In this post I review technical problems with the CDC COVID-19 primers and I describe how I generated a new set of primers and probes without those problems.
Note that this was based on available outbreak whole-genome sequence data obtained from the NCBI Blast NT database, downloaded from NCBI on 2020-03-02. I will try to re-run this pipeline every few days to update the set of candidate primers in light of newly-available sequence data. Please check these primers yourself before using them for the basis of any diagnostic kit.
This blog post describes technical problems with some of the COVID-19 primer-probe sets that are being promoted by the CDC to diagnose cases of COVID-19 infections. Several of these primers have dimerization and hairpin loop issues, among others. Here, I describe a bioinformatic pipeline to design better candidate primers that pass stringent design criteria. Using this pipeline, I generated a new set of primers and probes without the aforementioned technical issues of the CDC COVID-19 primer probe sets, and tested their in silico precision and recall using the most recent set of COVID-19 complete genomes from NCBI. The ten best primer-probe pairs have perfect classification performance (recall, precision, and F1-score all 1.0). I provide these candidate primers for free, under the Creative Commons Attribution 4.0 International (CC BY 4.0) license.
My hope is that this blog post will help start a conversation about the design process for these critical diagnostic primers. Starting with these primers, and with input from other scientists, we can develop a set of candidate primers for COVID-19 that could be used by health agencies and private diagnostic kit manufacturers alike to not only generate more diagnostic kits, but better ones.
Primer Design Issues
As the COVID-19 pandemic spread from country-to-country over the past month, I felt that I should try to help in some way. I had recently been working on primer design as part of my consultancy, and I thought that perhaps I can analyze the primers developed by the CDC and others, in part to see how their primers stack up against the ones that I had developed for a different project.
As others have noted, there are flaws in the COVID-19 diagnostic primers from the CDC. They released four primer-probe sets; three that target the N gene, which encodes for a nucleocapsid phosphoprotein, and one that targets a human RNA polymerase gene (as part of laboratory controls).
Using Primer3, a respected software program for predicting performant primer probe sets using thermodynamic calculations, I analyzed the three N gene primer probe sets. I found the following problems with each set:
Here is primer probe set N1, shown in Boulder format (as used by Primer3):
SEQUENCE_PRIMER=GACCCCAAAATCAGCGAAAT SEQUENCE_INTERNAL_OLIGO=ACCCCGCATTACGTTTGGTGGACC SEQUENCE_PRIMER_REVCOMP=TCTGGTTACTGCCAGTTGAATCTG
Here is the formatted analysis summary from Primer3 for N1 (note that in the terminology of Primer3, the forward primer is termed “Left”, the reverse primer is termed “Right”, and the hybridization probe is termed “Internal Oligo”):
OLIGO start len tm gc% any_th 3'_th hairpin seq LEFT PRIMER 28286 20 58.32 45.00 0.00 0.00 0.00 GACCCCAAAATCAGCGAAAT RIGHT PRIMER 28357 24 62.50 45.83 2.43 0.00 52.19 TCTGGTTACTGCCAGTTGAATCTG INTERNAL OLIGO 28308 24 69.19 58.33 13.48 0.00 42.14 ACCCCGCATTACGTTTGGTGGACC SEQUENCE SIZE: 29902 INCLUDED REGION SIZE: 29902 PRODUCT SIZE: 72, PAIR ANY_TH COMPL: 0.00, PAIR 3'_TH COMPL: 0.00
The reverse primer was rejected by Primer3 due to a predicted hairpin loop. These loop formations can cause problems during PCR, leading to lower amplification efficiency. The Primer3 output for the hairpin is as follows:
SEQUENCE_ID=CDC-N1-COVID-19 Reverse primer: Tm: 52.2°C dG: -1596 cal/mol dH: -34200 cal/mol dS: -105 cal/mol*K 5' TCTGGTTA* |||| | 3' GTCTAAGTTGACCGTC*
