Finally, the higher level of accuracy and reproducibility that your experiments deserve are within reach. The LoopSeq Microbiome sample prep kit generates robust and reliable data on relative species abundance for microbial metagenomics studies by utilizing single-molecule barcoding and de novo sequence assembly. With traditional shotgun sequencing methods, millions of short reads define your sample population, but it is impossible to trace any of those reads back to their original molecules, and even if you simply count the number of reads from a given species, there is no way to know if the abundance of reads reflects the actual number of original 16S molecules. PCR amplification bias will make some species appear more abundant when counting in this way, and the count can vary as the bias grows more or less pronounced between experiments. These issues collectively lead to poor reproducibility and thus low confidence in the relative abundance calls. The solution is to utilize single-molecule barcoding, which tags each molecule with a unique molecule ID before any amplification occurs, such that the true representation of relative species abundance can be consistently calculated from the final data. LoopSeq uses this approach.
To demonstrate the ability of LoopSeq Microbiome to generate reproducible measurements of relative species abundance, we sequenced technical replicates (eight replicates) of a defined microbial community standard. The relative species abundance of this standard across replicates within a single experiment as well as between experiments (conducted by different users) were both quantified. (Figure 1).
The LoopSeq Microbiome workflow allows up to 24 samples to be processed simultaneously into ready-to-run sequencing libraries. For the reproducibility study, we processed eight aliquots from the same stock of ZymoBIOMICS™ Microbial Community Standard, both in isolated single-sample conditions and as a multiplex pool of 24 samples done simultaneously per the LoopSeq design.
Comparison of the experimentally determined composition of the microbial community standard with the theoretical composition shows good agreement between the measured and theoretical values (Figure 1). In addition, the eight single-sample technical replicates show excellent relative species abundance reproducibility, with a standard deviation ranging from 0.1% to 1.1%.
Furthermore, when the two multiplexed pools that were processed by two different users were compared (Loop Kit #1 and Loop Kit #2), we again saw high reproducibility in the relative species abundances that were called. Essentially, the two replicate pools that contained the same sample types but processed by different researchers, were nearly indistinguishable from each other.
Also, the reproducibility of complex samples was demonstrated. Four complex samples from gut, each of which contain many unique species at different abundance levels, were prepared as multiplexed pools by two different users. The relative species abundances for all 4 samples tracked well between the runs (Figure 2), and this consistency was maintained for species with lower abundances.
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