Molbio - DNA Sequencing

DNA Fragmentation, Library Preparation, Amplification, Sequencing, Bioinformatics and Data Analysis

Steps of Next Generation Sequencing

DNA Fragmentation

Step in NGS where Targeted DNA is broken into short fragments, usually 100-300 bp in length (mechanical methods, enzymatic digestion)

Library Preparation

Step in NGS involving ligation of adaptors

Amplification

Step in NGS that uses bridge PCR or emulsion PCR

Sequencing

Step in NGS involving matrix such as flow cells in illumina and chips in ion torrent

Bioinformatics and data analysis

Step in NGS involving base calling, read alignment, and variant identification

Roche/454 Life Sciences

A Second Generation Sequencing Technique that uses the principle of pyrosequencing which is a detection of pyrophosphate that is released during DNA synthesis

Illumina/Solexa

A Second Generation Sequencing Technique that uses the principle of sequencing by synthesis

Applied Biosystems Supported Oligonucleotide Ligation and Detection

A Second Generation Sequencing Technique that uses the principle of sequencing by ligation

Ion Torrent and Illumina Compact Personal Genome Machine Sequencer

A Second Generation Sequencing Technique that uses semiconductor sequencing technology

Third Generation Sequencing

PCR not needed before sequencing which shortens DNA preparation time; signal is captured real time

whole genome sequencing

Scope of NGS wherein almost all nucleotide in the genome including chromosomal DNA and mitochondrial DNA are examined; used more often in research and less common in clinical setting

Exome sequencing

Scope of NGS wherein entire coding region of an organism is analyzed

RNA sequencing

Scope of NGS wherein assembly of RNA transcripts including mRNA, tRNA, and microRNA are studied

Targeted sequencing

Scope of NGS that is commonly used for cancer patients; interrogates hundreds of targeted genes

whole genome sequencing

Laboratory procedure that determines the order of bases in the genome of an organism in one process; provides a very precise DNA fingerprint that can help link cases to one another allowing an outbreak to be detected and solved sooner

DNA Extraction

1st step of WGS where scientists take bacterial cells from an agar plate and treat them with chemicals that break the, open, releasing the DNA. The DNA is then purified

DNA Shearing

2nd Step of WGS wherein DNA is cut into short fragments of known length either by using enzymes "molecular scissors" or mechanical disruption.

DNA Library Preparation

3rd step of WGS wherein Scientists make many copies of DNA fragment using a process called polymerase chain reaction (PCR).

DNA library

Pool of fragments generated in a PCR Machine

DNA Library Sequencing

4th step of WGS wherein DNA Library is loaded onto a sequencer. The combination of nucleotides (A,T,C,G) making up each individual fragment of DNA is determined and each result is called a "DNA read"

DNA Sequence Analysis

5th step of WGS wherein the sequencer produces millions of DNA reads and specialized computer programs are used to put them together in the correct order like pieces of a jigsaw puzzle.