bioinformatics lecture 2

What is Next-Generation Sequencing (NGS)?

large scale parallel sequencing where millions of DNA fragments are sequenced simultaneously.

Why did NGS replace Sanger sequencing for genome-scale projects?

It provides far higher throughput at much lower cost per base, enabling whole-genome and population-scale sequencing.

How does NGS connect to genome assembly concepts from Lecture 1?

NGS produces short reads, which makes genome assembly computationally challenging.

How did sequencing cost trends compare to Moore’s Law?

Sequencing costs dropped dramatically faster than Moore’s Law, which predicts computing power doubles every two years.

What biological advances became possible due to cheap sequencing?

Whole-genome sequencing, population genomics, and personalised medicine.

What tradeoff do most NGS technologies make compared to Sanger sequencing?

They trade long read length for massive throughput and speed.

How do Roche 454 and Illumina sequencing differ at a high level?

Roche 454 produces longer reads but lower throughput, while Illumina produces short reads with very high throughput.

What core features are shared by most NGS technologies?

Random DNA fragmentation, short reads (100–300 bp), amplification, sequencing-by-synthesis, and no gel electrophoresis.

Why is amplification required in NGS?

A single DNA molecule produces a signal too weak to detect reliably.

What are the main steps of Illumina sample preparation and amplification?

DNA fragmentation, adaptor ligation, size selection, and bridge amplification on a flow cell.

What is a flow cell in Illumina sequencing?

A surface where adaptor-ligated DNA binds and undergoes bridge amplification to form clusters.

What is a cluster in Illumina sequencing?

Thousands of identical copies of a single DNA fragment in one location on the flow cell.

What is bridge amplification?

A process where DNA fragments bend and replicate on the flow cell surface to form clusters.

What is reversible terminator technology in Illumina sequencing?

Modified nucleotides with fluorescent labels and 3′ blocking groups that allow only one base to be added per cycle.

Why does Illumina sequencing enforce one base incorporation per cycle?

To improve accuracy and ensure clear signal detection.

Describe one full Illumina sequencing-by-synthesis cycle.

Base incorporation, laser excitation, colour detection, cleavage of dye and blocker, then repetition.

How have Illumina read lengths changed over time?

They increased from about 35 bp to as long as 300 bp.

Why does read quality decrease toward the end of Illumina reads?

Due to phasing, where clusters lose synchrony during sequencing cycles.

What is phasing in Illumina sequencing?

The loss of synchronisation within a cluster, causing mixed signals and reduced accuracy.

What is paired-end sequencing?

Sequencing both ends of the same DNA fragment.

Why are paired-end reads useful?

They improve genome assembly, read alignment, and variant detection.

What is FastQC used for?

Assessing sequencing quality such as base quality, GC content, and adapter contamination.

What is GC content and why is it useful?

The percentage of G and C bases, used to identify genes and distinguish exons from introns.

What types of sequencing applications commonly use NGS?

Whole-genome sequencing, exome sequencing, and RNA sequencing (RNA-seq).

What major impacts has NGS had on genomics?

Increased data volume, reduced cost, routine resequencing, and population-scale studies.

What cost milestones were highlighted for genome sequencing?

The $1000 genome and the emerging $100 genome.

What are major technical challenges of NGS?

Short reads, higher error rates, phasing, and uneven sequencing depth.

Why do short reads make genome assembly difficult?

They provide limited context, especially in repetitive regions.

Which genome regions are particularly difficult to assemble using NGS?

Repetitive sequences, GC-poor regions, telomeres, and centromeres.

What type of sequencing error is most associated with Illumina platforms?

Substitution errors.

What key shift did NGS create in bioinformatics workflows?

The main challenge moved from data generation to data analysis.

What is a reference genome?

A standard genome sequence used for alignment, annotation, and variant calling.

Which human genome builds were listed and which is current?

hg19/hg37 are older; hg38/GRCh38 is the current standard.

Which databases provide access to reference genomes?

UCSC Genome Browser, Ensembl, and NCBI.

Compare Sanger sequencing and NGS in terms of speed, cost, read length, accuracy, and throughput.

Sanger is slow, expensive, long-read, highly accurate, and low-throughput; NGS is fast, low-cost, short-read, lower per-read accuracy, and massive throughput.