New England Biolabs, E6655S, NEBNext UltraShear® FFPE DNA Library Prep Kit

FFPE DNA poses many challenges for library preparation, including low input amounts and highly variable damage from fixation, storage, and extraction methods. Regions of interest are often enriched using hybrid capture-based approaches, and these workflows require a high input of diverse, uniform DNA library. Related Categories FFPE DNA,, Next Generation Sequencing Library Preparation Specification Materials Required but not Supplied 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 NEBNext Multiplex Oligos for Illumina® (www.neb.com/oligos) Magnetic rack/stand (NEB #S1515S, 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 NEBNext Unique Dual Index UMI Adaptor DNA Sets (NEB #E7395/E7874/E7876/E7878) FAQ Q: What sample types can I use with the NEBNext UltraShear FFPE DNA Library Prep Kit? A: The NEBNext UltraShear FFPE DNA Library Prep Kit is compatible with damaged samples, including formalin-fixed, paraffin-embedded (FFPE) DNA. This library preparation method is based on 3’-dA overhang-modified molecules ligating to adaptors containing a 5’-dT overhang. Q: How much starting material is required for the NEBNext UltraShear FFPE DNA Library Prep Kit? A: 5-250 ng of DNA is recommended. Please see NEB #E6650 for a version of this kit containing reagents to perform library prep downstream of mechanical or acoustic shearing. Q: When preparing samples using the NEBNext UltraShear FFPE DNA Library Prep Kit, can my input DNA be in EDTA-containing solutions? A: Yes, we recommend input DNA be in Tris-EDTA (TE) pH 8.0 for this kit. Alternatively, samples can be diluted in 10 mM Tris pH 8.0, 0.1X TE, or water. Q: Do I really need to vortex the NEBNext UltraShear enzyme mix before use? A: Yes, failure to vortex the NEBNext UltraShear enzyme mix may produce variable results. Q: What fragmentation time should be used with the NEBNext UltraShear FFPE DNA Library Prep Kit for FFPE DNA samples? A: For most FFPE samples diluted in 1X TE (validated on samples DIN 1.5 to 7.0), 5 minutes of fragmentation at 37°C is recommended for optimal yield, library quality, and library size. Fragmentation time may need to be optimized for FFPE samples of very high quality (DIN > 7.0). Q: Is size selection recommended? A: Size selection is not recommended for this workflow as the sample loss can be significant with highly damaged samples like FFPE DNA. Q: Which NEBNext Multiplex Oligos can be used with the NEBNext UltraShear FFPE DNA Library Prep Kit? A: The NEBNext UltraShear FFPE DNA Library Prep Kit is compatible with both the NEBNext truncated, non-indexed adaptor supplied with NEBNext index primer kits and the full-length unique dual index (UDI) unique molecular identifier (UMI) adaptors. For a complete list of all compatible oligos, please see the NEBNext Multiplex Oligos Selection Chart. Please note that the manual details different protocols for each adaptor type when used with FFPE DNA samples. Q: What input amount (nanograms) is recommended for standard versus target enrichment library prep with FFPE DNA samples? A: A minimal input amount of 100 ng FFPE DNA is recommended to achieve sufficient library yield and complexity from low quality FFPE samples going into target enrichment workflows. Higher input amounts will improve complexity. For standard library prep and sequencing, 5-250 ng input can be used, with higher inputs achieving better library complexity. Q: Can I use this NEBNext kit with adaptors and primers from vendors other than NEB? A: Yes. The adaptor must be a T overhang adaptor and the final library yield may be slightly lower than when using the NEBNext Adaptors for Illumina®. Guidelines for use (these apply for non-NEBNext, full length barcoded adaptors and P5 and P7 universal PCR primers.): Use 2.5 µl adaptor. Adaptors are typically used at 15-25 µM concentration. The optimal adaptor concentration and dilution would have to be determined experimentally. Skip the USER addition and the 15 min USER incubation. Add 3 µl water or TE to sample to make up volume difference for the next step. Sample bead cleanup conditions may have to be modified for other vendor oligos. PCR with NEBNext MSTC™ FFPE Master Mix: use PCR Primers supplied with the vendor adaptors used. Do NOT use NEBNext Index Oligo PCR primers. Q: Will treating my DNA with the FFPE DNA Repair Mix v2 as part of the NEBNext UltraShear FFPE DNA Library Prep Kit hurt my downstream reaction? A: We have not seen the NEBNext FFPE DNA Repair Mix v2 negatively affecting most downstream applications. The repair mix will act only on damaged bases, nicks and gaps. However, this workflow is not recommended for use in methylation detection studies. 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 I fragment my gDNA sample with NEBNext UltraShear® in buffers other than 1X TE (10mM Tris pH 8.0, 1mM EDTA)? A: gDNA samples can be fragmented with NEBNext UltraShear® with common gDNA extraction kits’ elution buffers, e.g. Monarch® DNA Elution Buffer, Monarch® gDNA Elution Buffer, Monarch® gDNA Elution Buffer II, Buffer EB and Buffer AE, in place of 1X TE pH 8.0. However, the fragmentation for NEBNext UltraShear is slower with gDNA samples fragmented in these elution buffers. Fragmentation conditions would need to be optimized and fragmentation may need to be incubated for extended times at 37˚C or 45 ˚C compared to gDNA samples in 1X TE pH 8.0. Monarch® DNA Elution Buffer (NEB #T1016): 10 mM Tris, 0.1 mM EDTA pH 8.5 Monarch® gDNA Elution Buffer (NEB #T3016): 10 mM Tris, 0.1 mM EDTA pH 9.0 Monarch® gDNA Elution Buffer II (NEB #T3056) or Buffer AE: 10 mM Tris-HCl, 0.5 mM EDTA pH 9.0 Qiagen Buffer EB: 10 mM Tris-HCl pH 8.5