Abstract
The efficiency of polymerase chain reaction (PCR) decreases under suboptimal conditions, such as low template concentration combined with high concentrations of similar sequences. Under these circumstances, mis-priming can occur, leading to the generation of erroneous copies. Specifically, in 16S amplicon sequencing of human intestinal biopsy samples, host off-target sequences are frequently generated and subsequently sequenced, particularly when the commonly used V3-V4 primers are employed. This issue not only introduces errors in data interpretation but also results in the unnecessary consumption of sequencing depth. In response to this challenge, we analysed over 1,300 publicly available V3-V4 amplicon sequences related to the human colon, profiling the colon microbiota while elucidating the biases introduced by host off-targets. Briefly, our findings reveal that unaddressed host DNA contamination can lead to false bacterial identifications and obscure significant differences in microbiota composition. Furthermore, we identified human sequences on chromosomes 5, 11, and 17 as the main contributors to the majority of off-target sequences. Finally, we suggest practical approaches to mitigate this issue without altering the original protocol design, retaining the widely used V3-V4 primers. In particular, using a C3 spacer-modified nucleotide targeting the off-target sequence is here proposed as a promising strategy acting upstream of the off-target generation.