PLoT-ME

Pre-classification of Long-reads for Memory Efficient Taxonomic assignment
Sylvain Riondet, K. Križanović, J. Marić and M, Šikić, Niranjan Nagarajan
NUS/SoC, Biopolis/GIS, Singapore

Tool in active development, any feedback or bug report is welcome, either through github or on twitter

Description

Pre-Processing

• Segmentation of NCBI RefSeq into clusters
• Building of taxonomic classifiers' indexes for each cluster

Classification

Taxonomic classification of mock communities / metagenomic fastq files

• Assignment of long DNA reads (Nanopore/PacBio) to each cluster
• Classification by the classifier with a subset of RefSeq
• Merging of reports

Kraken2 (Derrick E. Wood et al. 2019) and Centrifuge (D.Kim et al. 2016) are currently automated, and any classifier able to build its index on a set of .fna files with a provided taxid should work.

Take-aways

• High reduction in memory needs, defined by the number of clusters *
• Compatible and enhancing existing taxonomic classifiers
• Slight over-head of the pre-classification (currently ~3-5x in time, improvements for future releases)

* Mini Batch K-Means, Web-Scale K-Means Clustering D. Sculley 2010

Requirements

• Database of Genomes, in .fna / .fasta format, with an associated taxonomy id. Tested with NCBI RefSeq (ftp server)
• Taxonomic classifier, must be installed and added to PATH. Currently supported:
  • Kraken2
  • Centrifuge
  • (feel free to request support for more)
• Linux (tested on Ubuntu 18.04)
• Python >= 3.7

Package	Version
biopython	>= 1.72
ete3	>= 3.1.1
numpy	>= 1.17.3
pandas	>= 0.23
scikit-learn	>= 0.18
tqdm	>= 4.24.0

Installation

Create a Python 3 environment with conda or pyenv.
Installation is then done with pip:
python3 -m pip install plot-me
This will create 2 commands, plot-me.preprocess and plot-me.classify detailed in the 'Usage'.

It is also possible to clone PLoT-ME's repo, and launching commands directly with python path/to/PLoT-ME/parse_DB.py or classify.py

Usage

Pre-Processing

For the full help: plot-me.preprocess -h
Typical usage:
plot-me.preprocess <path/NCBI/refseq> <folder/for/clusters> <path/taxonomy> -k 4 -w 10000 -n 10 -o <OmitFoldersContainingString>

Pre-classification + classification

For the full help: plot-me.classify -h
Typical usage:
plot-me.classify <folder/with/clusters> <folder/reports> -i <fastq files to preclassify>

Example

/mnt/data
|-- mock_files
|   |-- mock_community_1.fastq
|   |   \-- minikm_b10_k3_s10000_oplant-vertebrate (one tmp file per cluster, generated by PLoT-ME)
|   \-- mock_community_2.fastq
|-- PLoT-ME
|   |-- k3_s10000
|   |   | -- kmer_counts
|   |   |    |-- counts.k3_s10000 (same tree as RefSeq, with <sequencing_name>.3mer_count.pd)
|   |   |    \-- all-counts.k3_s10000_oplant-vertebrate.csv
|   |   | -- minikm_b10_k3_s10000_oplant-vertebrate               <*>
|   |   |    |-- centrifuge       (10 folders with indexes)
|   |   |    |-- kraken2          (10 folders with indexes)
|   |   |    |-- RefSeq_binned    (10 folders with fna files)
|   |   |    |-- model.minikm_b10_k3_s10000_oplant-vertebrate.pkl
|   |   |    \-- segments-clustered.minikm_b10_k3_s10000_oplant-vertebrate.pd
|   |   \ -- minikm_b20_k3_s10000_oplant-vertebrate
|   |        \-- (same structure) 
|   |-- k4_s10000
|   |   ` --  (same structure)
|   \-- no-binning
|       |-- oAllRefSeq
|       \-- oplant-vertebrate
|           |-- centrifuge
|           \-- kraken2
|-- NCBI
|   \-- refseq
|-- reports
|   \-- mock_community_1 (one report per cluster)
\-- taxonomy

This <*> can be generated with:
plot-me.preprocess /mnt/data/NCBI/refseq /mnt/data/PLoT-ME /mnt/data/taxonomy -k 3 -w 10000 -n 10 -o plant vertebrate
And can be used with:
plot-me.classify /mnt/data/PLoT-ME/k3_s10000/minikm_b10_k3_s10000_oplant-vertebrate /mnt/data/reports -i /mnt/data/mock_files/mock_community_1.fastq

Technical details

Python 3 is the main programming language, with extensive use of libraries. Dependencies are resolved using PIP

Intermediate Data

Data is saved as pickle .pkl or Pandas DataFrame .pd

• Kmer counts Pandas DataFrames are saved under .../kmer_counts/counts.<param> and have the following columns:
  taxon category start end name description fna_path AAAA ... TTTT
• Cluster assignments segments-clustered.\<param\>.pd trade the nucleotides columns to a cluster column.
• RefSeq_binned is the clustering made by PLoT-ME, and holds one folder per cluster, with concatenated segments of genomes (one .fna file per taxa)
• Libraries generated by classifier, depends on each of them.

Final files

The model*.pkl and the folder kraken2 or centrifuge are needed for PLoT-ME to work. Folder tree needs to remain intact.

Work in progress

April 2021

• Implementation of Cython version of the kmer counter
• Adding reverse complement to forward strand

July 2020:

• pre-process Using large k (5+) and small s (10000-) yield very large kmer counts, costing high amounts of RAM (esp. when combining all kmer counts together, RAM needs to reach ~30GB or more).
• classify Merging of reports
• pre-process Cleaning of pre-processing files --clean

Future work

• classify Cleaning of pre-classification tmp files
• classify Multi cores
• classify/pre-process Speed up kmer counting
• pre-process Even sized bins
• pre-process Overlapping clusters or tricks for higher accuracy

Contact

Author: Sylvain Riondet, PhD student at the National University of Singapore, School of Computing
Email: sylvainriondet@gmail.com
Lab: Genome Institute of Singapore / National University of Singapore
Supervisors: Niranjan Nagarajan & Martin Henz

Thanks

Thanks for your support and supervision all along my PhD and this project: Martin Henz, Chenhao Li, Rafael Peres, D. Bertrand and the whole MTMS lab