key: cord-0890006-zugrtvyo
authors: Rose, R.; Nolan, D. J.; Moot, S.; Feehan, A.; Cross, S.; Garcia-Diaz, J.; Lamers, S. L.
title: Intra-host site-specific polymorphisms of SARS-CoV-2 is consistent across multiple samples and methodologies
date: 2020-04-29
journal: nan
DOI: 10.1101/2020.04.24.20078691
sha: c9eea28c201f1e5883a486766c8dec7aa0418a8e
doc_id: 890006
cord_uid: zugrtvyo

Despite the potential relevance to clinical outcome, intra-host dynamics of SARS-CoV-2 are unclear. Here, we quantify and characterize intra-host variation in SARS-CoV-2 raw sequence data uploaded to SRA as of 14 April 2020, and compare results between two sequencing methods (amplicon and RNA-Seq). Raw fastq files were quality filtered and trimmed using Trimmomatic, mapped to the WuhanHu1 reference genome using Bowtie2, and variants called with bcftools mpileup. To ensure sufficient coverage, we only included samples with 10X coverage for >90% of the genome (n=406 samples), and only variants with a depth >=10. Derived (i.e. non-reference) alleles were found at 408 sites. The number of polymorphic sites (i.e. sites with multiple alleles) within samples ranged from 0-13, with 72% of samples (295/406) having at least one polymorphic site. Correlation between number of polymorphic sites and coverage was very low for both sequencing methods (R2 < 0.1, p < 0.05). Polymorphisms were observed >1 sample at 66 sites (range: 2-38 samples). The minor allele frequency (MAF) at each shared polymorphic site was 0.03% - 48.5%. 33/66 sites occurred in ORF1a1b, and 37/66 changes were non-synonymous. At 10/66 sites, derived alleles were found in samples sequenced using both methods. Polymorphic amplicon samples were found at 10/10 positions, while polymorphic RNA-Seq samples were found at 7/10 positions. In conclusion, our results suggest that intra-host variation is prevalent among clinical samples. While mutations resulting from amplification and/or sequencing errors cannot be excluded, the observation of shared polymorphic sites with high MAF across multiple samples and sequencing methods is consistent with true underlying variation. Further investigation into intra-host evolutionary dynamics, particularly with longitudinal sampling, is critical for broader understanding of disease progression.

Researchers worldwide have engaged in a remarkable effort to generate and share genomic sequences of SARS-CoV-2, in particular to the GISAID database (gisaid.org), resulting in nearly 10,000 temporal and geo-located whole genomes available in mid-April 2020 [1] . These data, and sophisticated methods, have enabled a robust estimation of a relatively slow population evolutionary rate (~0.8 × 10-3 substitutions per site per year, [2] ). As additional genomes are uploaded each day, viral dynamics on a population level can be refined in real-time.

On the other hand, a similarly thorough investigation of the intra-host evolutionary rate has yet to be published, resulting in part from the scarcity of patient longitudinal samples. In addition, analyzing published datasets of raw sequence data is much more time-consuming than using consensus genomes, and the lack of a common sequencing protocol and analytical pipeline renders comparisons among samples difficult. Finally, much less raw data have been made available: for example, as of 14 April 2020, <1,000 raw sequence files were available from the Short Read Archive (SRA) at the National Center for Biotechnology Information (NCBI).

Despite these challenges, evidence from numerous angles suggests a potentially important role of intra-host variation. For example, a few studies have demonstrated the presence of intra-host variation of SARS on a population level, and more intriguingly, variation occurring at the same position in multiple individuals [3, 4] . These observations suggest that either that patients are becoming infected with a similarly diverse viral population, or that the virus is following a similar evolutionary trajectory in multiple patients, both of which may have clinical implications.

Furthermore, the evolution of shared variants has a well-established impact on viral replication and tropism in both human viruses (e.g. HIV-1) and other coronaviruses (e.g. feline).

Our goal in this study was to quantify and characterize intra-host variation in all SARS-CoV-2 raw sequence data available in SRA as of 14 April 2020, and compare results between two All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted April 29, 2020. . https://doi.org/10.1101/2020.04.24.20078691 doi: medRxiv preprint 4 sequencing methods (amplicon and RNA-Seq).. A search for "SARS-CoV-2" resulted in a total of 1173 samples. We downloaded all samples that were classified as "Wuhan seafood market pneumonia virus" or "Severe acute respiratory syndrome coronavirus 2"; host = Homo sapiens; derived from a primary isolate; sequenced using an Illumina platform, and downloaded successfully, resulting in a total of 441 samples (Supplemental File 1).

