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Using sequence reads from shotgun metagenomic analyses in both cattle and sheep, we describe how failures in mate pairing on Illumina sequencing can interact with bioinformatics pipelines to give spurious patterns among rare components of a metagenomic sample. We identified several different shotgun metagenomic datasets from different animals and different laboratories where the two members of the read pair matched a viral database at very different frequencies. We traced this bias to a set of poly-G reads of high quality that resulted from failures in generating read pairs during library preparation. These results reinforce the need to remove poly-G-rich reads when quality filtering shotgun metagenomic data.more » « lessFree, publicly-accessible full text available December 1, 2026
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