While lower than the default melting temperature cutoff, Primer3 found more problems with the primer probe set. Namely, it can form oligo-dimers with the reverse primer and the hybridization probe (with themselves), and the hybridization probe itself can form a hairpin loop:
Reverse primer self-dimer:
Tm: 2.4°C dG: -5495 cal/mol dH: -45600 cal/mol dS: -129 cal/mol*K 5' TCTGGTTACTGCCAGTTGAATCTG 3' |||| |||| 3' GTCTAAGTTGACCGTCATTGGTCT 5'
Hybridization probe self-dimer:
Tm: 13.5°C dG: -4045 cal/mol dH: -87400 cal/mol dS: -269 cal/mol*K 5' ACCCCGCATTACGTTTGGTGGACC 3' || | |||| | || 3' CCAGGTGGTTTGCATTACGCCCCA 5'
Hybridization probe 3’ hairpin loop:
Tm: 42.1°C dG: -274 cal/mol dH: -16800 cal/mol dS: -53 cal/mol*K 5' ACCCCGCA* || T 3' CCAGGTGGTTTGCAT*
Here is set N2:
SEQUENCE_PRIMER=TTACAAACATTGGCCGCAAA SEQUENCE_INTERNAL_OLIGO=ACAATTTGCCCCCAGCGCTTCAG SEQUENCE_PRIMER_REVCOMP=GCGCGACATTCCGAAGAA
Just from inspection, you can see that there is an issue with a poly-X run of five ‘C’s in the hybridization probe. Poly-X runs of five bases or longer are known to cause problems with non-specific priming (possibly leading to false positive readings), and are normally avoided.
Here is the formatted analysis summary from Primer3 for N2:
OLIGO start len tm gc% any_th 3'_th hairpin seq LEFT PRIMER 29163 20 58.75 40.00 0.00 0.00 34.76 TTACAAACATTGGCCGCAAA RIGHT PRIMER 29229 18 60.10 55.56 12.76 0.00 36.75 GCGCGACATTCCGAAGAA INTERNAL OLIGO 29187 23 68.15 56.52 8.63 0.00 49.09 ACAATTTGCCCCCAGCGCTTCAG SEQUENCE SIZE: 29902 INCLUDED REGION SIZE: 29902 PRODUCT SIZE: 67, PAIR ANY_TH COMPL: 0.00, PAIR 3'_TH COMPL: 0.00
Primer3 found self-dimer and hairpin loop issues with all of the oligos in the N2 primer-probe set, though at lower temeratures.
Here is set N3:
SEQUENCE_PRIMER=GGGAGCCTTGAATACACCAAAA SEQUENCE_INTERNAL_OLIGO=ATCACATTGGCACCCGCAATCCTG SEQUENCE_PRIMER_REVCOMP=TGTAGCACGATTGCAGCATTG
The forward primer, while having a C base in the last five base positions, has it at the 5’ end of that region, with a poly-A tail following. While runs of a single base of one to four base pairs in length are normally tolerated, it’s worrisome to place one at the 3’ end. It also had a predicted hairpin loop:
SEQUENCE_ID=CDC-N3-COVID-19 Reverse primer: Tm: 53.8°C dG: -1210 cal/mol dH: -23500 cal/mol dS: -72 cal/mol*K 5' TGTAGCACG* ||| | 3' GTTACGACGTTA*
Here is the formatted analysis summary from Primer3 for N3:
OLIGO start len tm gc% any_th 3'_th hairpin seq LEFT PRIMER 28680 22 60.53 45.45 0.00 0.00 0.00 GGGAGCCTTGAATACACCAAAA RIGHT PRIMER 28751 21 61.50 47.62 0.00 0.00 53.84 TGTAGCACGATTGCAGCATTG INTERNAL OLIGO 28703 24 67.77 54.17 0.00 0.00 41.98 ATCACATTGGCACCCGCAATCCTG SEQUENCE SIZE: 29902 INCLUDED REGION SIZE: 29902 PRODUCT SIZE: 72, PAIR ANY_TH COMPL: 0.00, PAIR 3'_TH COMPL: 0.00
Developing New Candidate Primers
Primer Design Criteria
In order to design better-performing COVID-19 primers, I developed a bioinformatic pipeline that generates primers using the following criteria:
- Target regions conserved perfectly among all COVID complete genomes (41 downloaded on 2020-03-02; check NCBI Nucleotide for the current list)
- Poly-X runs longer than four bases are not allowed
- Check that hybridization probes do not start with G (avoid quenching)
- Check for GC clamp on the forward and reverse primers (3’ end has one or two G’s or C’s in last five base pairs)
- GC% range between 40 and 60, with an optimum at 50
- Primer and probe size range between 18 to 27 bases
- Amplicon size from 75 to 200 bases
- Disallow any hairpins, self-dimers, or oligo-interactions at ANY temperature
- Exclude regions that are identical to the four known common cold-causing, human-associated coronaviruses(229E, NL63, OC43, and HKU1)
