Sample Preparation for Next Generation Sequencing
Next Generation Sequencing has been around for nearly two decades and is a much more high throughput analysis method for DNA sequencing than previous Sanger sequencing methods which originally emerged in 1977.1
There are several specific steps involved in getting samples prepared for next generation sequencing, which we will outline in this blog post.
Sample collection and storage
The first step in any DNA sequencing experiment is obtaining samples. As soon as samples have been collected, it is important to preserve these materials. CellCover acts as a cell fixation (cell fix) tool that liquid freezes samples so that downstream analyses can be performed on tissue (morphology), proteins (proteomics), RNA (transcriptomics), and DNA (genomics).
After cell fix with CellCover, samples can be analyzed using a variety of methods such as DNA FISH, single cell sequencing, microarray, immunostaining, or high throughput analysis of DNA via next generation sequencing. Cell fix is an important step in preserving sample quality for excellent results.
DNA Extraction and Amplification
In order to perform DNA sequencing on your samples, DNA must now be extracted from the preserved cells or tissue. There are several kits available on the market which achieve this, many of which can be automated to reduce hands on time.
If performing a single cell rna seq experiment, cells should be isolated prior to DNA extraction.2 Single cell sequencing generally requires unique cell be selected for individual high throughput analysis.
Amplification following DNA extraction is optional, and depends on the particular experiment design and the sample size. PCR is the most common technique used; isothermal amplification methods can be used as well.
Once DNA has been extracted and optionally amplified, several library preparation steps take place to prepare the sample for Next Generation Sequencing.
“In general, the core steps in preparing RNA or DNA for NGS analysis are: (i) fragmenting and/or sizing the target sequences to a desired length, (ii) converting target to double-stranded DNA, (iii) attaching oligonucleotide adapters to the ends of target fragments, and (iv) quantitating the final library product for sequencing.”3
The specifics of each of these steps is largely experiment- and platform-specific. Many NGS instrument and service providers have tools for scientists to design successful experiments taking into account these parameters.
Once library preparation is complete, you are ready to perform Next Generation Sequencing, followed by perhaps the most time consuming step – analyzing your results.
To get your experiment started off right, contact us for more information about CellCover for your upcoming DNA sequencing experiments.
1Jerzy K. Kulski (January 14th 2016). Next-Generation Sequencing — An Overview of the History, Tools, and “Omic” Applications, Next Generation Sequencing – Advances, Applications and Challenges, Jerzy K Kulski, IntechOpen, DOI: 10.5772/61964. Available from: https://www.intechopen.com/books/next-generation-sequencing-advances-applications-and-challenges/next-generation-sequencing-an-overview-of-the-history-tools-and-omic-applications
2Single-cell whole-genome sequencing reveals the functional landscape of somatic mutations in B lymphocytes across the human lifespanLei Zhang, Xiao Dong, Moonsook Lee, Alexander Y. Maslov, Tao Wang, Jan Vijg. Proceedings of the National Academy of Sciences Apr 2019, 116 (18) 9014-9019; DOI: 10.1073/pnas.1902510116
3Head SR, Komori HK, LaMere SA, et al. Library construction for next-generation sequencing: overviews and challenges. Biotechniques. 2014;56(2):61-passim. Published 2014 Feb 1. doi:10.2144/000114133