RATTLE

Reference-free reconstruction and error correction of transcriptomes from Nanopore long-read sequencing

Requirements

GCC with C++14 suppport

Installation

Clone the repository

git clone --recurse-submodules https://github.com/comprna/RATTLE

Build RATTLE

cd RATTLE
make

Running RATTLE

Warning All the commands and parameters are still highly experimental and subject to changes in future versions

Sample pipeline

Details on each command can be found below, but these are the most common commands used when running RATTLE.

Cluster cDNA/RNA Nanopore reads at gene level

$ ./rattle cluster -i reads.fq -c genes.out --fastq

Cluster cDNA/RNA Nanopore reads at isoform level

$ ./rattle cluster -i reads.fq -c transcripts.out --fastq --iso

View clustering summary (csv with read_id,cluster_id)

$ ./rattle cluster_summary -i reads.fq -c transcripts.out --fastq

Extract 1 fastq file per cluster in clusters/ folder

$ mkdir clusters
$ ./rattle extract_clusters -i reads.fq -c transcripts.out -o clusters --fastq

Correct reads (mafft in $PATH)

$ ./rattle correct -i reads.fq -c transcripts.out > corrected.fq

Extract 1 fastq file per corrected cluster in corrected_clusters/ folder

$ mkdir corrected_clusters
$ ./rattle extract_clusters -i corrected.fq -c transcripts.out -o corrected_clusters --fastq

Clustering

$ ./rattle cluster -h
    -h, --help
        shows this help message
    -i, --input
        input fasta/fastq file (required)
    --fastq
        whether input and output should be in fastq format (instead of fasta)
    -c, --clusters
        clusters file (default: clusters.out)
    -t, --threads
        number of threads to use (default: 1)
    -k, --kmer-size
        k-mer size for gene clustering (default: 14)
    -s, --score-threshold
        minimum score for two reads to be in the same gene cluster (default: 0.1)
    -v, --max-variance
        max allowed variance for two reads to be in the same gene cluster (default: 500)
    --iso
        perform clustering at the isoform level
    --iso-kmer-size
        k-mer size for isoform clustering (default: 7)
    --iso-score-threshold
        minimum score for two reads to be in the same isoform cluster (default: 0.25)
    --iso-max-variance
        max allowed variance for two reads to be in the same isoform cluster (default: 10)
    -B, --bv-start-threshold
        starting threshold for the bitvector k-mer comparison (default: 0.4)
    -b, --bv-end-threshold
        ending threshold for the bitvector k-mer comparison (default: 0.2)
    -f, --bv-falloff
        falloff value for the bitvector threshold for each iteration (default: 0.05)
    -r, --min-reads-cluster
        minimum number of reads per cluster (default: 0)

This clustering step will generate a file containing its clusters in binary format (by default clusters.out). To work with these clusters, the following commands are used.

Clustering summary

$ ./rattle cluster_summary -h
    -h, --help
        shows this help message
    -i, --input
        input fasta/fastq file (required)
    -c, --clusters
        clusters file (required)
    --fastq
        whether input and output should be in fastq format (instead of fasta)

This command will output a CSV file to stdout containing two columns:

read_id, cluster_id

Clustering extraction

$ ./rattle extract_clusters -h
    -h, --help
        shows this help message
    -i, --input
        input fasta/fastq file (required)
    -c, --clusters
        clusters file (required)
    -o, --output-folder
        output folder for fastx files (default: .)
    --fastq
        whether input and output should be in fastq format (instead of fasta)

This command will generate 1 fastq/fasta file per cluster in the specified folder. This is useful to analyze particular clusters, perform alignments...

Error correction

$ ./rattle correct -h
    -h, --help
        shows this help message
    -i, --input
        input fastq file (required)
    -c, --clusters
        clusters file (required)
    --mafft-path
        path to mafft (default: mafft)
    -t, --threads
        number of threads to use (default: 1)

This command will perform error correction based on a previous clustering using MSA from MAFFT [1]. Path to mafft can be specified using the --mafft-path argument if it is not in $PATH. Input must be fastq (quality values are used in the correction step).

Huanle/RATTLE