SEQUENCING Flashcards
What sequencing technique uses irreversible chain-termination?
Sanger Sequencing
Explain Sanger Sequencing methods, how you analyse the data and how you might apply this.
You add a mixture of di-deoxynucleotides and deocxynucleotides, primers and RNA polymerase to a mixture. Within the mixture there is a single-standed template strand, extracted from your gene of inerest.
The primer requires knowledge of the template sequence, because it has to be complimentary.
Primer binds to target region. Then PCR goes along the strand, termniating each time a ddNTP is incorporated into the sequence (binds to corresponding base).
You repeat this for each base (4 cycles in total)
Then you use gel electrophoresis to separate the different fragments, and each of the dNTPs are labelled with a fluorophore, so you identify this with a chromatogram. Thus you can tell the order of the bases by how far down the fragments travel (shortest will travel furthest).
Most accurate method, but can only be used to sequence singe genes because you will miss large indels - for example, testing whether someone has inherited a single mutation in a risk gene such as CFTR (cystic fibrosis)
How does NGS compare to SS?
stephen hawking!
NGS is:
- higher throughput because process is highly parallel
- SS = 500-800bp read length; NGS read length is ~300bp per reaction but generates 1000s of mbps per reaction
- NGS is more time and cost effective
- NGS can sequence a whole genome, for example to identify long-range indels missed by SS
- NGS is frequently used to identify mutant/abnormal embryos in IVF (indels or amplifications)
- NGS can identify locus of genetic neurological disorders e.g. ALS - lots of recent experiments showing that NGS can identify disease-associated variants in over 30 causative genes; SS would be used to identify the single variants within already known genes eg identifying SNPs in CRTF gene for CF
- NGS is slightly less accurate
- NGS uses reversible chain termination, SS uses irreversible chain termination
- NGS doesn’t require a priori knowledge of the genome of interest :)
- NGS can identify genome-wide SNPs; SS can only identify single SNPs within a gene
- NGS can detect genome-wide copy number changes for hundreds of cells, SS is only looking at one specific gene from one cell!
Outline the stages of NGS.
clue - reversible chain terminating , FG (detection)
- Sample prep (DNA extraction and fragmentation by enzymes, then adaptor regions are added - contain cluster regions and sequencing regions).
- Cluster Generation: Sample DNA fragments bind to complementary oligos on flow cell via H bonds between adapter region (fragment) and primer region (oligo). PCR creates ddDNA, then the template strand is cleaved leaving only ssDNA bound to the flow cell via the oligo. Amplified via bridge amplification the PCR of forward and reverse strand repeated until you have ssDNA corresponding to template strand. Within the same cluster, fragments are from the same section of DNA. This amplifies the fluoresent signal measured in step 3.
- Sequencing by Synthesis. Flow cell added to MiSeeq with buffer and PCR ingredients. Then fluorescent nucleotides with a fluorophore bind to the complementary bases on each fragment. These nucleotides have a chain terminator on them; only one fragment is incorporaed in each cycle. The light emitted by each fluorophore is measured with TIRF (wavelengths of each 4 colours correspond to specific base - this is 4 channel-sequencing). Then the chain-terminator is cleaved off (it is reversable), and the process is repeated until all bases have been identified.
- short overlapping regions between the fragments are aligned to put the entire sequence together
If you are looking for a particular SNP, the number of sequence reads containing the SNP will tell you if it’s het or homo. If it’s only present in one sequence it’s probably an artefact
What are the Short Range sequencing methods, and why is LRS sometimes more useful?
Sanger, 404 Pyrosequencing and NGS.
Sequencing long range samples (such as the human genome) with SRS requires lots of fragmentation and amplification which can introduce bias to the sequence. Also, there might be a lack of overlaps between regions. SRS misses SVs.
nanopore is better because it picks up on long deletions, over 10 thousand bps long, which SRS would often miss. This was the case with the 2.5kb heterozygous deletion on chromosome 10 of the human genome. These are known as structural variants (long range indels; translocations; substituions) and requires long reads to cover lots of repetitive regions (as opposed to single nucleotide polymorphisms - single nucleotide deletions/additions- which are picked up in SRS). This could help identify SVs in the genome of cancer patients, revealing personalised therapeutic targets.
Outline nanopore, and give an example of how you might use it.
- nanopore protein attached to DNA helicase which unravels dsDNA into ssDNA.
- membrane is negatively charged which attracts DNA through the nanopore protein; when bases pass through the nanopore they disrupt/block the ionic current in distinct/characteistic patterns (individual to each base; bases each different sructure)
- measuring the change in ionic current over time tells you the sequence of DNA
- nanopores read lengths are over 10kb. It is different from DNA sequencing methods because of the SS passing through the protein; there is no synthesis of new DNA.
the MinION is small and handheld, can generate reads as long as 800-900 kbs. This can be used for sequencing full-length genes used in taxonomy/epidemiology:
- Identifying SVs in the germplasm to map its genetic variability
- unlike SNPs, which often dont change the AA (and therefore the alleleic value), SVs can give rise to complex alleic values; this diversity would not be captured by SRS
- This would provide markers for ideal trait selection, or allow for genetic editing methods to modify germplasms to produce traits in a species which does not usually host those alleles.
- This paper used nanopore to locate and sequence the red flesh trait on the apple physical genome (2020 paper, published in Plant MEthods, Lopez et al)
What is phasing, and why is it useful?
Identifying inheritence - you can trace SNPs in coding and non-coding regions to see what bases are on the same chromosome
Haplotypes are regions of DNA inherited from both parents , so you can match up the haplotypes wth LRS (but not SRS) and find haplotype inheritence (mat or pat)
How might you confirm gene editing and identfy off-target effects?
Nanopore!
You can visualise any SVs not in the target region - this would be an off-target effect.
You can identify deletions which would mean NHEJ has happened. If not, proabably HR.
Also you could use Sanger if you know the specific gene you’re taregting, or a diagnostic PCR then separate with gel electrophoresis
what are the limitations of nanopore?
- takes a long time
- lower read acuracy than SRS
- not good for detecting SNV
what main advantage does nanopore have over NGS and SS?
Doesn’t depend on PCR , potetial for very long read lengths
what it read depth?
The number of bases sequenced and alligned at a given reference base position
note raw read depth can refer to just how many bases can be sequenced
