New England Biolabs, E7626L, NEBNext® ARTIC SARS-CoV-2 RT-PCR Module

The Small size of this product was discontinued on December 15, 2024. The Large size (NEB #E7626L) will continue to be available. Related Categories NEBNext® ARTIC products for SARS-CoV-2 sequencing,, RNA Library Prep for Illumina Specification Materials Required but not Supplied 80% Ethanol (freshly prepared) Nuclease-free Water DNA LoBind Tubes (Eppendorf® #022431021) Magnetic rack/stand (NEB #S1515, Alpaqua®, cat. #A001322 or equivalent) Thermocycler Vortex Mixer Microcentrifuge Agilent® Bioanalyzer® or similar fragment analyzer and associated consumables DNase RNase free PCR strip tubes (USA Scientific®1402-1708) SPRIselect® Reagent Kit (Beckman Coulter, Inc. #B23317) or AMPure® XP Beads (Beckman Coulter, Inc. #A63881) FAQ Q: What is the difference between the NEBNext VarSkip Short v2 SARS-CoV-2 Primer Mixes and the original NEBNext VarSkip Short SARS-CoV-2 Primer Mixes? A: The NEBNext VarSkip Short v2 SARS-CoV-2 Primer Mixes have been optimized to improve coverage of the Omicron variant. In these v2 mixes, 10 primers have been replaced, affecting 7 primer pairs. Q: What is the difference between the NEBNext VarSkip v2 Short SARS-CoV-2 Primer Mixes and the NEBNext ARTIC SARS-CoV-2 Primer Mixes? A: VarSkip Short v2 SARS-CoV-2 Mixes 1 and 2 for SARS-CoV-2 genome amplification are based on hCoV-2019/nCoV-2019 Version 3 (v3) sequences with balanced primer concentrations. NEBNext VarSkip Short v2 SARS-CoV-2 Mixes 1 and 2 for SARS-CoV-2 genome amplification were designed to reduce the impact of variants on amplification efficiency. NEBNext ARTIC SARS-CoV-2 amplicons are ~400 bp, whereas NEBNext VarSkip Short v2 SARS-CoV-2 amplicons are ~550 bp. Q: Where can I find primer sequence information for NEBNext VarSkip Short v2 SARS-CoV-2 Primer Mixes and NEBNext ARTIC SARS-CoV-2 Primer Mixes? A: NEBNext VarSkip Short v2 SARS-CoV-2 Primer sequences can be found here: https://github.com/nebiolabs/VarSkip NEBNext ARTIC SARS-CoV-2 Primer sequences can be found here: https://github.com/joshquick/artic-ncov2019/blob/master/primer_schemes/nCoV-2019/V3/nCoV-2019.tsv Q: Can the NEBNext ARTIC SARS-CoV-2 Primer Mixes and NEBNext VarSkip Short v2 SARS-CoV-2 Primer Mixes be added to the same targeted amplification reaction? A: No, the NEBNext ARTIC SARS-CoV-2 Primer Mixes and NEBNext VarSkip Short v2 SARS-CoV-2 Primer Mixes cannot be added to the same amplification reaction. Q: Which primer scheme should I use: NEBNext ARTIC SARS-CoV-2 Primer Mixes or NEBNext VarSkip Short v2 SARS-CoV-2 Primer Mixes? A: NEBNext VarSkip Short v2 SARS-CoV-2 Primer Mixes are recommended for known or expected SARS-CoV-2 variant samples. Q: Can the NEBNext VarSkip Short v2 SARS-CoV-2 Primers cover non-variant SARS-CoV-2 samples? A: Yes, they can also be used to amplify non-variant SARS-CoV-2 samples. Q: Which SARS-CoV-2 variants can the NEBNext VarSkip Short v2 SARS-CoV-2 Primers cover? A: The VarSkip Short SARS-CoV-2 Short primers were designed to be variant tolerant. We have confirmed that these primers provide high genome coverage for the following variants: B.1.351 (Beta) P.1. (Gamma) B1.617.1 (Kappa) B.1.617.2 (Delta) B.1.617.3 BA.1 (Omicron) BA.2 (Omicron) Q: How do I analyze VarSkip Short v2 