While DNA sequencing has been performed for many decades, the ability to sequence an entire human genome in hours versus years is a relatively recent event brought about by advanced sequencing technology- termed next-generation sequencing (NGS). The key to NGS is its massively parallel nature: DNA strands comprising an entire human genome can be captured on a substrate and millions of sequencing reactions performed simultaneously by flooding the DNA strands with a mix containing special nucleotides and a polymerase. Illumina is the NGS industry leader, and their NGS systems use fluorescent-labeled nucleotides that are detected by a high speed camera as they are incorporated into the DNA strands and excited by light of the proper wavelength. Software de-convolutes the imaging data, yielding millions of base reads that are then aligned to a known sequence.
The first step in any successful sequencing experiment is the preparation of the DNA to be sequenced. In a typical workflow, the DNA is first purified using any of a number of column clean-up methods and then mechanically sheared to yield thousands of short double-stranded molecules. At this point, a library must be produced that has the proper adaptors and indexes for use on the Illumina platform. To ensure the highest quality libraries for sequencing, especially when working with limiting (nanogram) quantities of DNA, the library preparation method must be highly efficient, accurate, and avoid purification steps where DNA can be lost.
Step 1. ThruPLEX technology features improved repair chemistry.
Step 2. The background is reduced using double-stranded adaptors with no single-stranded tails. Blunt end ligation occurs with high-efficiency. Blocked 5’ ends reduce adaptor-adaptor ligation.
Step 3. Background is reduced by destroying unused adaptors after ligation.
Overall, the time to complete the process is reduced by using compatible buffers and multiplexed enzymatic reactions that do not require intermediate purifications.
Learn more about ThruPLEX DNA-seq Kit
*Protected by US patent 7,803,550 and EP1924704
ThruPLEX DNA-seq Prep Kit’s Three-Step, Single Tube Workflow.