APHL COVIDSeq Training: Best Practice & Overview

Before We Start (Very Important) – Updated 02.21.2022

Reference: illumina-covidseq-test-reference-guide-ruo-1000000126053-04

*** Best Practice***

Always vortex/shake/pipette up and down, reaction tube or plate before ThermalCycling Step

Always spin down/centrifuge reaction tube after step 1 before ThermalCycling Step (except Tagmentation step)

Always centrifuge reaction after ThermalCycling Step

Avoiding Contamination:

Use proper laboratory practices to prevent nuclease and PCR product contamination. Nuclease and PCR product contamination can cause inaccurate and unreliable results.

Perform library preparation in a RNase/DNase-free environment. Thoroughly decontaminate work areas with a RNAse/DNase-inhibiting solution, such as RNAseZap and DNAzap.

Use fresh tips and fresh consumable labware between samples and dispensing reagents.

Use aerosol-resistant tips to reduce the risk of carry‐over and sample‐to‐sample cross‐contamination.

Due to the potential for contamination, take extreme care to make sure that well contents remain fully in the well. Do not splash contents.

Do not use aerosol bleach sprays when performing library preparation. Trace bleach contamination can lead to assay failure.

Use a unidirectional workflow when moving from pre-amplification to post-amplification environments.

Sealing and Unsealing the Plate

Always seal the 96-well plate before the following steps in the protocol:

Shaking steps

Vortexing steps

Centrifuge steps

Thermal cycling steps

To seal the plate, apply the adhesive cover to the plate and then seal with a wedge or rubber roller.

Make sure the edges and wells are completely sealed to reduce the risk of cross-contamination and evaporation.

Microseal 'B' adhesive seals are effective at -40°C to 110°C, and suitable for skirted or semiskirted PCR plates. Use Microseal 'B' for shaking, centrifuging, and long-term storage.

Before unsealing:

Briefly centrifuge the 96-well plate at 1000 × g for 1 minute. For bead steps, centrifuge at 500 x g for 1 minute.

Place the plate on a flat surface before slowly removing the seal.

Plate Transfers

When transferring volumes between plates, transfer the specified volume from each well of a plate to the corresponding well of the other plate.

If beads are aspirated into the pipette tips, dispense back to the plate on the magnetic stand and wait until the liquid is clear (~2 minutes).

Centrifugation

Centrifuge as needed at any step in the procedure to consolidate liquid or beads in the bottom of the well, and to prevent sample loss.

Handling Beads

Pipette bead suspension slowly to prevent splashing and bubbles. u When mixing, mix thoroughly.

To avoid sample loss, confirm that no beads remain in pipette tips after resuspension and mixing steps.

When washing beads:

Use the appropriate magnet for the plate.

Dispense liquid so that beads on the side of the wells are wetted.

Keep the plate on the magnet until the instructions specify to remove it

Do not agitate the plate while on the magnetic stand.

Do not disturb the bead pellet.

Library Preparation

Extract RNA:

This step extracts RNA from decontaminated viral transport medium tubes

Quick-DNA/RNA Viral MagBead Procedure

QIAamp Viral RNA Mini Kit Procedure

Anneal RNA:

During this process the extracted RNA is annealed using random hexamers to prepare for cDNA synthesis.

Synthesize First Strand cDNA

This step reverse transcribes the RNA fragments primed with random hexamers into first strand cDNA using reverse transcriptase.

SAFE STOPPING POINT If you are stopping, seal the plate and store at -25°C to -15°C for up to 7 days.

Amplify cDNA:

This step uses two separate PCR reactions to amplify cDNA.

SAFE STOPPING POINT If you are stopping, seal the plate and store at -25°C to -15°C for up to 3 days.

Tagment PCR Amplicons (Do not centrifuge after beads are added)

This step uses EBLTS HT to tagment PCR amplicons, which is a process that fragments and tags the PCR amplicons with adapter sequences.

If beads are adhered to the side or top of the 96-well plate, centrifuge at 500 × g for 1 minute, and then pipette to resuspend.

Post Tagmentation Clean Up (Do not overdry beads)

This step washes the adapter-tagged amplicons before PCR amplification

Amplify Tagmented Amplicons

This step amplifies the tagmented amplicons using a PCR program. The PCR step adds prepaired 10 base pair Index 1 (i7) adapters, Index 2 (i5) adapters, and sequences required for sequencing cluster generation.

Pool and Clean Up Libraries

This step combines libraries from each 96-well sample plate into one 1.7 ml tube. Libraries of optimal size are then bound to magnetic beads, and fragments that are too small or large are wash away.

SAFE STOPPING POINT If you are stopping, cap the tube and store at -25°C to -15°C for up to 30 days.

Quantify and Normalize Libraries

Analyze 2 µl library pool using a Qubit dsDNA HS Assay kit. If libraries are outside the standard range, dilute to 1:10 concentration, and analyze again.

Calculate the molarity value using the following formula.

Use 400 bp as the average library size.

Dilute each library pool to a minimum of 30 µl at a normalized concentration 4 nM using RSB HT.

Pool and Dilute Libraries

This step pools and dilutes libraries to the starting concentration for your sequencing system. After diluting to the starting concentration, libraries are ready to be denatured and diluted to the final loading concentration.

Denature and Sequence (specific to sequencing instrument) – No denaturing for Iseq (libraries are denatured automatically onboard the instrument