How do I remove High Molecular Weight (HMW) fragments from my DNA Library?

HMW peaks can be artifacts caused by overamplification resulting in hetero duplex bubble products and are more often seen in the protein library compared to the DNA libraries, or there can be panel-specific HMW byproducts seen in DNA libraries. Hetero duplex bubble products typically have broad, poorly defined peaks as seen on the BioAnalyzer. True HMW products typically have sharper peaks, see image at the end of the article.  

Removing bubble product peaks

Repeating the library PCR with fewer cycles (e.g., Protein library: 16 instead of 20 cycles, DNA library: 8 instead of 10 cycles) can reduce the appearance of HMW peaks.

If the bubble product persists, the libraries can still be sequenced. qPCR is recommended for quantifying libraries containing bubble products, as intercalating fluorometric quantification may be unreliable (Qubit/BioAnalyzer).

Example: Protein library after 20 cycle Library PCR:

Example: Protein library after 16 cycle Library PCR:

Additional information via illumina:

https://support.illumina.com/bulletins/2019/10/bubble-products-in-sequencing-libraries--causes--identification-.html

Removing HMW byproducts from DNA libraries

This protocol may be used for panels that routinely produce large amounts of HMW fragments that impact sequencing. 

1. Run the Tapestri DNA Library PCR with 12 cycles
2. Cleanup with AMPure XP beads (Following the standard v3 Tapestri protocol):
   1. 0.69x AMPureXP bead ratio
   2. 0.72x AMPureXP bead ratio
3. Elute in 50 uL nuclease-free water.
4. Add 32.5 uL (0.65x) AMPure XP beads.
5. Incubate at room temperature for 10 min.
6. Place on magnet for 5 min for the beads to separate from the solution.
7. Without removing the tube, transfer 75 uL supernatant to a new 0.2 mL PCR tube (Be careful not to transfer any beads, the beads bind the HMW fragments and are discarded). 
8. Add 58 uL (0.77x) of AMPure XP beads to the supernatant.
9. Incubate the tube at room temperature for 5 min.
10. Place on magnet for 5 min.
11. Without removing the tube from the magnet, remove the supernatant and discard.
12. Carefully add 200 uL of freshly prepared 80% ethanol, wait for 30 seconds, remove the ethanol without disturbing the AMPure XP beads.
13. Repeat Step 12 once for a total of two washes.
14. Keeping the tube on the magnet, remove all residual ethanol from the tube without disturbing the AMPure XP beads.
15. Dry AMPure XP bead pellets in the tube on the magnet for 2 to 5 min being careful not to overdry. 
16. Remove the tube from the magnet and add 12 uL of nuclease-free water. Vortex and quick-spin to collect the contents.
17. Incubate for 2 minutes.
18. Place on magnet and wait 2 minutes until the solution is clear. 
19. Transfer 10 uL of the purified PCR product from the tube to a new 0.2 mL PCR tube.
20. Run a 1:10 dilution of the product on a BioAnalyzer to check for library purity. 

Before HMW removal

After HMW removal