New England Biolabs, E7360S, NEBNext FFPE DNA Repair v2 Module

This new product further improves repair of FFPE DNA in next generation sequencing (NGS) workflows. Related Categories FFPE DNA,, Next Generation Sequencing Library Preparation Specification Materials Required but not Supplied NEBNext Ultra II DNA Library Prep Kit (NEB #E7645S, #E7645L, #E7103S or #E7103L) for Illumina, or other 80% Ethanol Nuclease-free Water 0.1X TE (1 mM Tris-HCl, pH 8.0, 0.1 mM EDTA) DNase- RNase-free PCR strip tubes DNA LoBind® Tubes (Eppendorf #022431021) SPRIselect® Reagent Kit (Beckman Coulter, Inc. #B23317) or AMPure® XP Beads (Beckman Coulter, Inc. #A63881) NEBNext Multiplex Oligos for Illumina (www.neb.com/oligos) Magnetic rack/stand (NEB #S1515, Alpaqua® cat. #A001322, or equivalent) Thermal cycler Agilent Bioanalyzer® or TapeStation® and associated reagents and consumables Adaptor Dilution Buffer NEB #B1430S or NEBNext Unique Dual Index UMI Adaptor Dilution Buffer supplied with NEB #E7395S/L FAQ Q: What is the difference between the NEBNext FFPE DNA Repair v2 Module (NEB #E7360) and the NEBNext FFPE DNA Repair Mix (NEB #M6630)? A: The NEBNext FFPE DNA Repair v2 Module contains the same repair activities as the NEBNext FFPE DNA Repair Mix but has been optimized for higher repair efficiency, a more streamlined workflow with NEBNext library preparation, and a more convenient reaction buffer containing all required buffer components for not only efficient DNA repair but downstream end repair and dA-tailing. Q: Will the FFPE DNA Repair v2 Module ligate my DNA fragments? A: The ligase present in the FFPE DNA Repair v2 Module effectively seals nicks in DNA, but it is not effective at ligating fragments at a mismatch or at ligating blunt ended DNA. We have not observed this ligase to ligate blunt ended DNA. Q: Will treating my DNA with the FFPE DNA Repair v2 Module hurt my downstream reaction? A: We have not seen the FFPE DNA Repair v2 Module to have a negative effect on most downstream applications. The repair mix will act only on damaged bases, nicks and gaps. However, in specialized applications, such as methylation detection in damaged samples, there is the chance that the patch of bases filled in during the nick translation function of repair could erase methylation signatures. This effect is more pronounced in low DNA input experiments. Q: Will the FFPE DNA Repair v2 Module blunt the ends of the DNA? A: No, the polymerase present in the NEBNext FFPE DNA Repair v2 Module will fill in 5′ overhangs, but will not remove 3’ overhangs. Furthermore, this polymerase may add a dA to the filled-in 5′ overhang. Q: Does the FFPE DNA Repair v2 Module insert random nucleotides into the sequence that it repairs? A: No, the polymerase in the mix adds the complementary bases that correspond to the template strand. The repair mix will not introduce artifacts into the sequence. Q: Does the FFPE DNA Repair v2 Module remove covalent modifications from DNA bases, such as biotin or digoxigenin? A: No, this repair mix will not remove covalent modifications in the DNA bases. Q: Does the FFPE DNA Repair v2 Mix repair DNA-protein crosslinks? A: No, the repair mix does not repair DNA-protein crosslinks. Q: Does the FFPE DNA Repair v2 Mix fix blocked 3′ ends? A: Yes, the repair mix will polish the 3′ end to a hydroxyl if there is a block at the 3′ end. Q: Can the FFPE DNA Repair v2 Mix repair damage in both single- and double-stranded DNA? Or, does it require double stranded DNA as a template? A: The full repair activity of the FFPE DNA Repair v2 Mix requires dsDNA for polymerization and ligation. However, damaged bases such as dU present in ssDNA will be excised during repair and not be incorporated into the sequencing library. Q: If I had a DNA template with mutation sites (i.e. 8-oxoguanine or deaminated cytosines) that are directly adjacent to each other on opposite strands would treatment with the FFPE DNA Repair v2 Mix cause a double strand nick/break? A: We have not tested the repair of adjacent mutation sites on opposite strands. In this scenario, if the two damaged bases are removed at the same time, then a double-stranded break could occur resulting in fragmented DNA. If, on the other hand, one reaction is faster than the other, and they do not occur simultaneously, then the FFPE DNA Repair v2 Mix should repair them. Q: What gap lengths can be repaired with the FFPE DNA Repair v2 Mix? A: The exact gap length is not known. Based on our experience, it is between 5-10 nucleotides. Q: Can the FFPE DNA Repair v2 Module repair bisulfite-treated DNA? A: Bisulfite treatment of DNA results in ssDNA containing uracils, which cannot be fully repaired by the polymerization and ligation activities of this mix. dU bases present in ssDNA will be excised but not fully repaired. Q: When working with fragmented DNA, will the ends be ligated together by the FFPE DNA Repair v2 Module? A: No, the ligase in the mix will only ligate where there is a nick in dsDNA. Q: How are abasic sites repaired by the FFPE DNA Repair v2 Module? A: For dsDNA, the repair mix recognizes the abasic site and cuts the DNA backbone. The polymerase replaces the missing base with the correct base and the ligase ligates the DNA. Q: Can I use the FFPE DNA Repair v2 Module to repair other types of low quality DNA other than FFPE DNA? A: Yes. The repair mix will repair many types of DNA damage that are commonly found in other DNA of low quality, such as UV or oxidative damage.