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+# 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)
+
+