Analysis bioinformatic pipeline for high-throughput assessment of resistance mutations. RM-seq is an amplicon-based, deep-sequencing technique using single molecule barcoding. We have adapted this method to identify and characterise antibiotic resistance mutation.
The complete description of the RM-seq workflow is available here
- To be able to us this pipeline you need to have sequenced amplicon library with molecular barcodes.
- It only supports paired-end FASTQ reads (including .gz compressed fastq files).
- It needs paired reads that are overlapping.
- It needs a reference fasta sequences of the sequenced gene (DNA sequence).
- It's written in Python3 and Perl.
pip3 install rmseq
RM-seq has the following package dependencies:
- EMBOSS >= 6.6 for
clustalo
,cons
,getorf
,diffseq
- clustal-omega >= 1.2.1
- bwa >= 0.7.15
- samtools >= 1.3
- bedtools >= 2.26.0
- pear >= 0.9.10
- cd-hit >= 4.7
- trimmomatic >= 0.36
- seqtk >= 1.3-r106 (only if you subsample reads)
- python modules:
plumbum
,Biopython
If you are using the OSX Brew or LinuxBrew packaging system:
brew tap homebrew/science
brew tap tseemann/bioinformatics-linux
brew install parallel; parallel --citation # please write will cite
brew install bedtools
brew install EMBOSS
brew install clustal-omega
brew install bwa
brew install samtools
brew install pear
brew install cd-hit
brew install trimmomatic
brew install seqtk
pip3 install plumbum
pip3 install biopython
Do
rmseq
usage: rmseq [-h] ...
Run RM-seq pipeline.
optional arguments:
-h, --help show this help message and exit
Commands:
run Run the pipeline.
version Print version.
check Check pipeline dependencies
test Run the test data set.
rmseq check
rmseq test
rmseq run -h
usage: rmseq run [options]
Run the pipeline
positional arguments:
R1 Path to read pair 1
R2 Path to read pair 2
refnuc Reference sequence that will be used for premapping
filtering and mutation annotation (fasta).
outdir Output directory.
optional arguments:
-h, --help show this help message and exit
-d, --debug_on Switch on debug mode.
-f, --force Force overwite of existing.
-b BARLEN, --barlen BARLEN
Length of barcode (default 16)
-m MINFREQ, --minfreq MINFREQ
Minimum barcode frequency to keep (default 5)
-q BASEQUAL, --basequal BASEQUAL
Minimum base quality threshold used for trimming the
end of reads (trimmomatic TRAILING argument) (default
30)
-c CPUS, --cpus CPUS Number of CPUs to use (default 72)
-t TRANSLATION, --translation TRANSLATION
Manually set the reading frame for translation (use 1,
2 or 3 - use getorf by default)
-r MINSIZE, --minsize MINSIZE
Minimum ORF size in bp used when annotating variants
(default 200)
-w WSIZE, --wsize WSIZE
Word-size option to pass to diffseq for comparison
with reference sequence (default 5)
-s SUBSAMPLE, --subsample SUBSAMPLE
Only examine this many reads.
-k, --keepfiles Keep the intermediate files (default remove)
-n, --noaln Skip reads alignment when generating consensus (to use
for indel quantification only) (default align)
rmseq version
RM-seq produces a tap-separated output file called amplicons.effect where each raw correspond to a consensus amplicon (a genetic variant in the sequenced population):
Column | Example | Description |
---|---|---|
barcode | GACACAACTGAGATTA | sequence of the barcode |
sample | Rifampicin1 | output folder name |
prot_mutation | H481N | annotation of the amino acid change (Histidine residue 481 substituted by Asparagine) |
prot_start | 481 | start coordinate of the mutation |
prot_end | 481 | end coordinate of the mutation |
nuc_mutation | C1443G | annotation of the nucleotide change |
nuc_start | 1443 | start coordinate of the nucleotide change |
nuc_end | 1443 | end coordinate of the nucleotide change |
prot | VRPPDKNNRFVGLYCTLV... | protein sequence of the consensus sequence |
dna | GGTTAGACCACCCGATAA... | dna sequence of the consensus sequence |
reference_barcode | CTGACACGTCCTGAAG | barcode of the identical consesnsus amplicon used for annotation |
The other files produced by RM-seq are:
File name | Description |
---|---|
amplicons.barcodes | Table with the count of each barcode sequence |
amplicons.fna | Multifasta file containing all the consensus nucleotide sequence (header of sequence is the barcode) |
amplicons.faa | Multifasta file containing all the consensus protein sequence (header of sequence is the barcode) |
amplicons.fna.cdhit | Multifasta file containing all the unique consensus nucleotide sequence (header of sequence is the barcode) |
amplicons.faa.cdhit | Multifasta file containing all the unique consensus amino acid sequence (header of sequence is the barcode) |
Please report problems to the Issues Page.
Romain Guerillot (github: rguerillot) | Torsten Seemann (github: tseemann) | Mark B Schultz (github: schultzm)
If you use this tool please cite: Guérillot R et al. Comprehensive antibiotic-linked mutation assessment by Resistance Mutation Sequencing (RM-seq). 2018. doi:10.1101/257915.