SARS-CoV-2 sequencing data? A: VarSkip Short v2 SARS-CoV-2 sequencing data can be analyzed using the same analysis methods as ARTIC multiplex amplicon sequencing. All necessary reference files for modifying ARTIC sequencing analysis pipelines for VarSkip Short v2 SARS-CoV-2 sequencing analysis can be found at https://github.com/nebiolabs/VarSkip. Q: Does my total RNA sample need to be DNA-free prior to starting the ARTIC workflow? A: No, the RNA does not need to be DNA-free for the ARTIC workflow. Q: Can coverage be improved for the recent variants? A: Yes, spike-in primers have been designed and tested to address low coverage areas caused by KP and JN variants. These primers can be provided free of charge upon request. Please contact info@neb.com. Alternatively, the sequences below can be used for custom primer synthesis from your preferred oligo provider. These spike-in primers should be used with the VarSkip Short v2 primers following the protocols below. VSSv2f Spike-in Mix Primer Sequence Target Concentration in 1xPool (µM) 1 varskip-0317-1_1_LEFT_ALT3 ATCTCTTGTAGATCTGTTCTCTAAACG 0.5 1 varskip-0317-1_05_RIGHT_ALT2 GACAATTTCACAAGCACAGGTTGAG 0.5 1 varskip-0317-1_07_LEFT_ALT2 CCTCTAAAAGCCCCAAAAGAAATTATC 0.5 1 varskip-0317-1_09_RIGHT_ALT2 GTTTACTTTCAGTTATAAATGGCTTAACTTC 0.5 1 varskip-0317-1_11_LEFT_ALT2 GTGGCACTACTGAAATGCTAGC 0.5 1 varskip-0317-1_13_RIGHT_ALT2 TTCATAAGAAAGTGTGCCCATGTAC 0.5 1 varskip-0317-1_15_RIGHT_ALT1 GCTCCAAAGACAACGTATACACC 0.5 1 varskip-0317-1_35_LEFT_ALT2 GATGATTATTTCAATAAAAAGGACTGGTATG 0.5 1 varskip-0317-1_45_RIGHT_ALT2 ATTGATCTCCAGGCGGTGGTTT 0.5 1 49_R_ALT2 GTAATAAGAACACCATTACGGGCAT 0.5 1 varskip-0317-1_53_RIGHT_ALT2 TGAGGATCTGAAAACTTTGTCAGG 0.5 1 55_L_ALT1 CCTACTTATTGTTAATAACGCTACTAATGTT 0.5 1 varskip-0317-1_57_LEFT_ALT6 CTCTGCTTTACTAATGTCTATGCAGA 0.5 2 varskip-0317-1_24_LEFT_ALT2 GCCTTTAATACTTTACTATTCCTTATGTC 0.5 2 varskip-0317-1_28_RIGHT_ALT2 AGGCCAAAGTAACAAGTACAAAAATAGC 0.5 2 varskip-0317-1_32_RIGHT_ALT2 CTGTTATTGCCTGACCAGTACC 0.5 2 varskip-0317-1_34_RIGHT_ALT2 ATCACAGAATTGTACTGTTTTTAACAAAGC 0.5 2 varskip-0317-1_54_RIGHT_ALT2 CTTCAAGGTCCATAAGAAAAGGCT 0.5 2 56L_ALT1 TTATTATGTGGGTTATCTTCAACCTAGG 0.5 2 varskip-0317-1_56_RIGHT_ALT2 CAGCCTGTAAAATCATCTGGTAATTTAT 0.5 Protocols for use of spike-in primers: For 96-reaction kits: Thaw VSSv2f Spike-in Mix 1, VSSv2f Spike-in Mix 2, VSSv2 Primer Mix 1. and VSSv2 Primer Mix 2. Mix and quick spin all four tubes. Add 3 μl of VSSv2f Spike-in Mix 1 to VSSv2 Primer Mix 1, vortex, spin-down, place on ice. Add 2.5 μl of VSSv2f Spike-in Mix 2 to VSSv2 Primer Mix 2, vortex, spin-down, place on ice. Use spiked VarSkip Short v2 Primer Mix 1 and 2 for RT-PCR protocol. For 24-reaction kits: Thaw VSSv2f Spike-in Mix 1, VSSv2f Spike-in Mix 2, VSSv2 Primer Mix 1. and VSSv2 Primer Mix 2. Mix and quick spin all four tubes. Add 2 μl of VSSv2f Spike-in Mix 1 to 2 μl 0.1x TE to make a ½ dilution of the VSSv2f Spike-in Mix 1. Add 1.5 μl of diluted VSSv2f Spike-in Mix 1 to VSSv2 Primer Mix 1, vortex, spin-down, place on ice. Add 2 μl of VSSv2f Spike-in Mix 2 to 2 μl 0.1x TE to make a ½ dilution of the VSSv2f Spike-in Mix 2. Add 1.25 μl of diluted VSSv2f Spike-in Mix 2 to VSSv2 Primer Mix 2, vortex, spin-down, place on ice. Use spiked VarSkip Short v2 Primer Mix 1 and 2 for RT-PCR protocol.