diff --git a/sequenced.md b/sequenced.md new file mode 100644 index 0000000..c5d7d8c --- /dev/null +++ b/sequenced.md @@ -0,0 +1,197 @@ +# Assemblies of putative SARS-CoV2-spike-encoding mRNA sequences for +vaccines BNT-162b2 and mRNA-1273. + +### (version 0.1.1Beta 03/23/21) + +Dae Eun Jeong, Matthew McCoy, Karen Artiles, OrkanIlbay, Andrew Fire*, Kari Nadeau, +Helen Park, Brooke Betts, Scott Boyd, Ramona Hoh, and Massa Shoura* + +Departments of Pathology, Genetics, Pediatrics, and Medicine, Stanford University School of +Medicine and Veterans Affairs Palo Alto Medical Center +*Correspondence: [afire@stanford.edu](afire@stanford.edu) and/or [massa86@stanford.edu](massa86@stanford.edu) + +RNA vaccines have become a key tool in moving forward through the challenges raised both +in the current pandemic and in numerous other publichealth and medical challenges. With +the rollout of vaccines for COVID-19, these synthetic mRNAs have become broadly +distributed RNA species in numerous human populations. Despite their ubiquity, sequences +are not always available for such RNAs. Standard methods facilitate such sequencing. In this +note, we provide experimental sequence informationfor the RNA components of the initial +[Moderna](https://pubmed.ncbi.nlm.nih.gov/32756549/)and [Pfizer/BioNTech](https://pubmed.ncbi.nlm.nih.gov/33301246/) +COVID-19 vaccines, allowing a working assembly of the former and a confirmation of previously +reported sequence information for the latter RNA. + +Sharing of sequence information for broadly used therapeutics has the benefit of allowing any +researchers or clinicians using sequencing approaches to rapidly identify such sequences as +therapeutic-derived rather than host or infectious in origin. + +For this work, RNAs were obtained as discards from the small portions of vaccine doses that +remained in vials after immunization; such portions would have been required to be +otherwise discarded and were analyzed under FDA authorization for research use. To obtain +the small amounts of RNA needed for characterization,vaccine remnants were +phenol-chloroform extracted using TRIzol Reagent (Invitrogen), with intactness assessed by +Agilent 2100 Bioanalyzer before and after extraction. + +Although our analysis mainly focused on RNAs obtained as soon as possible following +discard, we also analyzed samples which had been refrigerated (~4 °C) for up to 42 days with +and without the addition of EDTA. Interestingly