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



Procedures

Samples were obtained from the patient by nasopharyngeal swab at the community testing event, during the isolation and recovery period, and at hospital presentation. The swabs were transported to the Nevada State Public Health Laboratory (Reno, NV, USA) in viral transport medium or Aptima Multiswab Transport Media (Hologic, San Diego, CA, USA). Samples were transported on cold packs and stored in a refrigerator (4–8 ° C) for no more than 72 hours prior to nucleic acid extraction and subsequent real-time RT-PCR.

Nucleic acid extraction was performed using the Omega Biotek MagBind Viral DNA / RNA 96 kit (Omega Bio-tek, Norcross, GA, USA), according to the manufacturer̵

7;s instructions and with an elution volume of 100 μL. Aliquots of eluted RNA were subjected to real-time RT-PCR with the Taqpath COVID-19 Emergency Use Authorized (EUA) multiplex assay (ThermoScientific, Waltham, MA, USA; 10 μL aliquots) or with the US Centers for Control and Disease 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 analyzed by transcription-mediated amplification using the Aptima SARS-CoV-2 (Panther System) assay (Hologic, Marlborough, MA, USA). The tests were performed according to the respective EUA procedures, unless otherwise indicated. For the Taqpath real-time RT-PCR assay, the threshold for calling a positive sample was the reactivity of two of the three target sequences, each with reactivity at a cycle threshold of less than 40.00. A positive or negative Hologic Aptima test result was based on proprietary processes. The antibody test was performed with the Roche Elecsys Anti SARS-CoV-2 test (Roche Diagnostics, Indianapolis, IN, USA).

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

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

To ascertain the repeatability of the results, a second bioinformatics analysis was performed using an independent process and open source tools. 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.

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Fast reading alignment with Bowtie 2.