Next generation sequencing

Question 1

What is Next generation sequencing

Answer 1

group of automated techniques used for rapid DNA sequencing

• replaced Sanger sequencing for almost all sequencing techniques in the lab, mainly by these 2 methods:
• whole genome sequencing
• whole exome sequencing

Question 2

State the steps of NGS.

Answer 2

1) DNA library construction
2) Cluster Generation
3) Sequencing-by-synthesis
4) Data Analysis

Question 3

Describe the construction of the DNA library.

Answer 3

Shearing:

DNA sample from species of interest broken down into fragments either chemically, enzymatically or physically (sonication- sound waves fired at DNA breaking it up)

A-Tailing:

Adenine (A) nucleotide overhangs added to end of DNA fragments to repair end of sheared DNA fragments

Adapter Ligation:

Adapters with thymine (T) overhangs can be ligated to the DNA fragments

DNA Library:

Final DNA library containing billions of small, stable random DNA fragments representative of our original DNA sample

Question 4

What is the Importance of adapters in DNA library construction?

Answer 4

adapters on the ends of the fragments contain the essential components to allow the library fragments to be sequenced e..g:

• sequencing primer binding sites for hybridisation
• P5+P7 anchors for attachment of DNA library fragments to the flow cell

Question 5

Describe cluster generation.

Answer 5

• DNA library fragments hybridised to the flow cell
• Need to amplify the fragments to a bigger size to be able to visualise them:

Bridge Amplification:

Billions of DNA library fragment clusters generated originating from single DNA library molecules, now big enough to be visualised

Flow cell is now ready to be loaded onto the sequencing platform to perform the sequencing

Question 6

Describe Sequencing by Synthesis.

Answer 6

the synthesis of DNA is directly monitored to deduce the base sequence:

Modified bases (ACTG) incorporated with:

> chain terminators

> different fluorescent colour dye

• DNA polymerase incorporates one nucleotide
• Flow cell wash
• Image 4 bases (digital photograph) on surface of flow cell depending on colour of base
• Cleave chain terminator chemical group with enzyme
• Repeat (n) times for full length sequence

Question 7

What happens to the images produced in sequencing by synthesis?

Answer 7

Sequencing machine converts each cycle image into a DNA base call, which are short read sequences, one for each of our DNA clusters hence on for each of our library fragments on the flow cell

All of the short read sequences represent the original DNA library sequenced

Question 8

Describe data analysis.

Answer 8

The short read sequences (base calls) from the sequencing machine need to be re-assembled to find out the sequence of the original DNA sample from start to finish:

• Location of sequence reads on the reference genome are mapped to generate a consensus sequence of our original DNA sample
• We can compare our consensus sequence against the human genome reference and look for genetic variants
• Dedicated software and bioinformatics tools will achieve this

Question 9

What is the Difference in the readout between NGS and Sanger sequencing?

Answer 9

NGS:

• digital readout
• consensus sequence of many reads

Sanger Sequencing:

• analogue readout
• one sequence read

*The critical difference between Sanger sequencing and NGS is sequencing volume. While the Sanger method only sequences a single DNA fragment at a time, NGS sequences millions of fragments simultaneously per run.

Question 10

What is exome sequencing?

Answer 10

Exome Sequencing

• Target Enrichment used to capture target regions of interest (DNA library) with baits (RNA sequences complementary to exons)
• Baits hybridise with DNA library then these are captured with streptavidin coated magnetic beads which are attached to magnets to pull those fragments out
• Wash beads and digest RNA, leaving us with enriched library

Question 11

Give the applications of NGS.

Answer 11

Exome Sequencing

RNA Sequencing

Question 12

Describe exome data analysis.

Answer 12

After sequencing exome, align sequence reads obtained against the human reference genome to see if there are any genetic variants through variant calling

If there are any variants, there is variant annotation to locate the variant and find what they’re gene it is in

Question 13

What is RNA sequencing?

Answer 13

A method used to determine the actively expressed regions of a genome within some specific cell population, tissue sample, or organism:

• RNA converted to cDNA by reverse transcriptase prior to library construction
• Sample then sequenced as if it was DNA, but actually it is representing the RNA of our sample

Question 14

Describe RNA sequencing data analysis.

Answer 14

RNA sequence reads aligned to the reference genome to see where they map in terms of what genes

The number of sequencing reads produced from each gene can be used as a measure of gene abundance, allowing for the quantification of the expression levels (if gene has no reads, then it is not being expressed at all)

With appropriate analysis, RNA sequencing can be used to discover distinct isoforms for genes which are differentially regulated and expressed

Question 15

What is third Generation sequencing?

Answer 15

ability to sequence single molecules of DNA (single molecule sequencing)

Example:

-Oxford Nanopore sequencing

DNA passes through a nanopore and base sequence is converted into an electrical current

Question 16

What are the advantages of third generation sequencing?

Answer 16

No expensive machine required

Each flowcell is the machine itself

Scalable to required throughput

Question 17

What are the Disadvantages of Third Generation Sequencing?

Answer 17

Very Expensive

High error rates

Technology still developing