Loading [MathJax]/jax/output/HTML-CSS/jax.js

Genomics for statisticians 2: Next-generation sequencing, polymerase chain reaction

DNA sequencing lies at the core of modern genomic assays. Here, we discuss next-generation sequencing and the related technology of polymerase chain reaction.


Author

Affiliation

Tim Barry

 

Published

Aug. 11, 2020

DOI


Table of Contents


Introduction

DNA sequencing is the process of determining the nucleotide sequence of a DNA segment. DNA sequencing plays a prominent role in modern genomic assays. In this post we cover next-generation sequencing (also known, perhaps more accurately, as second generation sequencing). Next-generation sequencing (in contrast to first generation sequencing) is a type of sequencing based on DNA amplification and synthesis. The vast majority of sequencing data today are generated by next-generation sequencing machines. We also discuss the closely related technology of polymerase chain reaction (PCR), a method for cloning DNA. We start with PCR, as PCR is a crucial step in next-generation sequencing.

Polymerase chain reaction


A schematic of PCR.


Next-generation sequencing

Step 1: Library preparation



Figure 2: Library preparation. The pink and blue bars are short, artificial segments of DNA called adapters. Adapters contain primers for amplification and other subsequences for downstream sequencing. The DNA segment is fragmented, the adapters are ligated, and the fragments are amplified (or cloned) through PCR.


Step 2: Cluster amplification



Figure 3: Cluster amplification. Cluster amplification (in this example) produces four clusters, labeled 1-4. Fragments in a given cluster are clones of one another. Note: the color of the adapters and bound oligonucleotides in Figure 3 does not relate to the color of the adapters in Figure 2.


Step 3: Sequencing



Figure 4: Sequencing. All fragments within a cluster emit the same series of colors (as they are identical), allowing a computer to determine the sequence of the fragment. Note: the color of the adapters and bound oligonucleotides in Figure 4 do not relate to the color of the adapters in Figure 2.


Step 4: Alignment

References

  1. An introduction to next-generation sequencing technology (Illumina, 2015).

  2. Genetics: A conceptual approach (Pierce 2014).

  3. Image 1 source

  4. Images 2 - 5 source

Footnotes