Map#

Identify insertion sites for barcoded sequencing library with mbarq map#

Input/Output Files:#

Required Inputs

  • FASTQ file generated from sequencing barcoded mutant library

  • Genome FASTA file of the bacteria used to generate the library

  • Transposon construct structure add diagram.

Suggested inputs

  • Annotation file in GFF3 format (this will allow mapping insertion sites to genomic features).

  • Filtering parameter (-l, in our hands, filtering barcodes supported by less than 100 reads produced reliable library annotations. This of course is dependent on depth of the sequencing, and should be tested for each use case).

  • Report the closest gene (-c). If gff file is provided, by default, mbarq will only report features overlapping the insertion site. In addition, mbarq can report the location and distance of the closest downstream feature for barcodes that do not directly overlap any features.

Output files

library.annotated.csv: final library map with annotations library.map.csv: final library map without annotations library_mapping.log: log file library.blastn: blast output for each barcode: host sequence;
library.fasta: fasta files of barcodes and host sequences (>barcode\nhostsequence); library.output.bed: bedtools intersection of gff and barcode locations

Example Usage#

mbarq map -f <library_R1.fastq.gz> -g <host.fasta> -a <host.gff> -l 100 \ 
-n LibraryName -tn B17N13GTGTATAAGAGACAG

All Options#

Usage: mbarq map <options>

Options:
  -f, --forward FILE           input file for reads in forward orientation;
                               FASTQ formatted; gz is ok.  [required]
  -g, --genome FILE            reference genome in FASTA format  [required]
  -a, --gff FILE               annotation file in GFF format
  -n, --name STR               unique library name, by default will try to use
                               FASTQ filename
  -tn, --transposon STR         transposon construct structure, consisting of the following:
                                  1. barcode length, written as B[# of nt], eg. B17
                                  2. conserved sequence motif, usually part of transposons inverted repeat (IR), eg. GTGTATAAGAGACAG
                                  3. if there are extra nucleotides between barcode and
                                     conserved sequence motif, indicate with N[# of nt], eg. N13
                               The default represents the following construct:
                               ----------------------------------------------------------------------------
                               Read      ||AGTACTTTACTACTACT||TACCTGACCGTAA||GTGTATAAGAGACAG||TTACCTGACCGAC
                               ----------||-----------------||-------------||---------------||-------------
                               Components||     barcode     ||   spacer    ||    conserved  ||     host
                                         ||                 ||             ||    motif (IR) ||
                               ----------||-----------------||-------------||---------------||-------------
                               Encoding  ||       B17       ||     N13     ||GTGTATAAGAGACAG||
                               ----------------------------------------------------------------------------
                               Note: relative position of barcode and conserved sequence motif matters, 
                               i.e. if conserved sequence motif comes before the barcode,
                               it should be written as GTGTATAAGAGACAGN13B17.
                                [B17N13GTGTATAAGAGACAG]
  -o, --out_dir DIR            output directory [.]
  -l, --filter_low_counts INT  filter out barcodes supported by [INT] or less
                               reads [0]
  -ft, --feat_type STR         feature type in the GFF file to be used for
                               annotation, e.g. gene, exon, CDS [gene]
  --attributes STR[,STR]       Feature attributes to extract from GFF file
                               [ID,Name,locus_tag]
  --closest_gene               for barcodes not directly overlapping a
                               feature, report the closest feature [False]
  -h, --help                   Show this message and exit.

Re-annotate mapping file#

Library map file library.map.csv can be re-annotated (for example, if you would like to use different feature type or attributes) without re-mapping using mbarq annotate-mapped.

Example Usage#

mbarq annotate-mapped -i <library.map.csv> -a <host.gff> -ft gene --attributes ID,Name,locus_tag

All Options#

Usage: mbarq annotate-mapped <options>

Options:
  -i, --barcode_file FILE  unannotated barcode file produced by "map"
  -a, --gff FILE           annotation file in gff format
  -n, --name STR           unique library name, by default will try to use
                           FASTQ filename
  -o, --out_dir DIR        output directory [.]
  -ft, --feat_type STR     feature type in the gff file to be used for
                           annotation, e.g. gene, exon, CDS [gene]
  --attributes STR[,STR]   Feature attributes to extract from annotation file
                           [ID,Name,locus_tag]
  --closest_gene           for barcodes not directly overlapping a feature,
                           report the closest feature [False]
  -h, --help               Show this message and exit.