New England Biolabs, E7810S, NEBNext® Ultra™ II FS DNA Module

This module is part of the NEBNext Related Categories DNA Fragmentation & RNA Fragmentation,, Automation for NEBNext® NGS Library Prep,, Next Generation Sequencing Library Preparation Specification Materials Required but not Supplied Magnetic rack or plate (e.g., NEBNext ® Magnetic Separation Rack (NEB #S1515S), Alpaqua ® 96S Super Magnet Plate (#A001322), or equivalent) FAQ Q: When preparing samples using the NEBNext Ultra II FS DNA Library Prep Kit, can my input DNA be in EDTA-containing solutions? A: Yes, we recommend that the DNA be in 1X TE or low EDTA-containing TE. DNA can also be in 10 mM Tris pH 7.0-8.0 or H20. Q: What do I do if I see a precipitate in the Ultra II FS Reaction Buffer? A: It is not uncommon to see precipitation in the Ultra II FS Reaction Buffer. If this occurs, pipette the buffer several times to break up the precipitate, followed by a quick vortex to mix. You may still see a small amount of precipitation, but this will not affect performance. Q: Following the Fragmentation and End Prep steps of the NEBNext Ultra II FS workflow can the reactions be kept at 4° C? A: Yes, the fragmented, end-prepped DNA can be kept at 4˚C for several hours without any appreciable effect. However, after overnight storage at 4˚C a decrease in yield of approximately 20% can be expected. Q: Do I really need to vortex the Ultra II FS Enzyme Mix? A: Yes, failure to vortex the Ultra II FS Enzyme Mix may produce variable results. Q: What is the concentration of my index primers? A: NEBNext index primers are 10 µM concentration. Q: Which NEBNext ligation module should be used in conjunction with the NEBNext Ultra II FS DNA Module? A: The module is designed for use with the NEBNext Ultra II Ligation Module (NEB #E7595). Q: Can I use the NEBNext® Ultra™ II FS DNA Module for bisulfite conversion and Enzymatic Methyl-seq (EM-seq™) workflows? A: No, the NEBNext Ultra II FS DNA Module is not compatible with bisulfite conversion or EM-seq workflows. We recommend you use either NEBNext UltraShear™ (NEB #M7634) or mechanical shearing, followed by the NEBNext Ultra II DNA End Repair/dA-Tailing Module (NEB #E7546) for this application. After fragmentation with NEBNext UltraShear, you can go straight into End Repair/dA-Tailing reaction as described in section 2 of NEBNext UltraShear manual without a cleanup. Q: Does the fragmentation reagent included in NEBNext Ultra® II FS DNA kits contain NEBNext dsDNA Fragmentase®? A: No, the Ultra II FS (Fragmentation System) products contain a unique mix of enzymes that fragment, end repair, and dA tail DNA, in a single tube. Fragmentase is not included in the FS enzyme mix. Q: Is the NEBNext® Ultra™ II FS DNA Library Prep Kit for Illumina® compatible with all sample types? A: The NEBNext® Ultra™ II FS DNA Library Prep Kit for Illumina® has been tested in many sequencing applications including whole genome sequencing, hybrid capture, amplicon sequencing, and copy number assessment, etc. It has been designed to maximize the number of molecules incorporated into a sequencing library. As a result, this method is able to produce diverse libraries even from very small amounts of input DNA (< 1 ng). Please keep in mind the following: Modifications present in the DNA input, such as methylation and hydroxymethylation, can be replaced by their canonical form when using FS. This method should not be used for applications where modified bases are being studied (e.g. EM-seq and bisulfite sequencing). At very low frequency (typically less than 0.1%), FS may introduce artifacts identified as chimeras or tandem reads in downstream analysis. These artifacts can be filtered using fgbio FindSwitchbackReads. For FFPE samples, we have observed increased noise in some FS libraries. However, due to the diversity of FFPE damage, it is not possible to predict which FFPE samples will exhibit higher noise. For best data quality, we recommend using the NEBNext Ultrashear FFPE DNA Library Prep Kit (NEB #E6655) or the NEBNext FFPE DNA Library Prep Kit (NEB #E6650). We encourage users to carefully evaluate any library prep method to ensure it is appropriate for the specific sample type being studied. Please reach out to info@neb.com if you have questions about your specific application.