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
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%.
Optical duplicates of the PCR were marked using Picard MarkDuplicates in picard-slim version 2.22.5. Variants were called for both samples in concert using 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 genome sequence of each sample was constructed using coverage statistics from BBtools pileup.sh and applyvariants.sh version 38.86, whereby only variants supported by coverage of five or more reads were written in consensus version 1.10.2 of bcftools and all positions supported by one of five reads, both reference and alternate, have been replaced with Ns.
- Hartley P
- Tillett RL
- Xu Y
- et al.
the reference strain SARS-CoV-2 (MN908947.3) and a sequence derived from the USA-WA1 / 2020 isolate (Bei Resources, Manassas, VA, USA). After cutting six unnamed 5 ‘bases (Ns) from sample A and 98 Ns from sample B, the genomic sequences were aligned and correlated using NGPhylogeny.fr PhyML + SMS.
- Lemoine F
- Correia D
- Lefort V
- et al.
The sequences were then aligned first using MAFFT with automatic flavor selection.
Information regions were selected using Block Mapping and Gathering with Entropy, a sliding window size of 3 and a maximum entropy of 0.5.
The rootless trees were built by PhyML with Smart Model Selection, the Akaike information criterion and the pruning and reconstruction of substructures.
- Lefort V
- Longueville J-E
- Gascuel O
The Newick trees were visualized using Interactive Tree Of Live version 4 and rooted in the Wuhan reference strain.
Major memberships in the SARS-CoV-2 clade were predicted using Nextclade.
Population studies conducted by the NIST Forensics / Human Identity Project Team.