNA is pipetted into special chip

• enzyme unravels the DNA into single strands and guides the DNA towards a tiny pore in the membrane

• voltage is applied across the chip, pulling the DNA through the pore and into lower compartment

• as each base moves through the pore, flow of ions in the opposite direction is impeded

• each base is of a different size and shape so interferes with the flow of ions to different extents

• changes to the flow of ions is measured by a sensitive electrical current meter

• ATCG each induce characteristic change in the current

• sequence is read in real time as DNA moves through the pore creating a pattern in the ion flow

• computer calls the bases based on current pattern

• read length can be hundred of thousands of bases

how Oxford Nanopore sequencing works

• hairpin is added to one end of the target DNA so that both strands of the DNA can be read through the same pore

• second read of the same sequence improves the accuracy over a single read

Pacific Bioscience sequencing

• aims to achieve direct visualisation of fluorescence from a single nucleotide

• can’t be achieved in conventional systems due to excess background fluorescence

• use 4 different colours of fluorescent tag bound via phosphate group

• tag is cleaved and released when these incorporate into DNA

Pacific Bioscience sequencing uses zero mode wave guides

• uses thousands of tiny wells which they call zero mode wave guides

• lit from underneath by a laser which excites the fluorophores on the nucleotides

• because the wells are so narrow, light can only penetrate from very bottom nanometres of well

• single DNA polymerase enzyme is anchored to bottom of well

Pacific Bioscience sequences nucleotide incorporation in real

• as each base is incorporates, it is held within the active center of the DNA polymerase enzyme for several milliseconds

• long enough for the fluorescence of the bottom of the well to increase

• can be detected by increased fluorescence

camera monitors the sequence of the flashes of colour

• camera reads the flashes of colour that appear on the underside of the plate as each nucleotide is incorporated

• system can sequence at the same rate as nucleotide incorporation in real time

• reactions occur in thousands of wells per chip in parallel

• system is capable of very long read lengths

continuous circular sequencing with 2 hairpin use

• 2 hairpin adaptors are typically attached to each end of the target DNA

• allows continuous circular sequencing of the same DNA fragment, round and round again

benefits for single cells sequencing

• genome sequence of individual cells can be performed without the amplification step

• saves time and reagents and avoids the introduction of errors through the amplification process

overcoming the accuracy limitation

• simple way of getting around the Oxford Nanopore and Pacific Biosciences accuracy limitation

• simple repeat the read of the same target many times

• each time the read is repeated, accuracy goes up