The source of classification bias in marker gene metagenome sequencing?
a variability in the taxonomy classification of microbial communities when using different primer pairs (e.g. for 16S rDNA) is commonly known. However, the mismatches to these primers are not described as the major reason for this bias. My question is: what are other possible causes of this bias and which is now supposed to be the major one?
Rakesh Sharma
Incomplete Coverage of Variable Regions:Sequencing companies often do not sequence all nine hypervariable regions (V1-V9) of the 16S rRNA gene. Different primer sets target different regions, and incomplete coverage can lead to biases as some regions provide more taxonomically informative sequences than others. Primer Selection and Design:Different primer pairs have varying levels of specificity and efficiency. Primers designed for certain regions might preferentially amplify some taxa over others, leading to an imbalanced representation of the microbial community. PCR Amplification Bias:During PCR amplification, some sequences might be amplified more efficiently than others. This can be due to differences in GC content, secondary structures, or the presence of inhibitory substances. These biases can distort the relative abundance of taxa.
In marker gene metagenome sequencing, such as 16S rRNA sequencing used to profile microbial communities, several factors can contribute to classification bias. While primer mismatches can cause variability, they are not the primary source of bias. Here are some other significant causes:
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