New England Biolabs, E9642L, NEBNext® RSV Primer Module

NEBNext Related Categories Respiratory Syncytial Virus (RSV) Sequencing,, Infectious Diseases & ARTIC Sequencing Applications Polymerases for NGS Library Preparation FAQ Q: Which additional reagents are needed for cDNA synthesis and amplification with the NEBNext RSV Primer Module (NEB #E9642)? A: NEB recommends pairing the NEBNext RSV Primer Module with the LunaScript® Multiplex One-Step RT-PCR Kit (NEB #E1555). A protocol for the use of these reagents for RSV amplicon generation can be found here. Q: Are the NEBNext RSV primer sequences (NEB #E9642) available? A: Yes, the NEBNext RSV primer sequences can be found here: https://github.com/nebiolabs/rsvab-sequencing. Q: Which RSV strains can be targeted with the NEBNext RSV primers (NEB #E9642)? A: The NEBNext RSV Primer Mixes contained in the NEBNext RSV Primer Module target both RSV A and RSV B strains, by binding to regions that are conserved across RSV A and RSV B strains, as well as RSV A specific regions or RSV B specific regions. Q: Is it possible to target only RSV A or RSV B with the primer mixes included in the NEBNext RSV Primer Module (NEB #E9642)? A: No, both primer mixes included in the NEBNext RSV Primer Module contain some primer pairs that target both RSV A and RSV B. Q: Which library prep reagents and protocols can be used for sequencing amplicons made with the NEBNext RSV Primer Module (NEB #E9642)? A: The amplicons produced with the NEBNext RSV Primer Module are sequencing platform agnostic. However, given that the amplicons range from 400 bp to 1,400 bp in length, cDNA fragmentation may be required for short-read sequencing approaches. For sequencing on Illumina platforms, NEB recommends pairing the NEBNext RSV Primer Module with the NEBNext UltraExpress® FS DNA Library Prep Kit (NEB #E3340). A protocol (Section 2) for the use of these reagents for RSV amplicon sequencing can be found here. The protocol (Section 3, page 11) and reagents necessary for sequencing NEBNext RSV amplicons on Oxford Nanopore Technologies' platforms, can also be found here. Q: Is there a recommended Ct value-range for RSV inputs? A: NEB recommends inputs of at least 1,000 genome copies. As Ct values vary by assay type and the genome target of the assay, we do not have a specific Ct value range. However, as shown in the figure below sequencing of clinical samples with BioFire® assay Ct values of < 30 show high genome coverage. Refer to Figure 8 on the product page for data generated from clinical samples. Q: What is the minimum read depth needed when using the NEBNext RSV Primer Module (NEB #E9642)? A: NEB recommends a minimum read depth of 0.5 M PE reads per library for Illumina sequencing. For ONT sequencing on a MinION flow cell, NEB recommends a minimum read depth of 50,000 reads per library. Q: What sample types are supported when using the NEBNext RSV Primer Module (NEB #E9642)? A: The protocols provided have been validated with high quality commercially available gRNA templates. Additional sample types that have been tested include cell culture RNA isolates and RNA extracted from clinical samples (e.g. Nasopharyngeal swabs) (data shown in Figure 8 on the product page). Q: What extraction methods can be used to isolate RSV gRNA ahead of RT-PCR? A: RNA isolated using NEB’s Monarch® Mag Viral DNA/RNA Extraction Kit (NEB #T4010S) and Monarch Total RNA Miniprep Kit (NEB #T2010) can be used. Other bead based and column-based RNA extraction approaches can also be used. Q: Are there recommendations for cleaning up RSV amplicons prior to library prep when using the NEBNext RSV Primer Module (NEB #E9642)? A: The accompanying library preparation protocols for sequencing NEBNext RSV amplicons use diluted RSV amplicons as inputs for library prep. However, if a cleanup of amplicon cDNA is preferred, 0.8X cleanup with SPRIselect or AMPure beads is recommended. For example, if amplicon cDNA volume is 25 µl, 20 µl (0.8X) of SPRIselect/AMPure beads would be used to bind the cDNA, followed by two washes with 200 µl of 80% EtOH, and elution in 15-30 µl of water. Q: Are there recommended RSV cDNA amplicon input (after using the NEBNext RSV Primer Module (NEB #E9642)) for Oxford Nanopore Technologies End Prep reactions for low-plexity or high-plexity library prep? A: NEB recommends utilizing a minimum of 6 ONT Native Barcodes per sequencing run. For runs utilizing 6 to 12 barcodes, we recommend inputs of 350 ng of amplicons within the 400-1,400 bp size range per sample going into the end prep reaction for ONT library prep. If utilizing more than 48 barcodes, 20 ng – 40 ng of amplicons within the 400-1,400 bp size range per sample can be used as input for the end prep reaction for ONT library prep. Q: Can I minimize non-specific barcode ligation amongst pooled samples, after using the NEBNext RSV Primer Module (NEB #E9642)? A: Yes, add 2 ul of Thermolabile Proteinase K (NEB #P8111S) to each sample following the ONT barcode ligation reaction, then incubate at 37°C for 15 minutes. Following Thermolabile Proteinase K treatment, add 2 ul of EDTA provided in Oxford Nanopore Technologies Native Barcoding V14 Kit(s) to each barcoded sample and proceed with pooling and barcoded sample pool cleanup.