Overview

We use a synthetic RNA spike-in as a control in each of our wells. The sequence is meant to mimic the actual SARS CoV-2 amplicon (same amplicon structure except for a 6 nt unique stretch to distinguish). The use of this synthetic spike in has two important advantages.

  1. This serves as a positive reaction control. If we can see reads from the spike-in in each well, we know that the RT-PCR reaction for the viral sequence configuration worked and more confident in a negative result (in conjunction with reads from a human RNA sample as well).
  2. The use of a ratio to measure viral copy numbers is much more robust to small changes in experimental conditions due to differences in samples, noise and amplification biases. This allows us to detect more dilute viral samples.
  3. The spike in ensures there is amplicon in each well, and thus the endpoint pcr acts as a normalization to avoid downstream normalizing for sequencing.

Design/Construction

We RT-PCRed synthetic SARS CoV-2 RNA from Twist, adding on a T7 promoter in order for us to in vitro transcribe the spike RNA. For the primers used in spike construction, look here. Below are .gb files of the T7 spike in templates. NOTE: Please consult the primer contamination page about our experience getting contaminated oligos when ordering the T7 template primer for the N1 spike.

s2_synthetic-spike_t7-template.gb

n1_synthetic-spike_t7-template.gb

For in vitro transcription, we used the NEB HiScribe kit. The protocol we used is below:

After IVT, we DNase treated the reactions, purified the RNA with a Zymo RNA clean and concentrator column, and quantified with an Agilent TapeStation.

Implementation

We added 100 copies of the spike RNA per reaction into our mastermix before aliquoting. We didn't spend much time optimizing spike concentration, but we wanted to include it at a high enough concentration to ensure accurate quantitation but also not too high to waste sequencing reads.

Effect on Normalization

Before:

https://s3-us-west-2.amazonaws.com/secure.notion-static.com/f8524ac1-f477-407a-b2cd-e5ab62bfdc44/preview(131).pdf

After: