Dominance of Alpha and Iota variants in SARS-CoV-2 vaccine breakthrough infections in New York City

  1. Ralf Duerr
  2. Dacia Dimartino
  3. Christian Marier
  4. Paul Zappile
  5. Guiqing Wang
  6. Jennifer Lighter
  7. Brian Elbel
  8. Andrea B. Troxel
  9. Adriana Heguy

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Abstract

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  1. Version published to 10.1172/jci152702
  2. ScreenIT

    SciScore for 10.1101/2021.07.05.21259547: (What is this?)

    Please note, not all rigor criteria are appropriate for all manuscripts.

    Table 1: Rigor

    EthicsIRB: Study Approval: This study was approved by the NYU Langone Health Institutional Review Board, protocol number i21-00493. cDNA synthesis, library preparation and sequencing: Total RNA (11 μl) was converted to first strand cDNA by random priming using the Superscript IV first-strand synthesis system (Invitrogen, ref# 180901050).
    Sex as a biological variablenot detected.
    RandomizationThe control group consisted of full-genome sequenced SARS-CoV-2 positive cases in our health system who were randomly selected for SARS-CoV-2 genomic surveillance, had Ct ≤30, and were collected in the same time period as the breakthrough infections.
    Blindingnot detected.
    Power Analysisnot detected.

    Table 2: Resources

    Software and Algorithms
    SentencesResources
    Sequenced read processing: Sequencing reads were demultiplexed using the Illumina bcl2fastq2 Conversion Software v2.20 and adapters and low-quality bases were trimmed with Trimmomatic v0.36 27.
    Trimmomatic
    suggested: (Trimmomatic, RRID:SCR_011848)
    BWA v0.7.17 28 was utilized for mapping reads to the SARS-CoV-2 reference genome (NC_045512.2, wuhCor1) and mapped reads were soft-clipped to remove SNAP tiled primer sequences using Primerclip v0.3.8 29.
    BWA
    suggested: (BWA, RRID:SCR_010910)
    Maximum likelihood IQ trees were performed using the IQ-TREE XSEDE tool, multicore version 2.1.2, on the Cipres Science gateway v.3.3 33. GTR+I+G was chosen as the best-fit substitution model.
    IQ-TREE
    suggested: (IQ-TREE, RRID:SCR_017254)
    Propensity-score matching was implemented using the nearest neighbor strategy using a 1:1 ratio without replacement, with the MatchIt algorithm in RStudio version 1.4.1106 39.
    RStudio
    suggested: (RStudio, RRID:SCR_000432)
    Libraries were prepared using Swift Normalase Amplicon Panel (SNAP) SARS-CoV-2 and Sar-Cov-2 additional Genome Coverage (Cat# SN-5×296 core kit, 96rxn), using 10 μl of first strand cDNA, following the manufacturer’s instruction: https://swiftbiosci.com/wp-content/uploads/2021/06/PRT-028-Swift-Normalase-Amplicon-Panels-SNAP-SARS-CoV-2-Panels-Rev-9.pdf).
    SNAP
    suggested: (SNAP, RRID:SCR_007936)
    The BWA-MEM algorithm of the alignment program, BWA v0.7.17 (Li and Durbin 2009), was utilized for mapping reads of each sample to the SARS-CoV-2 reference genome (NC_045512.2, wuhCor1) with the ‘-M’ flag for marking shorter split hits as secondary and the parameter ‘-U’ set to a 17 alignment score penalty for an unpaired read pair.
    BWA-MEM
    suggested: (Sniffles, RRID:SCR_017619)
    Name-sorted mapped reads in SAM alignment format were soft-clipped to remove primer sequences specific to the short overlapping (tiled) amplicon design of the Swift Normalase Amplicon SARS-CoV-2 Panel using Primerclip v0.3.8 (Swift Biosciences: https://github.com/swiftbiosciences/primerclip), and the Picard v2.18.20 “AddOrReplaceReadGroups” tool (Broad Institute: http://broadinstitute.github.io/picard/) was used in converting the primer-trimmed SAM files to coordinate-sorted and indexed BAM format with read-groups added for downstream analysis.
    Picard
    suggested: (Picard, RRID:SCR_006525)
    The variant caller, BCFtools v1.9 (Li et al. 2009), was utilized to detect genetic mutations of the collected viral samples from BAM input: bcftools mpileup -r NC_045512.2 --count-orphans --no-BAQ --max-depth 50000 --max-idepth 500000 –annotate FORMAT/AD,FORMAT/ADF,FORMAT/ADR,FORMAT/DP,FORMAT/SP,INFO/AD,INFO/ADF,INFO/A DR --output-type v | bcftools call --ploidy 1 --keep-alts --multiallelic-caller --output-type v | bcftools norm --multiallelics -any --output-type v | bcftools filter -i “(DP4[0]+DP4[1])<(DP4[2]+DP4[3]) && ((DP4[2]+DP4[3])>0) & FORMAT/AD[0:1]>20” --output-type v | bcftools filter -e “IMF<0.5” --output-type v To generate viral sequences, the output in VCF format was applied to the reference sequence using ‘bcftools consensus’ while the BEDTools v2.30.0 utilities (Quinlan and Hall 2010), “genomecov” (with the ‘-bga’ option to report depth at each base including zero coverage sites), “merge” (for creating a BED file of low depth sites with <1000x coverage to be masked), and “maskfasta” in conjunction with the multiple sequence alignment program, MAFFT v4.471 (Katoh et al. 2002) in ‘--auto’ mode, were used in the production of final consensus sequences with all bases below 1000x coverage masked and both non-targeted ends of the sequences trimmed.
    BCFtools
    suggested: (SAMtools/BCFtools, RRID:SCR_005227)
    BEDTools
    suggested: (BEDTools, RRID:SCR_006646)
    MAFFT
    suggested: (MAFFT, RRID:SCR_011811)

    Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

    Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:
    Compared to the large number of SARS-CoV-2 infections among unvaccinated individuals, the recorded breakthrough cases between February and April 2021 (n=67) remained at∼1% of total infections, with the caveat that both breakthrough cases as well as unvaccinated controls were not exhaustively screened and covered. Despite the overall effectiveness of vaccination, our full spike mutation analysis revealed a broad set of spike mutations (n=21) to be elevated in the vaccine breakthrough group. It indicates that adaptive selection is in progress that may subsequently come into full effect. At this point, the breakthrough cases and differences in mutation rates between case and control groups are still too low to draw meaningful conclusions. However, the overrepresentation of functionally important spike mutations including NTD deletions ΔH69-V70 and ΔY144 together with RBD mutations K417X, S477X, E484X, and N501X as well as P681X (near the S1/S2 interface) in the absence of a clear elevation of VOCs or VOIs may indicate a starting sieve effect at individual or combinations of functional mutations. Spike mutations and deletions reported to confer neutralization escape in vitro23, 43, 44 might thus be responsible for a sieve effect in a real-life situation, i.e., among vaccinated individuals. During the time of our sample and data collection, there were two major variants arising in the New York City metro area constituting about two thirds of all cases, B.1.1.7, which was first rep...

    Results from TrialIdentifier: No clinical trial numbers were referenced.

    Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

    Results from JetFighter: We did not find any issues relating to colormaps.

    Results from rtransparent:
    • Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
    • Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
    • No protocol registration statement was detected.

    Results from scite Reference Check: We found no unreliable references.

    About SciScore

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  3. Version published to 10.1101/2021.07.05.21259547 on medRxiv