a substantial fraction of the RNA remained +intact in these preparations. We note that the formulation of the vaccines includes numerous +key chemical components which are quite possibly unstable under these conditions-- so these +data certainly do not suggest that the vaccine as a biological agent is stable. But it is of +interest that chemical stability of RNA itself is not sufficient to preclude eventual +development of vaccines with a much less involved cold-chain storage and transportation. + +For further analysis, the initial RNAs were fragmentedby heating to 94°C, primed with a +random hexamer-tailed adaptor, amplified through atemplate-switch protocol (Takara +SMARTerer Stranded RNA-seq kit), and sequenced usinga MiSeq instrument (Illumina) with +paired end 78-per end sequencing. As a reference material in specific assays, we included +RNA of known concentration and sequence (from bacteriophage MS2). + + +From these data, we obtained partial information on strandedness and a set of segments that +could be used for assembly. This was particularly useful for the Moderna vaccine, for which +the original vaccine RNA sequence was not available at the time our study was carried out. +Contigs encoding full-length spikes were assembled from the Moderna and Pfizer datasets. +The Pfizer/BioNTech data [Figure 1] verified the [reported sequence for that vaccine](https://berthub.eu/articles/posts/reverse-engineering-source-code-of-the-biontech-pfizer-vaccine/), while +the Moderna sequence [Figure 2] could not be checked against a published reference. + +RNA preparations lacking dsRNA are desirable in generating vaccine formulations as these +will minimize an otherwise dramatic biological (and non-specific) response that vertebrates +have to double stranded character in RNA (https://www.nature.com/articles/nrd.2017.243). +In the sequence data that we analyzed, we found that the vast majority of reads were from the +expected sense strand. In addition, the minorityof antisense reads appeared different from +sense reads in lacking the characteristic extensions expected from the template switching +protocol. Examining only the reads with an evident template switch (as an indicator for +strand-of-origin), we observed that both vaccines overwhelmingly yielded sense reads +(>99.99%). Independent sequencing assays and otherexperimental measurements are +ongoing and will be needed to determine whether this template-switched sense read fraction +in the SmarterSeq protocol indeed represents the actual dsRNA content in the original +material. + +This work provides an initial assessment of two RNAs that are now a part of the human +ecosystem and that are likely to appear in numerous other high throughput RNA-seq studies +in which a fraction of the individuals may have previously been vaccinated. + +ProtoAcknowledgements: Thanks to our colleagues for help and suggestions (Nimit Jain, +Emily Greenwald, Lamia Wahba, William Wang, Amisha Kumar, Sameer Sundrani, David +Lipman, Marc Salit, Bijoyita Roy). + +### Figure 1: Spike-encoding contig assembled from BioNTech/PfizerBNT-162b +vaccine. + Although the full coding region is included,the nature of the methodology used for +sequencing and assembly is such that the assembled contig could lack some sequence from +the ends of the RNA. Within the assembled sequence, this hypothetical sequence shows a +perfect match to the corresponding sequence from documents available online derived from +manufacturer communications with the World Health Organization [as reported by +https://berthub.eu/articles/posts/reverse-engineering-source-code-of-the-biontech-pfizer-vaccine/]. The 5’ end for +the assembly matches the start site noted in these documents, while the read-based assembly +lacks an interrupted polyA tail (A30(GCATATGACT)A70) that is expected to be present in the +mRNA. + +### Figure 2: Spike-encoding contig assembled from Moderna mRNA-1273 vaccine.** +This is a partial sequence of the vaccine RNA. Although the full coding region is included, the +assembled contig could lack some sequence from the ends of the RNA. + + +**Figure 1: Spike-encoding contig assembled from BioNTech/PfizerBNT-162b vaccine.** + +GAGAATAAACTAGTATTCTTCTGGTCCCCACAGACTCAGAGAGAACCCGCCACCATGTTCGTGTTCCTGGTGCTGCTGCCTCTGGTGTCCAGCCAGTGTGTGAACCTGACCACCAGAACACAGCTGCCTCCAGCCTACACCAACAGCTTTACCAGAGGCGTGTACTACCCCGACAAGGTGTT +CAGATCCAGCGTGCTGCACTCTACCCAGGACCTGTTCCTGCCTTTCTTCAGCAACGTGACCTGGTTCCACGCCATCCACGTGTCCGGCACC +AATGGCACCAAGAGATTCGACAACCCCGTGCTGCCCTTCAACGACGGGGTGTACTTTGCCAGCACCGAGAAGTCCAACATCATCAGAGGCT +GGATCTTCGGCACCACACTGGACAGCAAGACCCAGAGCCTGCTGATCGTGAACAACGCCACCAACGTGGTCATCAAAGTGTGCGAGTTCCA +GTTCTGCAACGACCCCTTCCTGGGCGTCTACTACCACAAGAACAACAAGAGCTGGATGGAAAGCGAGTTCCGGGTGTACAGCAGCGCCAAC +AACTGCACCTTCGAGTACGTGTCCCAGCCTTTCCTGATGGACCTGGAAGGCAAGCAGGGCAACTTCAAGAACCTGCGCGAGTTCGTGTTTA +AGAACATCGACGGCTACTTCAAGATCTACAGCAAGCACACCCCTATCAACCTCGTGCGGGATCTGCCTCAGGGCTTCTCTGCTCTGGAACC +CCTGGTGGATCTGCCCATCGGCATCAACATCACCCGGTTTCAGACACTGCTGGCCCTGCACAGAAGCTACCTGACACCTGGCGATAGCAGC +AGCGGATGGACAGCTGGTGCCGCCGCTTACTATGTGGGCTACCTGCAGCCTAGAACCTTCCTGCTGAAGTACAACGAGAACGGCACCATCA +CCGACGCCGTGGATTGTGCTCTGGATCCTCTGAGCGAGACAAAGTGCACCCTGAAGTCCTTCACCGTGGAAAAGGGCATCTACCAGACCAG +CAACTTCCGGGTGCAGCCCACCGAATCCATCGTGCGGTTCCCCAATATCACCAATCTGTGCCCCTTCGGCGAGGTGTTCAATGCCACCAGA +TTCGCCTCTGTGTACGCCTGGAACCGGAAGCGGATCAGCAATTGCGTGGCCGACTACTCCGTGCTGTACAACTCCGCCAGCTTCAGCACCT +TCAAGTGCTACGGCGTGTCCCCTACCAAGCTGAACGACCTGTGCTTCACAAACGTGTACGCCGACAGCTTCGTGATCCGGGGAGATGAAGT +GCGGCAGATTGCCCCTGGACAGACAGGCAAGATCGCCGACTACAACTACAAGCTGCCCGACGACTTCACCGGCTGTGTGATTGCCTGGAAC +AGCAACAACCTGGACTCCAAAGTCGGCGGCAACTACAATTACCTGTACCGGCTGTTCCGGAAGTCCAATCTGAAGCCCTTCGAGCGGGACA +TCTCCACCGAGATCTATCAGGCCGGCAGCACCCCTTGTAACGGCGTGGAAGGCTTCAACTGCTACTTCCCACTGCAGTCCTACGGCTTTCA +GCCCACAAATGGCGTGGGCTATCAGCCCTACAGAGTGGTGGTGCTGAGCTTCGAACTGCTGCATGCCCCTGCCACAGTGTGCGGCCCTAAG +AAAAGCACCAATCTCGTGAAGAACAAATGCGTGAACTTCAACTTCAACGGCCTGACCGGCACCGGCGTGCTGACAGAGAGCAACAAGAAGT +TCCTGCCATTCCAGCAGTTTGGCCGGGATATCGCCGATACCACAGACGCCGTTAGAGATCCCCAGACACTGGAAATCCTGGACATCACCCC +TTGCAGCTTCGGCGGAGTGTCTGTGATCACCCCTGGCACCAACACCAGCAATCAGGTGGCAGTGCTGTACCAGGACGTGAACTGTACCGAA +GTGCCCGTGGCCATTCACGCCGATCAGCTGACACCTACATGGCGGGTGTACTCCACCGGCAGCAATGTGTTTCAGACCAGAGCCGGCTGTC +TGATCGGAGCCGAGCACGTGAACAATAGCTACGAGTGCGACATCCCCATCGGCGCTGGAATCTGCGCCAGCTACCAGACACAGACAAACAG +CCCTCGGAGAGCCAGAAGCGTGGCCAGCCAGAGCATCATTGCCTACACAATGTCTCTGGGCGCCGAGAACAGCGTGGCCTACTCCAACAAC +TCTATCGCTATCCCCACCAACTTCACCATCAGCGTGACCACAGAGATCCTGCCTGTGTCCATGACCAAGACCAGCGTGGACTGCACCATGT +ACATCTGCGGCGATTCCACCGAGTGCTCCAACCTGCTGCTGCAGTACGGCAGCTTCTGCACCCAGCTGAATAGAGCCCTGACAGGGATCGC +CGTGGAACAGGACAAGAACACCCAAGAGGTGTTCGCCCAAGTGAAGCAGATCTACAAGACCCCTCCTATCAAGGACTTCGGCGGCTTCAAT +TTCAGCCAGATTCTGCCCGATCCTAGCAAGCCCAGCAAGCGGAGCTTCATCGAGGACCTGCTGTTCAACAAAGTGACACTGGCCGACGCCG +GCTTCATCAAGCAGTATGGCGATTGTCTGGGCGACATTGCCGCCAGGGATCTGATTTGCGCCCAGAAGTTTAACGGACTGACAGTGCTGCC +TCCTCTGCTGACCGATGAGATGATCGCCCAGTACACATCTGCCCTGCTGGCCGGCACAATCACAAGCGGCTGGACATTTGGAGCAGGCGCC +GCTCTGCAGATCCCCTTTGCTATGCAGATGGCCTACCGGTTCAACGGCATCGGAGTGACCCAGAATGTGCTGTACGAGAACCAGAAGCTGA +TCGCCAACCAGTTCAACAGCGCCATCGGCAAGATCCAGGACAGCCTGAGCAGCACAGCAAGCGCCCTGGGAAAGCTGCAGGACGTGGTCAA +CCAGAATGCCCAGGCACTGAACACCCTGGTCAAGCAGCTGTCCTCCAACTTCGGCGCCATCAGCTCTGTGCTGAACGATATCCTGAGCAGA +CTGGACCCTCCTGAGGCCGAGGTGCAGATCGACAGACTGATCACAGGCAGACTGCAGAGCCTCCAGACATACGTGACCCAGCAGCTGATCA +GAGCCGCCGAGATTAGAGCCTCTGCCAATCTGGCCGCCACCAAGATGTCTGAGTGTGTGCTGGGCCAGAGCAAGAGAGTGGACTTTTGCGG +CAAGGGCTACCACCTGATGACTCGAGCTGGTACTGCATGCACGCAATGCTAGCTGCCCCTTTCCCGTCCTGGGTACCCCGAGTCTCCCCCGACCTCGGGTCCCAGGTATGCTCCCACCTCCACCTGCCCCACTCACCACCTCTGCTAGTTCCAGACACCTCCCAAGCACGCAGCAATGCAGCTCAAAACGCTTAGCCTAGCCACACCCCCACGGGAAACAGCAGTGATTAACCTTTAGCAATAAACGAAAGTTTAACTAAGCTATACTAACCCCAGGGTTGGTCAATTTCGTGCCAGCCACACCCTGGAGCTAGCA + + +Cyan: Putative 5’ UTR +Green: Start Codon +Yellow: Signal Peptide +Orange: Spike encoding region +Red: Stop codon(s) +Purple: 3’ UTR +Blue: Start of polyA region (incomplete) + + +**Figure 2: Spike-encoding contig assembled from Moderna mRNA-1273 vaccine.** + +GGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGACCCCGGCGCCGCCACCATGTTCGTGTTCCTGGTGCTGCTGCCCCTGGTGAGCAGCCAGTGCGTGAACCTGACCACCCGGACCCAGCTGCCACCAGCCTACACCAACAGCTTCACCCGGGGCGTCTACTACCCCGACAAGGT +GTTCCGGAGCAGCGTCCTGCACAGCACCCAGGACCTGTTCCTGCCCTTCTTCAGCAACGTGACCTGGTTCCACGCCATCCACGTGAGCGGC +ACCAACGGCACCAAGCGGTTCGACAACCCCGTGCTGCCCTTCAACGACGGCGTGTACTTCGCCAGCACCGAGAAGAGCAACATCATCCGGG +GCTGGATCTTCGGCACCACCCTGGACAGCAAGACCCAGAGCCTGCTGATCGTGAATAACGCCACCAACGTGGTGATCAAGGTGTGCGAGTT +CCAGTTCTGCAACGACCCCTTCCTGGGCGTGTACTACCACAAGAACAACAAGAGCTGGATGGAGAGCGAGTTCCGGGTGTACAGCAGCGCC +AACAACTGCACCTTCGAGTACGTGAGCCAGCCCTTCCTGATGGACCTGGAGGGCAAGCAGGGCAACTTCAAGAACCTGCGGGAGTTCGTGT +TCAAGAACATCGACGGCTACTTCAAGATCTACAGCAAGCACACCCCAATCAACCTGGTGCGGGATCTGCCCCAGGGCTTCTCAGCCCTGGA +GCCCCTGGTGGACCTGCCCATCGGCATCAACATCACCCGGTTCCAGACCCTGCTGGCCCTGCACCGGAGCTACCTGACCCCAGGCGACAGC +AGCAGCGGGTGGACAGCAGGCGCGGCTGCTTACTACGTGGGCTACCTGCAGCCCCGGACCTTCCTGCTGAAGTACAACGAGAACGGCACCA +TCACCGACGCCGTGGACTGCGCCCTGGACCCTCTGAGCGAGACCAAGTGCACCCTGAAGAGCTTCACCGTGGAGAAGGGCATCTACCAGAC +CAGCAACTTCCGGGTGCAGCCCACCGAGAGCATCGTGCGGTTCCCCAACATCACCAACCTGTGCCCCTTCGGCGAGGTGTTCAACGCCACC +CGGTTCGCCAGCGTGTACGCCTGGAACCGGAAGCGGATCAGCAACTGCGTGGCCGACTACAGCGTGCTGTACAACAGCGCCAGCTTCAGCA +CCTTCAAGTGCTACGGCGTGAGCCCCACCAAGCTGAACGACCTGTGCTTCACCAACGTGTACGCCGACAGCTTCGTGATCCGTGGCGACGA +GGTGCGGCAGATCGCACCCGGCCAGACAGGCAAGATCGCCGACTACAACTACAAGCTGCCCGACGACTTCACCGGCTGCGTGATCGCCTGG +AACAGCAACAACCTCGACAGCAAGGTGGGCGGCAACTACAACTACCTGTACCGGCTGTTCCGGAAGAGCAACCTGAAGCCCTTCGAGCGGG +ACATCAGCACCGAGATCTACCAAGCCGGCTCCACCCCTTGCAACGGCGTGGAGGGCTTCAACTGCTACTTCCCTCTGCAGAGCTACGGCTT +CCAGCCCACCAACGGCGTGGGCTACCAGCCCTACCGGGTGGTGGTGCTGAGCTTCGAGCTGCTGCACGCCCCAGCCACCGTGTGTGGCCCC +AAGAAGAGCACCAACCTGGTGAAGAACAAGTGCGTGAACTTCAACTTCAACGGCCTTACCGGCACCGGCGTGCTGACCGAGAGCAACAAGA +AATTCCTGCCCTTTCAGCAGTTCGGCCGGGACATCGCCGACACCACCGACGCTGTGCGGGATCCCCAGACCCTGGAGATCCTGGACATCAC +CCCTTGCAGCTTCGGCGGCGTGAGCGTGATCACCCCAGGCACCAACACCAGCAACCAGGTGGCCGTGCTGTACCAGGACGTGAACTGCACC +GAGGTGCCCGTGGCCATCCACGCCGACCAGCTGACACCCACCTGGCGGGTCTACAGCACCGGCAGCAACGTGTTCCAGACCCGGGCCGGTT +GCCTGATCGGCGCCGAGCACGTGAACAACAGCTACGAGTGCGACATCCCCATCGGCGCCGGCATCTGTGCCAGCTACCAGACCCAGACCAA +TTCACCCCGGAGGGCAAGGAGCGTGGCCAGCCAGAGCATCATCGCCTACACCATGAGCCTGGGCGCCGAGAACAGCGTGGCCTACAGCAAC +AACAGCATCGCCATCCCCACCAACTTCACCATCAGCGTGACCACCGAGATTCTGCCCGTGAGCATGACCAAGACCAGCGTGGACTGCACCA +TGTACATCTGCGGCGACAGCACCGAGTGCAGCAACCTGCTGCTGCAGTACGGCAGCTTCTGCACCCAGCTGAACCGGGCCCTGACCGGCAT +CGCCGTGGAGCAGGACAAGAACACCCAGGAGGTGTTCGCCCAGGTGAAGCAGATCTACAAGACCCCTCCCATCAAGGACTTCGGCGGCTTC +AACTTCAGCCAGATCCTGCCCGACCCCAGCAAGCCCAGCAAGCGGAGCTTCATCGAGGACCTGCTGTTCAACAAGGTGACCCTAGCCGACG +CCGGCTTCATCAAGCAGTACGGCGACTGCCTCGGCGACATAGCCGCCCGGGACCTGATCTGCGCCCAGAAGTTCAACGGCCTGACCGTGCT +GCCTCCCCTGCTGACCGACGAGATGATCGCCCAGTACACCAGCGCCCTGTTAGCCGGAACCATCACCAGCGGCTGGACTTTCGGCGCTGGA +GCCGCTCTGCAGATCCCCTTCGCCATGCAGATGGCCTACCGGTTCAACGGCATCGGCGTGACCCAGAACGTGCTGTACGAGAACCAGAAGC +TGATCGCCAACCAGTTCAACAGCGCCATCGGCAAGATCCAGGACAGCCTGAGCAGCACCGCTAGCGCCCTGGGCAAGCTGCAGGACGTGGT +GAACCAGAACGCCCAGGCCCTGAACACCCTGGTGAAGCAGCTGAGCAGCAACTTCGGCGCCATCAGCAGCGTGCTGAACGACATCCTGAGC +CGGCTGGACCCTCCCGAGGCCGAGGTGCAGATCGACCGGCTGATCACTGGCCGGCTGCAGAGCCTGCAGACCTACGTGACCCAGCAGCTGA +TCCGGGCCGCCGAGATTCGGGCCAGCGCCAACCTGGCCGCCACCAAGATGAGCGAGTGCGTGCTGGGCCAGAGCAAGCGGGTGGACTTCTG +CGGCAAGGGCTACCACCTGATGAGCTTTCCCCAGAGCGCACCCCACGGAGTGGTGTTCCTGCACGTGACCTACGTGCCCGCCCAGGAGAAG +AACTTCACCACCGCCCCAGCCATCTGCCACGACGGCAAGGCCCACTTTCCCCGGGAGGGCGTGTTCGTGAGCAACGGCACCCACTGGTTCG +TGACCCAGCGGAACTTCTACGAGCCCCAGATCATCACCACCGACAACACCTTCGTGAGCGGCAACTGCGACGTGGTGATCGGCATCGTGAA +CAACACCGTGTACGATCCCCTGCAGCCCGAGCTGGACAGCTTCAAGGAGGAGCTGGACAAGTACTTCAAGAATCACACCAGCCCCGACGTG +GACCTGGGCGACATCAGCGGCATCAACGCCAGCGTGGTGAACATCCAGAAGGAGATCGATCGGCTGAACGAGGTGGCCAAGAACCTGAACG +AGAGCCTGATCGACCTGCAGGAGCTGGGCAAGTACGAGCAGTACATCAAGTGGCCCTGGTACATCTGGCTGGGCTTCATCGCCGGCCTGAT +CGCCATCGTGATGGTGACCATCATGCTGTGCTGCATGACCAGCTGCTGCAGCTGCCTGAAGGGCTGTTGCAGCTGCGGCAGCTGCTGCAAG +TTCGACGAGGACGACAGCGAGCCCGTGCTGAAGGGCGTGAAGCTGCACTACACCTGATAATAGGCTGGAGCCTCGGTGGCCTAGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGCAAAAAAAAA + + +Cyan: Putative 5’ UTR +Green: Start Codon +Yellow: Signal Peptide +Orange: Spike encoding region +Red: Stop codon(s) +Purple: 3’ UTR +Blue: Start of polyA region (incomplete) + +