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Higher off-target amplicon detection rate in MiSeq v3 compared to v2 reagent kits in the context of 16S-rRNA-sequencing.

Authors :
Odendaal ML
Groot JA
Hasrat R
Chu MLJN
Franz E
Bogaert D
Bosch T
de Steenhuijsen Piters WAA
Source :
Scientific reports [Sci Rep] 2022 Oct 01; Vol. 12 (1), pp. 16489. Date of Electronic Publication: 2022 Oct 01.
Publication Year :
2022

Abstract

One of the most widely used techniques in microbiota research is 16S-rRNA-sequencing. Several laboratory processes have been shown to impact sequencing results, especially in low biomass samples. Low biomass samples are prone to off-target amplification, where instead of bacterial DNA, host DNA is erroneously amplified. Knowledge on the laboratory processes influencing off-target amplification and detection is however scarce. We here expand on previous findings by demonstrating that off-target amplification is not limited to invasive biopsy samples, but is also an issue in low bacterial biomass respiratory (mucosal) samples, especially when below 0.3 pg/μL. We show that off-target amplification can partly be mitigated by using gel-based library purification methods. Importantly, we report a higher off-target amplicon detection rate when using MiSeq reagent kit v3 compared to v2 (mean 13.3% vs 0.1% off-target reads/sample, respectively), possibly as a result of differences in reagents or sequencing recipes. However, since after bioinformatic removal of off-target reads, MiSeq reagent kit v3 still results in a twofold higher number of reads when compared to v2, v3 is still preferred over v2. Together, these results add to the growing knowledge base on off-target amplification and detection, allowing researchers to anticipate this problem in 16S-rRNA-based microbiome studies involving low biomass samples.<br /> (© 2022. The Author(s).)

Details

Language :
English
ISSN :
2045-2322
Volume :
12
Issue :
1
Database :
MEDLINE
Journal :
Scientific reports
Publication Type :
Academic Journal
Accession number :
36183009
Full Text :
https://doi.org/10.1038/s41598-022-20573-1