Next generation sequencing

Question 1

State the basic steps involved in sangar sequencing

Answer 1

1. DNA templates go through enzymatic reaction
2. Products of enzymatic reaction separated by size via gel or capillary electrophoresis
3. Detection of fluorescently labelled ddNTPs at end of each elongated strand sequence
4. Re-contruction of original template sequence using fluorescently labelled ddNTPs

Question 2

Explain the process of the enzymatic reaction involved in sangar sequencing

Answer 2

1. Clonal population of DNA templates are placed in a reaction mixture with an oligonucleotide primer, DNA-dependent DNA polymerase and deoxyribonucleoside triphosphates (dNTPs) and fluoresecently labelled dideoxyribonucleoside triphosphates, ddNTPs.
2. Reaction mixture heated up to dentaure the DNA templates to make them single stranded
3. Oligonucleotide primer binds to the single stranded DNA primers to form a partial duplex
4. DNA-dependent DNA polymerase binds to partial duplex to form initiation complex
5. DNA-dependent DNA polymerase then begins to synthesise new complementary strand from the position of the primer by incorporating dNTPs into new strand
6. Eventually DNA-dependent DNA polymerase will incorporate one of the fluorescently-labelled ddNTPs at which point it won’t be able to add any other nucleotides into the newly sythesised strand
7. Elongation of other strands continues until a ddNTP has been incorporated into every newly sythesised strand thus stopping the reaction.

Question 3

Explain the processes that occur after the enzymatic reaction in Sangar sequencing

Answer 3

1. After enzymatic reaction you separate the products of the reaction by size using capillary/gel electrophoresis
2. Each fragment that is produced represents a newly sythesised strand with a terminal fluorescently-labelled ddNTP at a different position so each fragement is scanned to identify teh colour fluorescence it gives off
3. Once you identify the colour fluorescence you can identify which ddNTP was incorporated at that particular position and use that information to re-contruct the DNA template sequence

Question 4

Name the 4 steps involved in next generation sequencing

Answer 4

• DNA library Construction
• Cluster Generation
• Sequencing-by-synthesis
• Data analysis

Question 5

What is a DNA library?

Answer 5

A collection of random DNA fragments of a specific sample to be used for further study, e.g. next generation sequencing

Question 6

Explain the process of DNA library construction

Answer 6

1. Obtain DNA sample, usually from a patients blood, and then fragment that DNA into small pieces in a process called shearing
2. Shearing of the DNA produces single stranded overhangs at the end of the DNA fragments which are repaired in a process called end-repair
3. You then add Adenine nucleotides to opposite ends of each strand of the DNA fragment, to produce adenine overhangs, in a process called A-tailing
4. These Adenine overhangs then allow for adapters with thymine overhangs to ligate to the both strands of the DNA fragments

Question 7

What structural elements do the adaptors have that allow the DNA libraty fragemnts to be sequenced using next generation sequencing?

Answer 7

• P5 and P7 anchors on the end to allow for attachment of library fragments to the flow cell
• Sequencing primer binding sites which allow for the hybridisation of primers to the DNA library fragment

Question 8

Explain the process of cluster generation

Answer 8

• Involves Hybridisation of the DNA library fragments to the flow cell
• On the bottom of the flow cell there are oligonucleotides which match one of the 2 anchor sequences on the end of the adapator sequences attached to the DNA fragments either P5 or P7.
• These oligonucleotides bind to the complementary adaptor sequence on the DNA fragments causing the DNA fragments to become immobilised on the flow cell.

Question 9

Why do the DNA fragments need to be amplified when hybridising to the flow cell?

Answer 9

DNA fragemnts need to be amplified because without amplification they are too small to be seen/measured on the flow cell

Question 10

What is the name of the process used to amplify the DNA fragements on the flow cell?

Answer 10

Bridge amplification

Question 11

How does bridge amplification lead to the amplification of the DNA fragements on the flow cell?

Answer 11

1. Once the DNA fragments are attached, the fragment bends over and the adaptor on top of the fragment attaches to its complementary oligonucleotide, on the flow cell, to form a bridge-like shape.
2. A primer and a DNA polymerase bind to the fragment and the polymerase produces a strand complementary to the DNA fragment (reverse strand)
3. Once the reverse strand is produced you now have a double stranded DNA strand. This double stranded DNA strand is denatured so now you have the forward strand (original strand) and the reverse strand (complementary strand
4. The 2 strands then separately attach to their complementary oligonucleotides on the flow cell
5. This process happens across the flow cell creating clusters of amplified DNA fragments

Question 12

Explain the process of sequencing-by-synthesis

Answer 12

1. Before this process begins, all of the reverse strands (complementary strands) are washed away leaving only the forward strands on the flow cell
2. Primers bind to the forward strand and a DNA polymerase adds a single fluorescently-labelled deoxyribonucleoside triphosphate (dNTP) which extends the sequence by one nucleotide from the primer
3. Once the dNTPs have been added to the forward strand the flow cell is washed
4. The fluorescence emitted from every single position on the flow cell is scanned by the machine so it is able to identify which dNTP has been added to each newly synthesised sequence
5. The flow cell is then washed with an enzyme that cleaves the terminator chemical group allowing for the cycle to be repeated
6. This cycle is then repeated until every single sequence on the flow cell has been scanned entirely

Question 13

What data is produced from the next generation sequence machine about each sequence that it “re-constructs?”

