CONTINUING EDUCATION :: COVID-19 UPDATE


Participant characteristics We enrolled adults (18 years or older) in Los Angeles County and Riverside County who recently tested positive for SARS- CoV-2 by PCR (Curative, San Dimas, CA). Participants were required to have a positive SARS-CoV-2 RT-PCR result with a cycle threshold value less than or equal to 30 cycles within 5 days of enrollment and sample collection. Trained healthcare workers instructed participants to self-collect anterior nares specimens under direct observation. Individuals meeting eligibility criteria and providing written consent were enrolled in the study. Subjects considered vulnerable including pregnant women, nursing home residents or other institutionalized people, pris- oners, and persons without decisional capacity were excluded from the study. Written informed consent was obtained from each eligible participant prior to enrollment in the study and any specimen collection. The study was approved by Advarra Institutional Review Board under Pro00053729 on May 10, 2021. All research was performed in accordance with relevant regula- tions in accordance with the Declaration of Helsinki. Informed consent was obtained from all participants.


Sample transportation Specimens were placed into 10 mL collection tubes containing DNA/RNA Shield Stabilization Solution (Zymo Research, Irvine, CA). Samples were transported to the sequencing laboratory at 2°C to 8°C within 5 hours of specimen collection and stored at 4°C for up to 7 days before library preparation. Ribonucleic acid (RNA) isolation RNA extraction was performed using chaotropic agents/ silica-based methods. Either manual silica column-based ex- traction,33


or a modified automated magnetic silica beads-based


extraction method were used. RNA was eluted in 60 mL of 10 mM Tris (pH 7.4). 34 Library preparation and sequencing The libraries were prepared using COVIDSeq protocol.35


On a


single 96-well plate, samples were processed alongside a positive control (SARS-CoV-2 BEI NR-52287 genotype A) and a negative control of human nasal specimen without SARS-CoV-2 RNA. Next, 96 indexed sample libraries from each plate were pooled together and quantified using a fluorometer. Four 96-well plates were combined at equimolar concentrations to a total of 384 samples and sequenced. Dilution and loading were performed as per the manufacturer’s instructions. Dual-indexed paired-end sequencing was performed for 100 cycles or 200 cycles to get a deeper sequencing depth. Sequencing aimed to have 1 to 2 million reads per sample.37 Quality control of reads Paired-end reads were filtered and trimmed to reduce low quality base calls in the analysis and to eliminate the presence of primers and adapters. A minimum quality score of 30 was selected (pTrimer-1.3.4 -a Primers.bed -q 30 -t pair).14 Mapping Paired-end reads were then mapped against the ‘Wuhan seafood market pneumonia virus isolate” Wuhan-Hu-1 genome (Accession number: NC_045512.2),15 using bwa.16 Each read was aligned (bwa aln -t 8 NC_045512.2.fasta), then alignments were paired with the sampe option. Alignment files were then subsetted using samtools view to consider only proper pairs with a quality score larger than 12 (samtools view -bS -q 12 -f 0X2).17


Validation To validate the SARS-CoV-2 sequencing assay, BEI SARS-CoV-2 samples were used as control samples.18


were prepared by two operators. Each operator would prepare 16 replicates of BEI 52287, which would be used as the positive control in clinical sample testing, and one library for each of the other 13 BEI standards. All extracted nucleic acids from the BEI samples were verified to contain SARS-CoV-2 RNA. Ct values were between 26 and 29 for each sample before proceeding to cDNA synthesis. cDNA synthesis and library were prepared. Finished libraries were enzymatically normalized and pooled. Pooled libraries were quantified with KAPA qPCR and pooled together in equimolar concentrations to make a standardized final pooled library. 38


The final pooled library was sequenced and analyzed with the protocol listed later.


Variant calling and consensus genome generation, mutation identification, and variant identification Variable sites were generated, regardless of coverage depth assuming a haploid genome using bcftools (bcftools call -mv -Ov).17


(20x) and minimum allele frequency (0.25). Finally, the consensus genome was generated using the VCFcons.py script (VCFCons. py --input_depth TEMP.depth --input_vcf sample.vcf --vcf_type bcftools -c 10 -f 0.25 -q 20), part of a CoSa suite. 39


Consensus


genomes were then run on the command line version of both: pangolin and nextclade.19 Ad-hoc analysis Custom scripts were used to calculate sequencing, effort, and the percentage of reads used to assemble de novo genomes and base pair coverage. These can be found at (https://github.com/ curative/bioinformatics).


Analysis of consensus sequences mutations and identifying similar isolates A lineage comparison was done using Outbreak.info resource created by Scripps Research. A search for genomic sequences similar to the identified SARS-CoV-2 isolates was performed using Nucleotide BLAST 2.6.0+. Isolated were compared to all sequences available on the Global Influenza Surveillance & Re- sponse System (https://www.epicov.org/epi3/cfrontend#2c08bd). Data underwent alignment to identify gaps in generated con- sensus sequences and to match them with positions in amino acid sequences carrying hallmark mutations using software.40


Reporting Results were submitted to Global Influenza Surveillance & Re- sponse System EpiCoV database for widespread data sharing and surveillance, a public surveillance service.20,21


Variable sites were then filtered for quality (20) coverage


The accession


numbers were added to the Global Influenza Surveillance & Response System.


Useability From May 27, 2021, to January 11, 2022, 820 recently tested SARS- CoV-2 positive participants were enrolled and underwent speci- men collection. Of those enrolled, there were 408 (49.8%) females, 570 (69.5%) vaccinated, and 351 (42.8%) of Hispanic or Spanish origin. Median age of participant was 43 years (IQR: 33, 53). Of the cohort, 803 (97.9%) participants had symptoms at time of collection. The time from specimen collection to sequence result was reduced to three days. During the study period, we observed a decreased prevalence of Alpha, Gamma, Iota, Lambda, which was replaced by Delta, then Omicron variant of SARS-CoV-2 (Figure 1).


Sequencing libraries


The view from here In all, outpatient SARS-CoV-2 variant surveillance could be conducted by a private laboratory in a timely and accu-


MLO-ONLINE.COM JUNE 2022 9


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