Overview

TnAtlas is a Python package for identifying and annotating transposon integration events into genomes.

Given a set of sequencing reads, transposon sequences, and genomes, the TnAtlas package can:

• Looks for reads which contain genomic DNA preceded by transposon DNA.
• Annotate the reads with corresponding features from the genome.
• Produces a summary for a set of reads in excel format.

The package ships with 2 utilities, tnfind and tnmeta, which can be used to run analysis from the command line. 'tnfind' uses Blastn to align sequencing reads to a given transposon plasmid sequence to identify the transposon end and subsequently aligns the reads to a given genome. The annotations from the genome file are included in the output file results.xlsx. 'tnmeta' adds metadata to your results.xlsx file. A usual metadata added is the plate layout to identify each sequencing read.

Installing

Dependencies

• Python >= 3.8
• blastn >= 2.12

Optionally

Some parts of the pipeline also require

• fastqc (for sequencing quality control reports)
• sickle (for trimming based on sequencing quality)

From source code

1. Get the code: git clone https://github.com/BiocomputationLab/tnatlas

2. Install using pip:

   python3 -m pip install ./tnatlas

From PyPI (using pip)

python3 -m pip install tnatlas

Using Docker (recommended for Windows users to use trimming function with sickle)

docker pull biocomputationlab/tnatlas:latest

Usage

tnfind

tnfind sequencing_data path_to_results_folder -transposon transposon_file.gb -genome genome_file.gb -trim -sam

usage:

tnfind [-h] [-v] [--sequencing-type {sanger,solexa,illumina}] [--input-type {fasta,fastq}] [--input-ext INPUT_EXT] [-o OUTPUT_FILE] [-qc [PATH_TO_FASTQC]] [-trim [PATH_TO_SICKLE]] [--trim-quality TRIM_QUALITY] [--trim-length TRIM_LENGTH] [--trim-save] [-blastn PATH_TO_BLASTN] [-sam] -transposon TRANSPOSON [TRANSPOSON ...] [--transposon-type TRANSPOSON_TYPE] [--transposon-save] [--transposon-word-size TRANSPOSON_WORD_SIZE] [--transposon-evalue TRANSPOSON_EVALUE] -genome GENOME [GENOME ...] [--genome-type GENOME_TYPE] [--genome-save] [--genome-word-size GENOME_WORD_SIZE] [--genome-evalue GENOME_EVALUE] [--genome-prefix GENOME_PREFIX] [-config CONFIG] input_dir output_dir

Use a set of sequencing reads to identify the positions of likely transposon integration events in genomes. The integration events are identified using target sequences from the files provided in the transposon argument. Integration events are position in the genomes by alignment of the remainder of the sequencing read. Likely integration events annotated and saved in genbank format. A summary table is also produced containing detail on each likely event.

positional arguments:

input_dir The directory containing the reads to be processed

output_dir The directory in which to save results

options:

-h, --help show this help message and exit

-v --sequencing-type {sanger,solexa,illumina} The method from which the single end reads are obtained (default: sanger)

--input-type {fasta,fastq} The file format in which the single end reads are saved (default: fastq)

--input-ext INPUT_EXT The file extension of the single end read files (default: ab1 if INPUT_TYPE is fastq, fasta otherwise)

-o OUTPUT_FILE A filename for the output summary table (default: results.xlsx)

-qc [PATH_TO_FASTQC] If present, fastqc analysis of read quality is performed

-trim [PATH_TO_SICKLE] If present, sickle is used to trim the reads

--trim-quality TRIM_QUALITY The quality threshold passed to sickle for trimming (default: 20)

--trim-length TRIM_LENGTH The length threshold passed to sickle for trimming (default: 20)

--trim-save If present, the trimmed reads will be saved in OUTPUT_DIR

-blastn PATH_TO_BLASTN The blastn executable (default: blastn)

-sam If present, save the blastn output in SAM format in OUTPUT_DIR

-transposon TRANSPOSON [TRANSPOSON ...] The file or files containing the sequences used to identify integrations

--transposon-type TRANSPOSON_TYPE The file format of the file(s) given in TRANSPOSON (default: genbank)

--transposon-save If present, save reads annotated with transposon sequences in OUTPUT_DIR

--transposon-word-size TRANSPOSON_WORD_SIZE The word size passed to blastn for the transposon alignment (default: 10)

--transposon-evalue TRANSPOSON_EVALUE The evalue passed to blastn for the transposon alignment (default: 0.01)

-genome GENOME [GENOME ...] The file or files containing genomes for alignment

--genome-type GENOME_TYPE The file format of the file(s) given in GENOME (default: genbank)

--genome-save If present, save reads annotated with all genome alignments in OUTPUT_DIR

--genome-word-size GENOME_WORD_SIZE The word size passed to blastn for the genome alignment (default: 10)

--genome-evalue GENOME_EVALUE The evalue passed to blastn for the genome alignment (default: 0.01)

--genome-prefix GENOME_PREFIX The number of base pairs, after the integration site, of genomic DNA to include in the output summary table

-config CONFIG A configuration file in TOML format. Command line arguments are overrided with value in the configuration file, if present

i.e (in data folder) tnfind . ./results/results.xlsx -transposon transposons.gb -genome pputidakt2240.gb -trim -sam

tnmeta

tnmeta -o path_to_results_folder/results.xlsx 'PLATE-WELL-' output_file_name.xlsx metadata_file.xlsx

usage:

tnmeta [-h] [-o OUTPUT_FILE] plate_regex well_regex result_file metadata [metadata ...]

Attach well metadata to a results table from a plate map

positional arguments:

plate_regex A regular expression which matches plates in record names

well_regex A regular expression which matches wells in record names

result_file The spreadsheet to annotate with metadata

metadata The spreadsheets containing the metadata map of a plate

options:

-h, --help show this help message and exit

-o OUTPUT_FILE If given, output will be saved to OUTPUT_FILE, if not, RESULT_FILE will be overwritten

i.e (in data folder) tnmeta -o ./results.xlsx 'PLATE-WELL' ./results.meta.xlsx ./PLATE_metadata.xlsx

Contributing

Contributions of all kinds are welcomed, including:

• Code for new features and bug fixes
• Test code and examples
• Bug reports and suggestions for new features (e.g. opening a github issue)

If you plan to change the source code, please open an issue for discussing the proposed changes and fork the repository.

Citing

If you use this package as part of a publication, please cite:

Acknowledgements

This work was funded by grants BioSinT-CM (Y2020/TCS-6555) and CONTEXT (Atracción de Talento Program; 2019-T1/BIO-14053) projects of the Comunidad de Madrid, MULTI-SYSBIO (PID2020-117205GA-I00) and Severo Ochoa Program for Centres of Excellence in R&D (CEX2020-000999-S) funded by MCIN/AEI/10.13039/501100011033, and the ECCO (ERC-2021-COG-101044360) contract of the EU.