TELOMORE

Telomore is a tool for identifying and extracting telomeric sequences from Oxford Nanopore or Illumina sequencing reads of Streptomycetes spp. that have been excluded from a de novo assembly. It processes sequencing data to extend assemblies, generate quality control (QC) maps, and produce finalized assemblies with the telomere/recessed bases included.

Before running Telomore

Telomore does not identify linear contigs but rather rely on the user to provide that information in the header of the fasta-reference file.

Usage

telomore --mode <mode> --reference <reference.fasta> [options]

Required Arguments

• --mode Specify the sequencing platform. Options: nanopore or illumina.
• --reference Path to the reference genome file in FASTA format.

Nanopore-Specific Arguments

• --single Path to a single gzipped FASTQ file containing Nanopore reads.

Illumina-Specific Arguments

• --read1 Path to gzipped FASTQ file for Illumina read 1.
• --read2 Path to gzipped FASTQ file for Illumina read 2.

Optional Arguments

• --coverage_threshold Set the threshold for coverage to stop trimming during consensus trimming (Default is coverage=5 for ONT reads and coverage=1 for Illumina reads).
• --quality_threshold Set the Q-score required to count a read position in the coverage calculation during consensus trimming (Default is Q-score=10 for ONT reads and Q-score=30 for Illumina reads).
• --threads Number of threads to use (default: 1).
• --keep Retain intermediate files (default: False).
• --quiet Suppress console logging.

Process overview

The process is as follows:

1. Map Reads: Reads are mapped against all contigs in a reference using either minimap2 or Bowtie2.
2. Extract Extending Reads Extending reads that are mapped to the ends of linear contigs are extracted.
3. Build Consensus The terminal extending reads from each end is used to construct a consensus using either lamassemble or mafft + EMBOSS cons
4. Align and Attach consensus The consensus for each end is aligned to the reference and used to extend it.
5. Trim Extended Replicon In a final step, all terminally mapped reads are mapped to the new extended reference and used to trim away spurious sequence, based on read-support.

Outputs

At the end of a run Telomore produces the following outputs:

├── {fasta_basename}_{seqtype}_telomore
│   ├── {contig_name}_telomore_extended.fasta
│   ├── {contig_name}_telomore_ext_{seqtype}.log
│   ├── {contig_name}_telomore_QC.bam
│   ├── {contig_name}_telomore_QC.bam.bai
│   ├── {contig_name}_telomore_untrimmed.fasta
│   └── {fasta_basename}_telomore.fasta
└── telomore.log # log containing run information.

In the folder there is a number of files generated for each contig considered:

Additionally, there is a fasta-file collecting all tagged linear contigs as they appear in {contig_name}_telomore_extended.fasta together with all non-linear contigs in the order they appear in the original file.

Inspecting the {contig_name}_QC.bam-file in IGV (Integrative Genomics Viewer) can be informative in evaluating the extended contig.

Dependencies (CLI-tools)

• Bowtie2
• Emboss tools (cons specifically)
• Lamassemble
• LAST-DB
• Mafft
• Minimap2, version 2.25 or higher
• Samtools

These can be installed using the conda recipe in this repo:

conda env create -f environment.yml -y

This repo can then be downloaded using git clone, the conda enviroment activated and the tool installed

# Activate telomore conda env
conda activate telomore

# Clone telomore repo
git clone https://github.com/dalofa/telomore && cd telomore

# Install package
pip install -e '.[dev]'