Analysis of SARS-CoV-2 reads in sequencing of 2018-2019 Antarctica samples in PRJNA692319

The samples analyzed here are described in this preprint, which is a pre-print by Istvan Csabai and co-workers that describes SARS-CoV-2 reads in samples from Antarctica sequencing in China. I was originally alerted to the pre-print by Carl Zimmer on Dec-23-2021. Istvan Csabai and coworkers subsequently posted a second pre-print that also analyzes the host reads.

Repeating key parts of the analysis

The code in this repo independently repeats some of the analyses.

To run the analysis, build the conda environment in environment.yml and then run the analysis using Snakefile. To do this on the Hutch cluster, using run.bash:

sbatch -c 16 run.bash

The results are placed in the ./results/ subdirectory. Most of the results files are not tracked due to file-size limitations, but the following key files are tracked:

• results/alignment_counts.csv gives the number of reads aligning to SARS-CoV-2 for each sample. This confirms that three accessions (SRR13441704, SRR13441705, and SRR13441708) have most of the SARS-CoV-2 reads, although a few other samples also have some.
• results/variant_analysis.csv reports all variants found in the samples relative to Wuhan-Hu-1.
• results/variant_analysis_to_outgroup.csv reports the variants found in the samples that represent mutations from Wuhan-Hu-1 towards the two closest bat coronavirus relatives, RaTG13 and BANAL-20-52. Note that some of the reads contain three key mutations relative to Wuhan-Hu-1 (C8782T, C18060T, and T28144C) that move the sequence closer to the bat coronavirus relatives. These mutations define one of the two plausible progenitors for all currently known human SARS-CoV-2 sequences (see Kumar et al (2021) and Bloom (2021)).

Archived links after initially hearing about pre-print

I archived the following links on Dec-23-2021 after hearing about the pre-print from Carl Zimmer:

• https://assets.researchsquare.com/files/rs-1177047/v1_covered.pdf
• https://www.frontiersin.org/articles/10.3389/fmicb.2020.573302/full
• https://bg.copernicus.org/articles/16/4113/2019/
• https://www.ncbi.nlm.nih.gov/bioproject/PRJNA692319/
• https://trace.ncbi.nlm.nih.gov/Traces/sra/?run=SRR13441700
• https://trace.ncbi.nlm.nih.gov/Traces/sra/?run=SRR13441701
• https://trace.ncbi.nlm.nih.gov/Traces/sra/?run=SRR13441702
• https://trace.ncbi.nlm.nih.gov/Traces/sra/?run=SRR13441703
• https://trace.ncbi.nlm.nih.gov/Traces/sra/?run=SRR13441704
• https://trace.ncbi.nlm.nih.gov/Traces/sra/?run=SRR13441705
• https://trace.ncbi.nlm.nih.gov/Traces/sra/?run=SRR13441706
• https://trace.ncbi.nlm.nih.gov/Traces/sra/?run=SRR13441707
• https://trace.ncbi.nlm.nih.gov/Traces/sra/?run=SRR13441708
• https://trace.ncbi.nlm.nih.gov/Traces/sra/?run=SRR13441709
• https://trace.ncbi.nlm.nih.gov/Traces/sra/?run=SRR13441710
• https://trace.ncbi.nlm.nih.gov/Traces/sra/?run=SRR13441711

Deletion of some samples from SRA

On Jan-3-2022, I received an e-mail one of the pre-print authors, Istvan Csabai, saying that three of the samples (appearing to be the ones with the most SARS-CoV-2 reads) had been removed from the SRA. He also noted that bioRxiv had refused to publish their pre-print without explanation; the file he attached indicates the submission ID was BIORXIV-2021-472446v1. I confirmed that three of the accessions had indeed been removed from the SRA as shown in the following archived links:

• https://www.ncbi.nlm.nih.gov/sra?linkname=bioproject_sra_all&from_uid=692319
• https://trace.ncbi.nlm.nih.gov/Traces/sra/?run=SRR13441704
• https://trace.ncbi.nlm.nih.gov/Traces/sra/?run=SRR13441705
• https://trace.ncbi.nlm.nih.gov/Traces/sra/?run=SRR13441708

I also e-mailed Richard Sever at bioRxiv to ask why the pre-print was rejected, and explained I had repeated and validated the key findings. Richard Sever said he could not give details about the pre-print review process, but that in the future the authors could appeal if they thought the rejection was unfounded.

Details from Istvan Csabai

On Jan-4-2022, I chatted with Istvan Csabai. He had contacted the authors of the pre-print, and shared their reply to him. The authors had prepped the samples in early 2019, and submitted to Sangon BioTech for sequencing in December, getting the results back in early January.

Second pre-print from Csabai and restoration of deleted files

Istvan Csabai then worked on a second pre-print that analyzed host reads and made various findings, including co-contamination with African green monkey (Vero?) and human DNA. He sent me pre-print drafts on Jan-16-2022 and on Jan-24-2022, and I provided comments on both drafts and agreed to be listed in the Acknowledgments.

On Feb-3-2022, Istvan Csabai told me that the second pre-print had also been rejected from bioRxiv. Because I had previously contacted Richard Sever when I heard the first pre-print was rejected, I suggested Istvan could CC me on an e-mail to Richard Sever appealing the rejection, which he did. Unfortunately, Richard Sever declined the appeal, so instead Istvan posted the pre-print on Resarch Square.

At that point on Feb-3-2022, I also re-checked the three deletion accessions (SRR13441704, SRR13441705, and SRR13441708). To my surprise, all three were now again available by public access. Here are archived links demonstrating that they were again available:

• still missing from this overview page: https://www.ncbi.nlm.nih.gov/Traces/study/?acc=SRP301869&o=acc_s%3Aa
• again active: https://trace.ncbi.nlm.nih.gov/Traces/sra/?run=SRR13441704
• again active https://trace.ncbi.nlm.nih.gov/Traces/sra/?run=SRR13441705
• again active https://trace.ncbi.nlm.nih.gov/Traces/sra/?run=SRR13441708

I confirmed that the replaced accessions were identical to the deleted ones.

Inquiry to authors of PRJNA692319

On Feb-8-2022, I e-mailed the Chinese authors of the paper to ask about the sample deletion and restoration. They e-mailed back almost immediately. They confirmed what they had told Istvan: they had sequenced the samples with Sangon Biotech (Shanghai) after extracting the DNA in December 2019 from their samples. The suspect that contamination of the samples happened at Sangon Biotech. They deleted the three most contaminated samples from the Sequence Read Archive. They do not know why the samples were then "un-deleted."