Package: rmseq
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 availablehereIs this the right tool for me?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.InstallationInstall RM-seq pipelinepip3 install rmseqDependenciesRM-seq has the following package dependencies: * EMBOSS >= 6.6 forclustalo,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,BiopythonIf you are using theOSX BreworLinuxBrewpackaging 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 biopythonQuick startDormseqHelpusage: 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.To check dependencies are installedrmseq checkTo run the test datasetrmseq testTo run analysis pipeline, follow the steps inrmseq 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)To check the versionrmseq versionOutputsRM-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):ColumnExampleDescriptionbarcodeGACACAACTGAGATTAsequence of the barcodesampleRifampicin1output folder nameprot_mutationH481Nannotation of the amino acid change (Histidine residue 481 substituted by Asparagine)prot_start481start coordinate of the mutationprot_end481end coordinate of the mutationnuc_mutationC1443Gannotation of the nucleotide changenuc_start1443start coordinate of the nucleotide changenuc_end1443end coordinate of the nucleotide changeprotVRPPDKNNRFVGLYCTLV…protein sequence of the consensus sequencednaGGTTAGACCACCCGATAA…dna sequence of the consensus sequencereference_barcodeCTGACACGTCCTGAAGbarcode of the identical consesnsus amplicon used for annotationThe other files produced by RM-seq are:File nameDescriptionamplicons.barcodesTable with the count of each barcode sequenceamplicons.fnaMultifasta file containing all the consensus nucleotide sequence (header of sequence is the barcode)amplicons.faaMultifasta file containing all the consensus protein sequence (header of sequence is the barcode)amplicons.fna.cdhitMultifasta file containing all the unique consensus nucleotide sequence (header of sequence is the barcode)amplicons.faa.cdhitMultifasta file containing all the unique consensus amino acid sequence (header of sequence is the barcode)IssuesPlease report problems to theIssues
Page.AuthorsRomain Guerillot (github: rguerillot) | Torsten Seemann (github:
tseemann) | Mark B Schultz (github: schultzm)CitationIf 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.