GeneFinder is written in Python 2.7.5 and requires the following dependencies:

• lxml 3.2.3,
• numpy 1.7.1
• yaml 1.1
• pysam 0.7.5
• Biopython 1.61
• Samtools 1.0.18
• Bowtie 2.1.0
• Clustalw 2.1

The nucleotide sequences of genes of interest need to be stored in a multi FASTA format with only the names of genes in headers and MUST be designated ‘reference.fasta’. Gene names must NOT include underscores [‘_’]; these are reserved to designate variant sequences of a given gene (e.g gyrA_1, gyrA_2) which are treated by gene_finder as a sub-group with only the best matching variant selected for further processing and reporting. Before running gene_finder, an additional tab delimited table need to be prepared. This MUST be named ‘workflow.txt’ and should include for each gene sought seven description fields (see example below). Both files need to be located in the same folder which will be referred to in this document by ‘gene file directory’.

gene_finder.py –1 <fastq_forward> -2 <fastq_reverse> -gf <reference_file_directory> -o <output_directory>

• -1 full path to Illumina paired fastq forward
• -2 full path to Illumina paired fastq reverse
• -o full path to output directory
• -gf full path to gene file directory which is expect to include two files called ‘reference.fasta’ and ‘workflow.txt’ prepared as described above

GeneFinder output results for each sample in an XML format

Gene_finder XML result includes two global control metrics: coverage_control Reports the average depth of reads for chromosomal genes, calculated from all genes labeled by mode ‘mutant’ in ‘workflow.txt’, return 'failed’ if no chromosomal gene is detected or included in reference sequences Example value="122.68" Reports the estimated average of read depth expected for chromosomal genes, thus present in one copy in the genome

mix_indicator Reports the number of positions with mixture of alleles and total number of chromosomal positions inspected with values separated by colon, return 'failed' if chromosomal genes are not detected or included in the reference sequences Example value="4:13928" Example reporting 4 positions with mixture of nucleotides amongst 13928 positions representing the total number of positions of all chromosomal genes inspected.

For each gene sought, thereby specified in workflow.txt, gene_finder reports the following additional metrics: gene Returns gene name as defined in the FASTA reference header

mode Returns mode of analysis (e.g variant, mutant or regulator) as defined in the workflow.txt

detection Reports gene detection with three designations:

• ‘D’ for detected when coverage is 100 % and nucelotide similarity is greater than similarity cutoff value defined for the gene in the ‘workflow.txt’ file.
• ‘ND’ for not detected if similarity is below similarity cutoff value
• ‘U’ for uncertain if similarity is greater than similarity cutoff value but coverage is below 100%

alteration Reported only for gene ‘labeled ‘variant’ in ‘workflow.txt’ and returns amino acid changes with corresponding positions, including replacement or absence of translational termination with ‘early_stop’ or ‘no_stop’ or ‘None’ if no variations are detected for all positions listed in ‘positions of interest’ of workflow.txt, return all variations if no positions of interest are defined or ‘ND’ if reference sequence have less than 100% coverage.

mutation Reported for gene labeled ‘mutant’ in ‘workflow.txt’ and returns amino acid changes with corresponding positions or ‘None’ if no variations are detected for all positions listed in ‘positions of interest’ of workflow.txt, return ‘ND’ if no positions of interest are defined or if reference sequence have less than 100% coverage or undetected.

modification Reported for gene labeled ‘regulator’ in ‘workflow.txt’ and returns nucleotide variations with corresponding positions or ‘None’ if no variations are detected for positions listed in workflow.txt, return ‘ND’ if no positions of interest are defined or ‘ND’ if reference sequence have less than 100% coverage or undetected.

description Returns gene description as defined in workflow.txt

report_type Returns ‘required’ or ‘conditional’ and reporting position as defined in workflow.txt

coverage Reports the proportion of the reference sequence covered by a minimum acceptable depth of reads (see option –c setting before running gene_finder)

similarity Reports percentage of nucleotide identities relative to the reference sequence

Depth Reports the average, minimum and maximum depth of reads detected across all nucleotide positions of the gene with values separated by colons Example value=30.59:15:55; reporting an average of 30.59, minimum of 15 and a maximum of 55 reads detected across the total mapped positions of the gene

coverage_distribution Reports sections of the reference sequence covered by a minimum acceptable depth of reads Example value=[‘1-50’, ‘100-953’]; reporting coverage with an acceptable read depth from position 1 to 50 and from 100 to 953. The absence of coverage between positions 50 and 100 could suggest a low coverage compared to the average depth, a high level of variability or deletion.

insertions Reports insertion types and their corresponding positions Example value=68:+3GGT; reporting an insertion of three nucleotides ‘GGT’ at reference sequence position 68

deletions Deletion types and corresponding positions Example value= 842:-1A Example reporting a deletion of one nucleotide ‘A’ at reference sequence position 842

mix Positions with mixed nucleotides, their natures and relative percentages Example value=="108:C-T-0.4-0.6” ; reporting mixture of C and T with a proportion of 40 and 60%, respectively amongst a total of 120 reads mapped at position 108.

large_indels Positions with potentials large-scale indels Example value="[(99, '19:88.89'), (100, '15:100.0')]”, reporting the proportion of reads starting or ending (88.89 and 100%) of the total number of reads (19 and 15) mapped at position 99 and 100 and thereby indicating the presence of a possible large insertion sequence between these two positions.

mismatch Positions with mismatches Example value=573:T-A;576:C-T;608:T-Y; reporting a substitution of T to A and C to T at reference sequence positions 573 and 576 whereas in position 608 the mix of C and T is reported using iupac code