Learning Outcomes - Week 10 - Next generation sequencing

Question 1

Explain basics of next generation sequencing (NGS)

Answer 1

Question 2

What are the main differences between Sanger sequencing and NGS

Answer 2

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 is massively parallel, sequencing millions of fragments simultaneously per run. This process translates into sequencing hundreds to thousands of genes at one time

Question 3

Explain what short-read sequencing and technologies are and how they work

Answer 3

Short-read sequencing is currently the most commonly used form of next-generation sequencing (NGS) and has a wide range of diagnostic applications. In these types of sequencing, the genome is broken into small fragments (usually 50 to 300 bases) before being sequenced.

Question 4

Define and describe Paired-end short read sequencing workflow

Answer 4

Paired-end sequencing allows users to sequence both ends of a fragment and generate high-quality, alignable sequence data. Paired-end sequencing facilitates detection of genomic rearrangements and repetitive sequence elements, as well as gene fusions and novel transcripts.

Question 5

What are the advantages and disadvantages of short reads

Answer 5

Advantages:

• Largest throughput
• Minimum cost per base
• Reads of high accuracy Q30 > 75% (what does that mean?)
• Adaptors used to hold the fragment to the substrate
• Barcodes can be added enable multiplexing (samples or cells or individual reads)

Disadvantages:

• Cost– but it’s the cheapest per base
• Turn around time few days
• Lots of data
  Problems?
• Main issue is reassembly of the data

Question 6

What are the long read technologies and applications

Answer 6

While short reads can capture the majority of genetic variation, long-read sequencing allows the detection of complex structural variants that may be difficult to detect with short reads. These include large inversions, deletions, or translocations, some of which have been implicated in areas like genetic disease.

Example Technologies:
* PacBio
* Oxford Nanopore
Typical Characteristics
* Higher error rate ~ 4% on raw data
* Errors are typically random (not clustered)
* Typically no G/C or A/T bias

Question 7

What are the advantages and limitations of long read sequencing

Answer 7

Question 8

Answer 8

e.

Question 9

(Example of a question that is very detailed and will not be asked)

Which of the following is an example of a next-generation sequencing system?

(a) ABI 373 DNA Sequencing Unit.
(b) BigDye Terminator Sequencer.
(c) Spectrum Compact CE Sanger Sequencing System.
(d) NovaSeq 6000 Sequencing System

Answer 9

d