- Primers designed to cover the viral RNA polymerase gene, as it tends to be highly conserved within an RNA viral species, and different from the RNA polymerase sequence of different RNA viral species (as per Tom Slezak, former head of biodefense program at LLNL, private communication)
The New Candidate Primers Files
The set of candidate primers (last generated on 2020-03-11) can be downloaded here in XLSX spreadsheet format, and here as a comma-separated value (“CSV”) file. These are released as Creative Commons Attribution 4.0 International 4.0 (CC BY 4.0). I recommend focusing on the top ten primer-probe sets, as they have the greatest performance (based on the F1-score).
Below find selected details about how the primers were designed and validated using an ‘e-PCR’ approach.
Primer Performance Validation in silico
Even if you design your primers using recommended settings with trusted software like Primer3, there’s still the chance that you’ll have off-target binding of the oligos to other stretches of DNA in your sample, or even off-target amplicons if a forward and reverse primer bind close enough to one another in the correct orientation. For that reason, I analyzed the in silico performance of the newly designed primers to determine their predicted recall and precision. To do so, I performed a sequence homology search using NCBI Blast+ (version 2.10.0) against the NT database (downloaded 2020-03-02). Complete genomes (whether prokaryotic or viral) where the forward and reverse primers, and the hybridization probe, all had gapless alignments with 90% or greater sequence identity, and in the proper orientation, were counted as a hit. I used all complete viral genomes in NT with NCBI Taxonomy Database identifier 2697049 (the identifier for the COVID-19 sequences) as the “gold standard” for assessing the performance of these primer probe sets as diagnostics for COVID-19 presence (i.e., these are the sequences that the primers should hit without fail).
The spreadsheet shows that many of the newly-designed primers have five false negatives, but this is actually a bug in my current pipeline. It’s due to an obscure detail about how NCBI represents identical genomes in its Blast databases. Those genomes are all identified, just under a different identifier. So the top ten primer-probe pairs actually have perfect performance: precision, recall, and F1-score all 1.0.
Thank you for reading this far. My hope is that I can start up a conversation among scientists about how to design and validate candidate primer probes /in silico/, to allow for faster and more efficient wet-bench validation of the candidate primers. I hope this will lead to better, more accurate COVID-19 diagnostic kits being designed and successfully deployed around the country.
Please feel free to follow up with me via email (blog-at-me.tomeraltman.net) or Twitter (@tomeraltman). I look forward to constructive feedback about how to improve this analysis. I am working on releasing the source code for the bioinformatic pipeline, and writing up the methodology in more detail. Thanks again for your time, and thanks in advance for any help that you might provide.
- Perform same analysis on WHO recommended COVID-19 primers
- Release software pipeline code
- Complete secondary structure code blocks for low-temperature entries
- Fix false negative reporting bug due to NCBI Blast databases [FIXED: see update post]
- Prepare technical manuscript detailing the methodology
My deepest gratitude to the following individuals for helping me by reviewing this post:
[2020-03-06] Incorporated suggestions and fixes from Nathan Walsh and Nora Callahan. Thanks!
[2020-03-11] Released latest version of COVID-19 candidate primers. Updated file links. See this update post for more information.
[2020-03-12] The formatted analysis summary for N3 was accidentally copied from N2. Fixed.