Indexed PentAdapters™ for next generation sequencing on Illumina and Ion Torrent sequencing platforms. Using PentaBase’s outperforming technologies, we have made indexed adapters ready-to-use and ready-to-ship, for the sensitive sequencing of multiplexed samples.
Single Indexed PentAdapter™ design for Illumina Sequencing
The illustration below, shows how the PentAdapter top strand includes a part that can bind to the flow cells used in Illumina’s sequencing instruments as well as a reverse primer binding site. This is followed by a complementary region and finally a T-tail overhang. The bottom strand includes a binding site for universal priming, an index region, a region complementary to the top strand, and finally a 5′-phosphate used for sample ligation. The special PentaBase modifications are not shown in this illustration for proprietary reasons.
Protocol – From sample to NGS library
Step 1: The first step is to shear your DNA into smaller pieces. There are several different methods for doing that, including sonication and enzymatic cleavage.
Step 2: The ends of the DNA pieces are “repaired” with enzymes and buffers. 2A: The 5′-ends of the sheared DNA pieces are phosphorylated. The blue screw driver illustrates the phosphorylation by the polymerase and kinase mixture often used. 2B: The 3′-ends are added a single A-tail. The red screw driver illustrates the Klenow fragment often used for adding dAMP to the 3′-ends of dsDNA.
Step 3: The indexed labeled PentAdapters™ are annealed using a DNA ligase. The PentAdapters™ are ligated to both ends of the sheared and end repaired DNA. The phosphorylation of the 5′-ends of the DNA, the phosphorylation of the PentAdapters™, the preparation of the 3′-end A-tail and the stabilizing chemistry from PentaBase are all important factors for successful ligation. The purple glue bottle illustrate the ligase.
Step 4: The library comprising up to billions of different DNA fragments are ready to be mixed with other samples likewise comprising up to billions of different DNA fragments labeled with different indexed PentAdapter™ for multiplexing the readouts.
DNA analysis after preparation using different DNA preparation kits
Instructions from manufactures were followed. DNA purification on beads was used on all samples except lane 4 where gel based purification was performed. From left: Lane 1: DNA ladder. Lane 2: Illumina’s DNA prep + Competitor I’s Adapter. Lane 3: Illumina’s DNA prep + PentAdapter™. Lane 4: Illumina’s DNA prep + Competitor I’s Adapter (size select on gel). Lane 5: Kapa Biosystem’s DNA prep + PentAdapter™. Lane 6: NEB’s DNA prep + Custom adapter from PentaBase. Lane 7: NEB’s DNA prep + PentAdapter™.