4. Reference Creation
EPITOME creates references from a set of input sequences that have passed the initial QC filters. Below is a descript of each step of the reference creation process.
The first step of reference creation is to split quality filtered sequences into closely related clusters (controlled using --dist_threshold). This is accomplished using a multi-step process, which was designed to handle very large data sets. A general overview of this process is described below:
- Hierarchal clustering
- A subset of sequences are randomly selected (controlled using
--max_cluster). - The subset is split into clusters using the "complete linkage" of pairwise average nucleotide identity (ANI) between sequences (controlled using
--dist_threshold).
- Assign clusters
- Representatives of each cluster are selected.
- Remaining sequences are assigned to one of the clusters by comparing to the representatives using the pairwise ANI.
- Unassigned sequences are passed to step 1.
This process is repeated until all sequences have been assigned into a cluster.
Note
Average nucleotide identity is estimated using sourmash.
