TBpore
Mycobacterium tuberculosis genomic analysis from Nanopore sequencing data
Table of Contents
Synopsis
tbpore is a tool with two main goals.
First is to process Nanopore Mycobacterium tuberculosis sequencing data to describe variants with respect to the
canonical TB strain H37Rv and predict antibiotic resistance (command tbpore process).
Variant description is done by decontaminating reads, calling variants with
bcftools and filtering variants.
Antibiotic resistance is predicted with mykrobe.
Second, tbpore can be used to cluster TB samples based on their genotyping and a given distance threshold (command
tbpore cluster).
Installation
conda
Prerequisite: conda (and bioconda channel correctly set up)
$ conda install tbporepip
The python components of tbpore are availble to install through PyPI.
pip install tbporeHowever, you will need to install the following dependencies, which cannot be installed through PyPI.
Dependencies
rasusa-
psdmversion 0.1 -
samtoolsversion 1.13 -
bcftoolsversion 1.13 -
mykrobeversion ≥ 0.11 -
minimap2version 2.22 -
seqkitversion 2.0
We make no guarentees about the performance of tbpore with versions other than those specified above. In particular, the bcftools version is very important. The latest versions of the other dependencies can likely be used.
Container
Docker images are provided through biocontainers.
singularity
Prerequisite: singularity
$ URI="docker://quay.io/biocontainers/tbpore:<tag>"
$ singularity exec "$URI" tbpore --helpsee here for valid values for <tag>.
docker
Prerequisite: Docker
$ docker pull quay.io/biocontainers/tbpore:<tag>
$ docker run quay.io/biocontainers/tbpore:<tag> tbpore --helpsee here for valid values for <tag>.
Configuring the decontamination database index
When you run your first tbpore process, you will get this error:
ERROR | Decontamination DB index tbpore/data/decontamination_db/tbpore.remove_contam.fa.gz.map-ont.mmi does not
exist, please follow the instructions at https://github.com/mbhall88/tbpore#configuring-the-decontamination-database-index
to download and configure it before running tbpore
This means you need to download the minimap2 decontamination database index before proceeding. You can download this index here or by running:
wget https://figshare.com/ndownloader/files/36708444 -O tbpore.remove_contam.fa.gz.map-ont.mmi.gzOnce the download is complete, you can:
- Ensure that the compressed index was transferred correctly by checking its
md5sum:
md5sum tbpore.remove_contam.fa.gz.map-ont.mmi.gz
82d050e0f1cba052f0c94f16fcb32f7b tbpore.remove_contam.fa.gz.map-ont.mmi.gz- Decompress the index:
gunzip tbpore.remove_contam.fa.gz.map-ont.mmi.gz- Check the md5sum of the decompressed index:
md5sum tbpore.remove_contam.fa.gz.map-ont.mmi
810c5c09eaf9421128e4e52cdf2fa32a tbpore.remove_contam.fa.gz.map-ont.mmi- Move the decompressed index to
<tbpore_dir>/data/decontamination_db/tbpore.remove_contam.fa.gz.map-ont.mmi
Once these four steps above are done, you should be able to run tbpore on an example isolate by going into the
tbpore dir and running:
just test-runPerformance
tbpore process
Benchmarked on 151 TB ONT samples with 1 thread:
- Runtime:
2103s avg,4048s max (s = seconds); - RAM:
12.4GB avg,13.1GB max (GB = Gigabytes);
tbpore cluster
Clustering 151 TB ONT samples:
- Runtime:
286s; - RAM:
<1GB;
Usage
General usage
Usage: tbpore [OPTIONS] COMMAND [ARGS]...
Options:
-h, --help Show this message and exit.
-V, --version Show the version and exit.
-v, --verbose Turns on debug-level logger. Option is mutually exclusive
with quiet.
-q, --quiet Turns off all logging except errors. Option is mutually
exclusive with verbose.
Commands:
cluster Cluster consensus sequences
process Single-sample TB genomic analysis from Nanopore sequencing data
process subcommand
Usage: tbpore process [OPTIONS] [INPUTS]...
Single-sample TB genomic analysis from Nanopore sequencing data
INPUTS: Fastq file(s) and/or a directory containing fastq files. All files
will be joined into a single fastq file, so ensure they're all part of the
same sample/isolate.
Options:
-o, --outdir DIRECTORY Directory to place output files [default:
tbpore_out]
-r, --recursive Recursively search INPUTS for fastq files
--tmp DIRECTORY Specify where to write all (tbpore)
temporary files. [default: <outdir>/.tbpore]
-S, --name TEXT Name of the sample. By default, will use the
first INPUT file with any extensions
stripped
-t, --threads INTEGER Number of threads to use in multithreaded
tools [default: 1]
-A, --report_all_mykrobe_calls Report all mykrobe calls (turn on flag -A,
--report_all_calls when calling mykrobe)
-d, --cleanup / -D, --no-cleanup
Remove all temporary files on *successful*
completion [default: no-cleanup]
--help Show this message and exit.
cluster subcommand
Usage: tbpore cluster [OPTIONS] [INPUTS]...
Cluster consensus sequences
Preferably input consensus sequences previously generated with tbpore
process.
INPUTS: Two or more consensus fasta sequences. Use glob patterns to input
several easily (e.g. output/sample_*/*.consensus.fa).
Options:
-T, --threshold INTEGER Clustering threshold [default: 6]
-o, --outdir DIRECTORY Directory to place output files [default:
cluster_out]
--tmp DIRECTORY Specify where to write all (tbpore)
temporary files. [default: <outdir>/.tbpore]
-t, --threads INTEGER Number of threads to use in multithreaded
tools [default: 1]
-d, --cleanup / -D, --no-cleanup
Remove all temporary files on *successful*
completion [default: no-cleanup]
--help Show this message and exit.
