Genomic evidence for reinfection with SARS-CoV-2: a case study

Genomic evidence for reinfection with SARS-CoV-2: a case study
Genomic evidence for reinfection with SARS-CoV-2: a case study


The samples were taken from the patient with a nasopharyngeal swab at the group examination, during the isolation and recovery period, and when presented to the hospital. The swabs were transported to the Nevada State Public Health Laboratory (Reno, NV, USA) either with viral transport medium or with Aptima Multiswab Transport Media (Hologic, San Diego, CA, USA). The samples were transported on cold packs and stored refrigerated (4-8 ° C) for no longer than 72 hours before nucleic acid extraction and subsequent real-time RT-PCR.

Nucleic acid extraction was carried out using the Omega Biotek MagBind kit for viral DNA / RNA 96 (Omega Bio-tek, Norcross, GA, USA) according to the manufacturer’s instructions and with an elution volume of 100 μl. l carried out. Aliquots of eluted RNA were subjected to real-time RT-PCR, either using the Taqpath COVID-19 Multiplex Assay (ThermoScientific, Waltham, MA, USA; 10 μL aliquots) or the US Centers for Disease Control and Prevention (CDC)) 2019-nCoV real-time RT-qPCR diagnostic panel (CDC, Atlanta, GA, USA; 5 μL aliquots). Samples transported on Aptima Multiswab Transport Media were tested by transcription-mediated amplification using the Aptima SARS-CoV-2 assay (Panther System) (Hologic, Marlborough, MA, USA). The tests were performed according to the respective EUA procedures, unless otherwise stated. For the Taqpath real-time RT-PCR test, the sample recall threshold was positive for the reactivity of two out of three target sequences, each with a reactivity at a cycle threshold of less than 40.00. A positive or negative result from the Hologic Aptima assay was based on proprietary processes. The antibody test was carried out with the Roche Elecsys Anti-SARS-CoV-2 test (Roche Diagnostics, Indianapolis, IN, USA).

For viral genome sequencing, the total RNA was extracted from nasopharyngeal swabs as described. 70 µ l extracted RNA were treated with Qiagen DNase I (Qiagen, Germantown, MD, USA) for 30 min at room temperature and then purified and purified with silica spin columns (Qiagen RNeasy MinElute; Qiagen) with a 12. l concentrated water elution. A portion (7 μl) of this RNA was bound to an rRNA inhibitor (Qiagen FastSelect rRNA HMR; Qiagen) and then reverse transcribed (cDNA) using random hexamers. The synthesized DNA was strand-ligated and amplified isothermally to form micrograms of DNA (Qiagen FX Single Cell RNA Library Kit; Qiagen). A portion (1 µg) of this amplified DNA was sheared and ligated to Illumina-compatible sequencing adapters, followed by six cycles of PCR amplification (KAPA HiFi HotStart library amplification kit; Roche Sequencing and Life Science, Kapa Biosystems, Wilmington, MA, USA) ) for library molecules with adapters at both ends. Next, these sequencing libraries were enriched to a sequence specific for SARS-CoV-2 using biotinylated oligonucleotide baits (myBaits Expert Virus, Arbor Biosciences, Ann Arbor, MI, USA). A further eight to 16 PCR cycles were carried out after enrichment (98 ° C for 45 s, 98 ° C for 15 s, 60 ° C for 30 s, repetition for eight to 16 cycles, then 72 ° C for 60 s and 4 ° C), and these SARS-CoV-2 -enriched sequencing libraries were pooled and sequenced with an Illumina NextSeq 500 (Illumina, San Diego, CA USA) as paired 2×75 base pair reads using the NextSeq version 2.5 Medium power 150 stroke kit (Illumina).

For the bioinformatic analysis of the two SARS-CoV-2 agents (here referred to as sample A and sample B), after sequencing each library, FASTQ files were transferred to the CLC Genomics Workbench Version 20.0.4 (Qiagen A / S, Vedbæk, Denmark) with the CLC Microbial Genomics Module, the CLC Genome Finishing Module and the Biomedical Genomics Analysis. In short, the readings were imported, cropped and assigned to the SARS-CoV-2 reference sequence MN908947.3 of the National Center for Biotechnology Information. The alignment was refined with the help of the InDels and Structural Variants module, followed by the Local Realignment module. Variants were identified with a minimum coverage of five reads, a minimum number of five, and a minimum frequency of 70.0%.

