  The only animals other than bats reported to have been infected with
SARS-CoV-2-related coronaviruses (SARS2r-CoVs) prior to the COVID-19 pandemic
are pangolins. In early 2020 multiple papers reported the identification of two
clades of SARS2r-CoVs, GD and GX, infecting pangolins. However the RNA-Seq
datasets supporting pangolin genome assembly were widely contaminated,
contained synthetic vectors or were heavily enriched or filtered with little
but coronavirus sequences left in the datasets. Here we investigate two
pangolin fecal samples sequenced by Li et al. (2021) provided in support of GD
PCoV infection of pangolins in Guangdong and find the read distribution
consistent with PCR amplicon contamination and SARS-CoV-2 contamination, and
further identify the presence of synthetic plasmid sequences. We also build
upon our previous work to further analyze the dataset GX/P3B by Lam et al.
(2020), which is the only non enriched/heavily filtered pangolin tissue dataset
sequenced by Lam et al. (2020). We identify synthetic vectors and confirm human
genomic origin samples in the dataset. Finally, we find human mitochondrial
sequences in all pangolin organ datasets and mouse and tiger mitochondrial
sequences in selected pangolin organ datasets sequenced by Liu et al. (2019).
We infer that human and mouse genomic origin sequences were probably sourced
from contamination prior to sequencing, while tiger origin sequence
contamination may have occurred due to index hopping during sequencing. These
observations are problematic for attributing pangolins as SARS2r-CoV hosts in
the datasets examined. The forensic methods developed and used here can be
applied to examine any third party SRA data sets.
