Beginner's Handbook of Next Generation Sequencing
Everything you need to know about starting a sequencing project
Massively parallel sequencing or next generation sequencing is becoming a ubiquitous technology in basic biology research and starting to make inroads in both diagnostic and clinical settings. Despite its popularity, understanding how it works usually requires reading several manuscripts, translating technology jargon and stitching together tidbits from presentations or discussions with colleagues. This handbook is designed to assist those new to next generation sequencing get from nucleic acid sample to next generation sequencing data on the first try. Avoiding sequencing redo’s saves a lot of time and research money!
Section I, Designing a Sequencing Run outlines what you should consider when you first decide you’d like to sequence your sample. Section II, Library Preparation goes into choosing a library prep kit, considerations for calculating the coverage you need for DNA or RNA applications and determining whether targeted or global whole genome sequencing makes more sense for your application. Section III, briefly summarizes considerations for how your DNA or RNA should be fragmented and best practices for isolation and shipping your sample to a NGS services provider. Section IV, Recommendations for Analyzing your Sequencing Data, highlights types of biotinformatics services and the providers available to perform analysis of your raw sequencing data.
This is a living handbook, meaning we actively update it to keep current with fast changing technology. If you’d like to add something, please send us a message at science@genohub.com.
I. Designing a Sequencing Run
- NGS project considerations for beginners
- Determining the coverage or read depth you need for your application
- Choosing an instrument – comparison of all NGS instruments
- Choosing an instrument - in-depth guide for each specific NGS instrument
- Optimizing flow cell loading and cluster densities
- Choosing between WGS and exome-sequencing
- Choosing between a gene panel and exome-sequencing
Instrument-Specific Considerations:
II. Library Preparation
Library prep considerations for:
- Whole genome and re-sequencing
- Exome sequencing
- ChIP-seq
- Transcriptome or mRNA
- Shotgun metagenomics
- Small RNA - microRNA
III. Preparing your Sample for Library Preparation
- Fragmenting your DNA or RNA sample before library preparation
- Best Practices for Shipping RNA/DNA/Tissue for NGS