Raw fastq files were quality filtered and trimmed using Trimmomatic and mapped to the WuhanHu1 reference genome (NC_045512.2) using Bowtie2. Since the reference sequence was one of the earliest samples from geographic origin of the current pandemic, this was considered the ancestral sequence. Bam files were sorted and indexed with samtools, and variants called with bcftools mpileup. Only variants with a depth >=10 were retained. Individual vcf files were merged, and allele frequencies were extracted using the vcfR package in R. The complete commands are available in Supplemental File 2.

While all raw data was generated using the Illumina short read technology, numerous experimental variables differed among studies, (e.g. assay type, library construction kit, instrument, etc). We sorted the dataset into two general groups based on their sequencing approach: those which sequenced just PCR amplicons of SARS-CoV-2 vs. those that used a general RNA-Seq metagenomic method which typically sequences all the RNA isolated from a sample unless specific RNA classes are excluded i.e. rRNA depletion, or enriched i.e. mRNA selection. Since an accurate estimation of intra-host variation requires sufficient genomic coverage, we first investigated sample quality by visualizing mapping metrics for all samples.

In general, coverage was higher for the amplicon data compared with the RNA-Seq data (Supplemental Figure 1) . The interquartile range of mean coverage was 239 -246 for the amplicon data, and 9-231 for the RNA-Seq data. The percent of the genome covered at 1x was high for both (amplicon IQR: 0.997 -0.998; RNA-Seq IQR: 0.916 -0.999). However, while the All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted April 29, 2020. . https://doi.org/10.1101/2020.04.24.20078691 doi: medRxiv preprint 5 percent of the genome covered at 10x and 25x remained high with the amplicon data (10x IQR: 0.987 -0.997; 25x IQR: 0.980 -0.997), the range was much broader for the RNA-Seq data (10x IQR: 0.240 -0.997; 25x IQR: 0.024 -0.993). This suggests that, particularly for studies investigating intra-host variation, amplicon-based sequencing may be preferable.

To ensure sufficient coverage, we excluded samples with 10X coverage for <90% of the Next we looked the minor allele frequency (MAF) in each sample for all of the 66 shared polymorphic sites (Figure 1B) . MAF per site/per sample ranged from 0.03% to 48.5%. The combined MAF for all polymorphic samples per site ranged from 0.08% to 604%. There was a moderate correlation between combined MAF and the number of polymorphic samples (R 2 = All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted April 29, 2020. . https://doi.org/10.1101/2020.04.24.20078691 doi: medRxiv preprint 6 0.43; Figure 1B) . However, two sites in particular were notable for high MAFs among multiple samples: 8782 and 17747. When these sites were removed, the correlation was reduced (R 2 = 0.23).

Of the 66 shared polymorphic positions, 50% occurred in ORF1a1b, including the two sites with the highest MAFs (8782 and 17747) In conclusion, our results suggest that intra-host variation is prevalent among clinical samples, most of which is masked when constructing consensus genomes. While this variation could be due to amplification and/or sequencing errors, evidence supporting real virus variation includes All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted April 29, 2020. . https://doi.org/10.1101/2020.04.24.20078691 doi: medRxiv preprint 7 nonrandom distribution of polymorphic sites, shared polymorphic sites among up to 38 samples; MAF up to 50%; and detection of polymorphisms at the same site using two very different sequencing methods. The high number of mutations that change the amino acid suggest a potential functional relevance. Further investigation into intra-host evolutionary dynamics, particularly with longitudinal sampling, is critical for broader understanding of disease progression.

RR, DN, SC, SM, and SLL are employed by Bioinfoexperts, LLC. The authors thank all researchers for making raw data publicly available.

SLL is the recipient of National Science Foundation SBIR funding (#1830867). All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted April 29, 2020. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted April 29, 2020. . https://doi.org/10.1101/2020.04.24.20078691 doi: medRxiv preprint 9 processed samples were monomorphic; double asterisk indicates >=1 RNA-Seq processed samples were polymorphic. All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted April 29, 2020. 

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Cryptic transmission of SARS-CoV-2 in Washington State. medRxiv

Genomic diversity of SARS-CoV-2 in Coronavirus Disease 2019 patients

All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted

All rights reserved. No reuse allowed without permission.(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint this version posted April 29, 2020. . https://doi.org/10.1101/2020.04.24.20078691 doi: medRxiv preprint