-
The VSK-PTC001 RNA library preparation kit
The VolTRAX PCR Tiling 1-12 COVID-19 Kit generates tiled PCR amplicons from the SARS-CoV-2 viral RNA for whole genome sequencing using the VolTRAX V2b platform to improve consistency in library preparation due to the precise fluid handling of the device.
This kit provides all the reagents required to generate reverse-transcribed and PCR-amplified material from SARS-CoV-2 RNA samples using VolTRAX automation to prepare the library, without the need to purchase further components. The PCR tiling generated overlapping amplicons across the whole SARS-CoV-2 genome before barcoding and pooling multiple samples together for sequencing.
Below is an overview of the library preparation stages using a VolTRAX V2b, blue cartridges and the VolTRAX PCR Tiling 1-12 COVID-19 Kit (VSK-PTC001).
-
Overview of the VolTRAX PCR Tiling 1-12 COVID-19 protocol
1. Loading a protocol
- Use the protocol selector in the UI to load the VSK-PTC001 protocol.
- When prompted, connect your VolTRAX V2b device, press Continue and await connection.
2. System QC - Cartridge Test
- When prompted, connect a cartridge to the VolTRAX device and press Continue to proceed to the Cartridge Test.
3. Loading the Priming Fluid and reagents into the cartridge
- Follow the instructions on the equipment required and pipetting technique.
- Prime the cartridge by twisting the oil container and removing the foil tab.
- Load the appropriate reagents and extracted RNA into the ports as instructed by the UI.
- The reagents will be manipulated by the VolTRAX V2b to prepare the library for nanopore sequencing.
4. Extracting the library from the cartridge
- Follow the extraction instructions, using UI as a visual guide, to slowly extract the library from the cartridge, then transfer it into an Eppendorf tube.
- Prepare the library for loading onto the flow cell.
-
VolTRAX PCR Tiling 1-12 COVID-19 Kit (VSK-PTC001) contents
Name Acronym Number of vials Fill volume per vial (µl) LunaScript RT SuperMix LS RT 1 10 Q5 HS Master Mix Q5 1 35 VX Midnight Primer A VMP A 1 15 VX Midnight Primer B VMP B 1 15 VX BC Frag Mix 01-12 VXB01-VXB12 12 10 VX AMPure XP Beads VXP 1 45 VX Rapid Adapter F VRA F 1 12 VX Elution Buffer VELB 1 50 VX Running Buffer VRB 1 250 Negative Control NEG 1 10 Flush Buffer FB 3 1170 Flush Tether FLT 1 200 -
VolTRAX PCR Tiling 1-12 COVID-19 Kit barcode sequences
Component Sequence VXB01 AAGAAAGTTGTCGGTGTCTTTGTG VXB02 TCGATTCCGTTTGTAGTCGTCTGT VXB03 GAGTCTTGTGTCCCAGTTACCAGG VXB04 TTCGGATTCTATCGTGTTTCCCTA VXB05 CTTGTCCAGGGTTTGTGTAACCTT VXB06 TTCTCGCAAAGGCAGAAAGTAGTC VXB07 GTGTTACCGTGGGAATGAATCCTT VXB08 TTCAGGGAACAAACCAAGTTACGT VXB09 AACTAGGCACAGCGAGTCTTGGTT VXB10 AAGCGTTGAAACCTTTGTCCTCTC VXB11 GTTTCATCTATCGGAGGGAATGGA VXB12 CAGGTAGAAAGAAGCAGAATCGGA -
VSK-PTC001 chemistry
The VolTRAX PCR Tiling 1-12 COVID-19 Kit generates overlapping amplicons in a tiled fashion across the SARS-CoV-2 genome. First, the RNA samples are reverse-transcribed and then amplified by tiled PCR using separate primer pools. The primer pools are combined and SPRI purified before moving into the rapid barcoding stage which attaches Rapid Barcodes to the DNA ends for samples to be pooled.