“Next Generation (NextGen) Sequencing” is a general term for all sequencing platforms that offer a higher throughput alternative to Sanger (Capillary) sequencing. NextGen sequencing has revolutionized genomics research by substantially increasing the amount of data generated from one experiment, decreasing the time it takes to sequence entire genomes and allowing researchers to circumvent the cloning process when sequencing genetically heterogeneous samples.
There are several NextGen sequencing platforms on the market. Each platform operates using a unique combination of molecular chemistry and engineering/fluidics designs. As a result, read length, total base output, cost and data quality of each sequencing run varies with each platform. Many platforms are capable of sequencing at various lengths and throughput which will decrease cost and sequencing run time.
The Center for Genome Innovation has three sequencing platforms available for use:
- Illumina: MiSeq (Available at both the Storrs and UConn Health locations)
- Illumina: NextSeq 550 (Available at Storrs)
- Illumina: NovaSeq 6000 (Available at Storrs)
- Oxford NanoPore MinION and PromethION
Choosing the right platform
Choosing a platform for your experiment will depend on the type of sequencing (i.e. RNA-Seq, de novo genome sequencing, amplicon sequencing, etc.), desired coverage and budget you have. There are other important considerations to make in terms of read length and how long the experiment will take from start to finish. Given the complicated nature of these experimental designs, the CGI’s staff will help you determine which platform is best suited to meet your goals. Upon request, Bo Reese will generate a platform-specific project budget and sequencing coverage analysis based on your experimental design ideas.
Here are links to the Specifications pages for each NGS Illumina Platform:
When you purchase a sequencing kit, the number of cycles you get in the kit will vary. To determine how much sequencing you can get from your kit, visit this page.
For more information about the PromethION system from Oxford Nanopore, visit their web page.
Protocols
The users of the CGI have adapted protocols and techniques for each of the NextGen platforms available for use in the Center in addition to the “standard” protocols provided by each company. Listed below are some of the most commonly requested library preparation/sequencing applications adapted by CGI users:
- Whole genome shotgun
- ddRAD-seq
- ChIP-seq
- RNA-seq (mRNA, total and small)
- amplicon sequencing for any target
- Methylation sequencing (whole genome and reduced representation)
- Single Cell mRNA-Seq
- Exome Sequencing