Kepler is a novel taxonomic profiling pipeline engineered to extract optimal value from metagenomic data. Combining the sensitivity of K-mer exact-matching and the precision and accuracy of probabilistic alignment, Kepler achieves robust identification and enumeration of microbial taxa spanning bacteria, viruses, fungi, and protists. Significantly, Kepler's accuracy and precision relies upon a meticulously curated biomarker database, where over 30,000 species across Bacteria, Viruses, Protists and Fungi are arranged in a phylogenetic tree-like structure.

The core of Kepler’s technology is patented in both the US Patent office (US10108778B2, US20200294628A1) and European Patent Office (ES2899879T3).

How does Kepler work?

The patented Kepler multi-kingdom taxonomic profiler is a comprehensive algorithm consisting of three interwoven pipelines: (1) pre-computation database construction, (2) K-mer based taxonomic identification and classification, and (3) abundance estimation and classification refinement. More details on the three steps are given below.

1. Pre-computational stage for curating and building a comprehensive biomarker database (GenBook™)
   The first iteration of database curation involves the selection of high quality microbial genomes based on high completeness/low contamination ratio, quality of genome assembly (genomes with high fragmentation/# of contigs discarded when better representatives for that clade are present), and prioritization of intra-species diversity instead of redundancy. Once the library of high quality genome assemblies are selected, the genome assemblies are then scrubbed clean of low complexity sequences, prophages, plasmids, and human/mouse contaminated regions to maximize for taxonomically informative signal to noise ratio. The final database (GenBook™) includes >30,000 species encompassing multiple microbial kingdoms including Bacteria, Viruses, Protists, Fungi, and Phages. Next, the second iteration of database building involves splitting the curated and cleaned collection of microbial genomes into n-mers of variable length. The n-mers are then categorized as either shared or unique biomarkers across individual genomes, which is facilitated by a phylogenetic tree-like data structure. The tree backbone represents shared genomic biomarkers between different taxa, while the tree leaves are individual microbial genomes with unique biomarkers.
2. K-mer based Taxonomic classification/Identification
   This first comparator phase splits the millions of sequencing reads for each sample into k-mer sets. It then looks for exact matches between sample k-mers and reference biomarkers that are dispersed across the different phylogenetic branches and leaves of the GenBook™ database. . By focusing on small genomic regions of large differential informational content, this phase eliminates 99% of genomes which are almost certainly not in the metagenomic sample and generates a short list of nearest reference strains. This highly efficient and accurate classification readily admits sub-species taxonomy to an ANI divergence less than 0.003. Classification sensitivity and accuracy is maintained through biomarker aggregation statistics, coverage depth estimation, and abundance estimation–but with an inhomogeneous variance that is parsed into Step (3).
3. Probabilistic Smith-Waterman based Abundance Estimation and Classification Refinement
   The second comparator phase uses an edit distance-scoring based probabilistic Smith-Waterman algorithm [1] to compare sequencing reads with a reference set of identified microbial taxa from Step (2). This involves a more computationally-intensive processing of the remaining 1% of strains to eliminate any false positives and achieve abundance estimations which are more homogeneous, more accurate, and have significantly less variance.
   Given an example scenario of 10 reads–where reads 1-8 belong to strain A, read 9 belongs to strain B, and read 10 belongs to both strain A and strain B with equal alignment score–Kepler’s will probabilistically split read 10’s abundance into strain A with an abundance of 8.89 and strain B with an abundance of 1.11. Given a more complicated metagenomic sample scenario, contested reads are apportioned according to implied priors from uncontested reads. An iterative Maximum Likelihood Estimation algorithm is then converged to the final read abundance estimates. To summarize, overall abundance precision and classification accuracy is achieved by running the comparators in sequence, scoring the entire read probabilistically against the reference set, and distinguishing homologous regions.

To learn more about Kepler and its benchmarking, please download the whitepaper from here.