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. 2020 Oct 1;10(1):16356.
doi: 10.1038/s41598-020-73403-7.

Non-specific amplification of human DNA is a major challenge for 16S rRNA gene sequence analysis

Sidney P Walker #  1   2   3   4 , Maurice Barrett #  3   4 , Glenn Hogan  1   2 , Yensi Flores Bueso  1   2   3 , Marcus J Claesson  3   4 , Mark Tangney  5   6   7
Affiliations

Non-specific amplification of human DNA is a major challenge for 16S rRNA gene sequence analysis

Sidney P Walker et al. Sci Rep. .

Abstract

The targeted sequencing of the 16S rRNA gene is one of the most frequently employed techniques in the field of microbial ecology, with the bacterial communities of a wide variety of niches in the human body have been characterised in this way. This is performed by targeting one or more hypervariable (V) regions within the 16S rRNA gene in order to produce an amplicon suitable in size for next generation sequencing. To date, all technical research has focused on the ability of different V regions to accurately resolve the composition of bacterial communities. We present here an underreported artefact associated with 16S rRNA gene sequencing, namely the off-target amplification of human DNA. By analysing 16S rRNA gene sequencing data from a selection of human sites we highlighted samples susceptible to this off-target amplification when using the popular primer pair targeting the V3-V4 region of the gene. The most severely affected sample type identified (breast tumour samples) were then re-analysed using the V1-V2 primer set, showing considerable reduction in off target amplification. Our data indicate that human biopsy samples should preferably be amplified using primers targeting the V1-V2 region. It is shown here that these primers result in on average 80% less human genome aligning reads, allowing for more statistically significant analysis of the bacterial communities residing in these samples.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Proposed mechanism for off target amplification of mammalian DNA by V3–V4 primers, as opposed to V1–V2. (A) DNA extracted from human biopsies is known to contain large proportions of human DNA. In these circumstances V3–V4 degenerate primers, which also align to region in human mitochondrial DNA as shown can bind and amplify human DNA. There is no such alignment for V1–V2 degenerate primers. (B) Off target amplification significantly alters the 16S rRNA gene sequencing profile of a sample.
Figure 2
Figure 2
The scale of the problem of off-target amplification. % of sequencing reads produced by Miseq 2 × 300 bp sequencing of amplicons produced by primers targeting the V3–V4 regions shown to align to the human genome.
Figure 3
Figure 3
Rarefaction curve generated by plotting observed species vs read depth on a per sample basis. (A) Rarefaction curve prior to removal of human genome aligning reads. (B) Rarefaction curve following removal of human genome aligning reads.
Figure 4
Figure 4
Pairwise comparison of matched samples using primers targeting the V1–V2 and V3–V4 regions of the 16S rRNA gene fragment. (A) Sample composition at the family level of paired samples. (B) Average Shannon Diversity comparison between samples amplified using V1–V2 primers (red) and V3–V4 primers (blue). (C) Percentage of total sequencing reads aligning to human genome. In both (B) and (C) statistical testing is performed using Wilcoxon signed-rank test.
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