README GENERAL INFORMATION September 2022 1. Title of Dataset: SARS-CoV-2 RNA detectable at least eight months after shedding in an isolation facility. 2. Author Information Corresponding Investigator of dataset Name: Kaze, Mo, University of California, Davis, Institution: University of California, Davis Email: mkaze@ucdavis.edu Co-investigators: Coil, David, University of California, Davis Bischel, Heather, University of California, Davis Shapiro, Karen, University of California, Davis Pechacek, Randi, University of California, Davis Zuniga-Montanez:, Rogelio, University of California, Davis Guerrero, Roque, University of California, Davis Eisen, Jonathan, University of California, Davis 3. Date of data collection: June - July, 2021 4. Geographic location of data collection: University of California, Davis, United States 5. Recommended citation for this dataset: Kaze, Mo et al. (2022), SARS-CoV-2 RNA detectable at least eight months after shedding in an isolation facility, Dryad, Dataset DATA & FILE OVERVIEW 1. Description of dataset This sequence data was generated to determine if ARS-CoV-2 RNA was detectable at least eight months after shedding in an isolation facility. No identifying information is included in this raw data. The download should be a total of 13 compressed raw reads in the fastq format. Naming schema: Sample_name | Sample_date | Purification Instrument | Sequencer lane ID | Read direction | File format 2. File List: File 1 Name: MZA_8_061421_KFE1_S3_R1_001.fastq.gz File 1 Description: Forward raw reads for sample MZA_6 File 2 Name: MZA_8_061421_KFE1_S3_R1_001.fastq.gz File 2 Description: Reverse raw reads for sample MZA_6 File 3 Name: MZA_8_061421_KFE1_S3_R1_001.fastq.gz File 3 Description: Forward raw reads for sample MZA_8 File 4 Name: MZA_8_061421_KFE1_S3_R1_001.fastq.gz File 4 Description: Reverse raw reads for sample MZA_8 File 5 Name: MZA_13_061421_KFE1_S1_R2_001.fastq.gz File 5 Description: Forward raw reads for sample MZA_13 File 6 Name: MZA_8_061421_KFE1_S3_R1_001.fastq.gz File 6 Description: Reverse raw reads for sample MZA_13 File 7 Name: MZA_51_061421_KFE1_S7_R1_001.fastq.gz File 7 Description: Forward raw reads for sample MZA_51 File 8 Name: MZA_51_061421_KFE1_S7_R2_001.fastq.gz File 8 Description: Reverse raw reads for sample MZA_51 File 9 Name: MZA_66_061421_KFE1_S5_R1_001.fastq.gz File 9 Description: Reverse raw reads for sample MZA_66 File 10 Name: MZA_66_061421_KFE1_S5_R2_001.fastq.gz File 10 Description: Reverse raw reads for sample MZA_66 File 11 Name: MZA_96_070621_KFE1_S2_R1_001.fastq.gz File 11 Description: Reverse raw reads for sample MZA_96 File 12 Name: MZA_96_070621_KFE1_S2_R2_001.fastq.gz File 12 Description: Reverse raw reads for sample MZA_153 File 13 Name: MZA_153_070621_KFE1_S4_R1_001.fastq.gz File 13 Description: Reverse raw reads for sample MZA_153 File 14 Name: MZA_153_070621_KFE1_S4_R2_001.fastq.gz File 14 Description: Reverse raw reads for sample MZA_96 Naming schema: Sample_name | Sample_date | Instrument for purification | Sequencer lane ID | Read direction METHODOLOGICAL INFORMATION Samples were collected using nylon fiber oral swabs with an ABS handle (Miraclean Technology Co. Ltd, China) that were pre-moistened in DNA/RNA Shield (Zymo Research, USA). An area of 10 cm x 10 cm (or equivalent) was swabbed for surface samples and a similar process was used to swab the exterior surface of the HEPA filter (~823 cm2) in a portable filtration unit. The same 10 cm x 10 cm area was swabbed every sampling time point until day 37. Afterward, a different 10 cm x 10 cm area was swabbed every time. RNA extraction and RT-qPCR were performed. Samples were extracted using the MagMAX Microbiome Ultra Nucleic Acid Isolation Kit (Applied Biosystems, USA) with a KingFisher Flex automated purification system (Thermo Fisher Scientific, USA). The MagMAX_Microbiome_Stool_Flex.bdz nucleic acid isolation protocol (Applied Biosystems, USA) was utilized, with modifications. In brief, the sample lysis step was not conducted as lysis was achieved through the use of DNA/RNA Shield and vortexing. All extracts were analyzed by RT-qPCR targeting the spike glycoprotein (S) gene of SARS-CoV-2 using the Luna Universal Probe One-Step RT-qPCR Kit (New England Biolabs Inc., USA). Eight samples from this room were chosen for sequencing. The samples across projects were chosen to represent a variety of Ct values and surface types to calibrate future sequencing efforts. Ten μl of extracted nucleic acid from each of the 6 samples was converted to cDNA using the LunaScript RT SuperMix Kit (New England Biolabs, Ipswich, MA) in a 20 μl reaction mix, which was incubated at 25 °C for 2 min followed by 55 °C for 10 min and heat-inactivated at 95 °C for 1 min. Subsequently, 10 μl of this 1st-strand cDNA was used as input for amplification of the SARS-CoV-2 viral genome, using the xGen SARS-CoV-2 Amplicon Panel (IDT, Coralville, IA) which consist of 345 amplicons covering 99.7% of the SARS-CoV-2 Wuhan-Hu-1 strain (NC_045512.2). The workflow uses a single tube of tiled primer pairs resulting in an average amplicon size of 150 bp. The amplicon libraries were generated according to the workflow for low viral load workflows (Ct > 20) which involves two rounds of PCR rounds, a multiplex PCR (4 + 24 cycles), and the indexing PCR (5 cycles) to generate sequence-ready libraries. Libraries were barcoded with 8 bp unique dual indices during the indexing PCR. Equimolar libraries were pooled and quantified by qPCR with the KAPA Library Quantification Kits (Roche, Basel, Switzerland). The pooled library was sequenced on one lane of Illumina Mid Output NextSeq 500 (Illumina, San Diego, CA) with paired-end 150 bp reads.