To determine the repeatability of the results, a second bioinformatics analysis was carried out using an independent process and open source tool. Potential reinfection sequence libraries were clipped using Trimmomatic version 0.39 with the ILLUMINACLIP adapter clipping setting 2: 30: 10: 2: keepBothReads. Sequence pairs were aligned to the SARS-CoV-2 reference genome (MN908947.3) using Bowtie 2 version 2.3.

Fast lift-read alignment with bowtie 2.

Optical PCR duplicates were marked with Picard MarkDuplicates in Picard-Slim version 2.22.5. Variants were called for both samples along with Freebayes version 1.0.2 with ploidy settings of 1, a minimum allele frequency of 0.70, and a minimum depth of five reads for each variant call. The genomic sequence of each sample was constructed using coverage statistics from BBtools and version 38.86, with only variants supported by coverage of five or more reads written to the bcftools consensus version 1.10.2 and all positions supported by less than five reads, whether reference or alternative, were replaced with Ns.


A statistical framework for SNP calls, mutation detection, association mapping and population genetic parameter estimation from sequencing data.

For the phylogenetic analysis, the entire genome sequences of the isolates (sample A and sample B) were compared with those of 171 simultaneous sequences from Nevada.
  • Hartley P.
  • Tillett RL
  • Xu Y.
  • et al.

Genomic monitoring revealed the prevalence of unique SARS-CoV-2 variants that carry a mutation in the RdRp gene in Nevada patients.

the SARS-CoV-2 reference strain (MN908947.3) and a sequence derived from isolate USA-WA1 / 2020 (Bei Resources, Manassas, VA, USA). After trimming six 5 ‘bases (Ns) from Sample A and 98 Ns from Sample B, the genomic sequences were aligned and related using PhyML + SMS.

  • Lemoine F.
  • Correia D.
  • Lefort V.
  • et al. New generation phylogenetic services for lay people.

The sequences were then first aligned using MAFFT with automatic flavor selection.


MAFFT Multiple Sequence Alignment Software Version 7: Improvements in performance and usability.

Informative regions were selected using block mapping and gathering with entropy, a sliding window size of 3 and a maximum entropy of 0.5.


BMGE (Block Mapping and Gathering with Entropy): A new software for the selection of phylogenetically informative regions from several sequence alignments.

Unrooted trees were created by PhyML using Smart Model Selection, the Akaike Information Criterion, and Subtree Pruning and Regrafting.

  • Lefort V.
  • Longueville I.
  • Gascuel O.

SMS: Smart Modellauswahl in PhyML.

Newick trees were visualized with Interactive Tree Of Live version 4 and rooted on the Wuhan reference stem.


Interactive Tree of Life (iTOL) v4: Current updates and new developments.

Important SARS-CoV-2-Clade memberships were predicted with Nextclade.

To confirm that Samples A and B were from the same individual, the original swab samples, transport media, and residual samples of extracted RNA that were delivered to the sequencing core facility were subjected to a brief tandem repeat analysis (STR) for identity comparison by the Washoe County Sheriff’s Forensic Department of Science (Reno, NV, USA). 2 µ 1 Extracted DNA was quantified using the Quantifiler Trio DNA Quantitation Kit (Applied Biosystems, Foster City, CA, USA) on the 7500 Real-Time PCR System and analyzed with 7500 HID software version 1.3 (Applied Biosystems). The amplification of 24 GlobalFiler STR markers (Thermo Fisher Scientific, Waltham, MA, USA) was carried out on the ProFlex PCR instrument (Thermo Fisher Scientific) for 29 cycles. The 3500xL Genetic Analyzer (Applied Biosystems) was used for fragment analysis of the amplified STR marker regions in conjunction with the HID Data Collection software version 4.0.1 (Applied Biosystems) and the Genemapper ID-X software version 1.6 (Thermo Fisher Scientific). Statistical interpretation of the STR data was achieved using allele frequencies stored in the population database of the National Institute for Standards and Technology.

National Institute for Standards and Technology
Population studies by the NIST Forensics / Human Identity Project team.

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