Answer 13

• ID number which shows the position of each cluster on the flow cell - normally a coordinate
• DNA sequence itself
• Whether strand is positive or negative
• Thread quality score - how confident the machine is that it has chosen the correct base call

Question 14

How can Next generation sequencing data be analysed?

Answer 14

• Short read sequences from the sequencing machine are re-assembled to form the entirety of the original DNA sequence that was extracted from the patient
• Location of Short read sequences within the reference genome can also be identified
• Short read sequences can also be used to generate a consensus sequence, a sequence that tells you the nucleotide most likely to be present at every position within the sequence.
  
  • This consensus sequence can then be compared with the reference genome and any positions that are different between the 2 show the presence of a genetic variant within the orginal DNA sequence from the patient

Question 15

What are some of the applications of Next generation sequencing?

Answer 15

• Whole genome sequencing
• Whole exome sequencing
• RNA-seq

Question 16

Why do you usually use whole exome sequencing when trying to identify a common variant associated with disease rather than whole genome sequencing

Answer 16

• Around 80% of all pathogenic mutations are protein coding so if we are looking for a particular mutation within a person’s DNA it is more than likely going to be within their exome
• It is more efficient to sequence the exome rather than entire genome
• It’s less expensive

Question 17

Target enrichment is the first step involved in whole exome sequencing. Explain the process of Target enrichment.

Answer 17

1. Take DNA fragments from the DNA library and hybridise them with baits made out of RNA which are complementary to exons of the DNA fragments. Buffer also added to the reaction mixture
2. During hybridisation the baits will bind to their complementary exon sequences on the DNA fragments
3. Once hybridisation occurs the bait-bound DNA fragments will be “captured” using streptavidin coated beads
4. These beads are magnetic so using a magnet will separate the fragments containing exon sequences from the DNA fragments that don’t have these sequences
5. The beads are then washed away and the RNA baits are digested leaving only the DNA fragments containing exons
6. Once you have conducted target enrichment whole exome sequencing proceeds just as next generation sequencing would

Question 18

How can whole exome sequencing data be analysed?

Answer 18

• Short sequence reads produced are aligned against the reference genome sequence to locate their positions within the reference genome
• The short read sequences are then compared with the reference genome to see if there are any differences between them - areas where the short read sequences don’t match the reference genome sequence are areas that contain genetic variants
• Genetic variants analysed to see for exaample, what gene they’re in and whether or not they change the amino acid sequence (non-synonymous)

Question 19

What is RNA-seq?

Answer 19

• Technique used to determine which genes are actively expressed or not being expressed within a biological sample
• You are also able to see changes in gene expression - upregulation and down regulation.

Question 20

Explain the process of RNA-seq

Answer 20

1. RNA is extracted from a collection of cells/tissues
2. The RNA that’s extracted is then filtered for RNA with 3’ poly(A) tails to include only mRNA, filtered RNA doesn’t contain ribosomal RNA (rRNA), and/or filtered for RNA that binds specific sequences
3. This filtered RNA is then converted to cDNA using reverse transcription via the enzyme reverse transcriptase.
4. This cDNA is used to construct a library and specific samples from the library can then be sequenced using next generation sequencing

Question 21

How can RNA-seq data be analysed?

Answer 21

• RNA-seq reads are aligned to the reference genome to identify what genes are expressed in order to produce the RNA extracted from the sample
• Can also be used to quantify the expression levels of a particular gene.
• This is because the amount of reads aligned to each gene is proportional to the expression level of that gene

Question 22

What is third generation sequencing?

Answer 22

• Third generation sequencing involves reading DNA sequences at the single molecule level (Single-molecule sequencing) so you are sequencing the smaple directly rather than having to extract DNA or RNA from it and then sequence that.

Question 23

What are the advantages of third generation sequencing technologies?

Answer 23

• No expensive machine required
• Each flow cell is the machine itself
• Throughput can be changed to desired level (Can be made to sequnce a small or large amounts of a sample at any one time)

Question 24

What are the disadvantages of third generation sequencing?

Answer 24

• Very expensive
• High error rates
• Technology is still developing