CallGenes

Script: callgenes.sh Package: prok Class: CallGenes.java

Finds orfs and calls genes in unspliced prokaryotes. This includes bacteria, archaea, viruses, and mitochondria. Can also predict 16S, 18S, 23S, 5S, and tRNAs.

Basic Usage

callgenes.sh in=contigs.fa out=calls.gff outa=aminos.faa out16S=16S.fa

The only required parameter is in. All other parameters are optional and have default values.

Parameters

CallGenes provides gene calling capabilities with parameters organized by function.

File parameters

in=<file>: A fasta file; the only required parameter. Can also use infna, fnain, fna, or ref.
out=<file>: Output GFF file for gene annotations.
outa=<file>: Amino acid output file (FASTA format). Also accepts outamino, aminoout, outaa, aaout, or amino.
out16s=<file>: 16S rRNA output file (FASTA format). Also accepts 16sout.
out18s=<file>: 18S rRNA output file (FASTA format). Also accepts 18sout.
model=<file>: A PGM file or comma-delimited list. If unspecified, a default model will be used. Can use auto or default to use the built-in model. Also accepts pgm or gm.
stats=stderr: Stats output destination (may be stderr, stdout, a file, or null). Also accepts outstats.
hist=null: Gene length histogram output file. Also accepts outhist, lengthhist, lhist, or genehist.
compareto=: Optional reference GFF file to compare with the gene calls. 'auto' will name it based on the input file name.
ingff=: Input GFF file for additional gene annotations. Also accepts gffin.

Formatting parameters

json=false: Print stats in JSON format. Also accepts json_out.
binlen=21: Histogram bin length. Also accepts binlength or histdiv.
bins=1000: Maximum number of histogram bins.
pz=f: (printzero) Print histogram lines with zero count.
ordered=false: Maintain output order matching input order (may reduce performance).
trd=f: (trimreaddescription) Set to true to trim read headers after the first whitespace. Necessary for IGV compatibility.

Gene calling parameters

minlen=60: Don't call genes shorter than this length. Also accepts minlength. Default 60 in shell, but Java uses 80.
maxoverlapss=80: Maximum overlap allowed between genes on the same strand. Also accepts overlapss, overlapsamestrand, moss, or maxOverlapSameStrand.
maxoverlapos=110: Maximum overlap allowed between genes on opposite strands. Also accepts overlapos, overlapoppositestrand, moos, or maxOverlapOppositeStrand.
minStartScore=-0.10: Minimum score required for start codons.
minStopScore=-0.5: Minimum score required for stop codons.
minInnerScore=0.02: Minimum score for inner kmers. Also accepts minKmerScore.
minOrfScore=50: Minimum overall ORF score. Higher values increase specificity but reduce sensitivity. Also accepts minScore.
minAvgScore=0.08: Minimum average kmer score across the ORF.
breakLimit: Maximum number of consecutive low-scoring kmers allowed before breaking an ORF.
clearcutoffs=false: Clear all score cutoffs to very permissive values. Also accepts clearfilters.

Processing parameters

merge=f: For paired reads, merge overlapping reads before calling genes.
ecco=f: Error-correct overlapping paired reads without merging.
passes=1: Number of passes for iterative model refinement. Also accepts 2pass or twopass for 2 passes.
translate=true: Set amino acid output mode to translation (default).
detranslate=f: Output canonical nucleotide sequences instead of amino acids. Also accepts retranslate.
recode=f: Re-encode nucleotide sequences over called genes using canonical codons, leaving non-coding regions unchanged.
plus=true: Process the plus strand.
minus=true: Process the minus strand.

rRNA detection parameters

setbias16s: Set detection bias for 16S rRNA genes.
setbias18s: Set detection bias for 18S rRNA genes.
setbias23s: Set detection bias for 23S rRNA genes.
setbias5s: Set detection bias for 5S rRNA genes.
setbiastRNA: Set detection bias for tRNA genes.
setbiasCDS: Set detection bias for protein-coding sequences.
min16SIdentity: Minimum identity threshold for 16S rRNA detection. Also accepts min16SId.
min18SIdentity: Minimum identity threshold for 18S rRNA detection. Also accepts min18SId.
min23SIdentity: Minimum identity threshold for 23S rRNA detection. Also accepts min23SId.
min5SIdentity: Minimum identity threshold for 5S rRNA detection. Also accepts min5SId.

rRNA alignment parameters

align16s=: Enable 16S sequence alignment. Also accepts load16SSequence.
align18s=: Enable 18S sequence alignment. Also accepts load18SSequence.
align23s=: Enable 23S sequence alignment. Also accepts load23SSequence.
align5s=: Enable 5S sequence alignment. Also accepts load5SSequence.
16sstartslop: Start position tolerance for 16S detection. Also accepts ssustartslop.
16sstopslop: Stop position tolerance for 16S detection. Also accepts ssustopslop.
23sstartslop: Start position tolerance for 23S detection. Also accepts lsustartslop.
23sstopslop: Stop position tolerance for 23S detection. Also accepts lsustopslop.
5sstartslop: Start position tolerance for 5S detection.
5sstopslop: Stop position tolerance for 5S detection.

Kmer parameters

load16skmers: Load kmers for 16S detection. Also accepts load18skmers or loadssukmers.
load23skmers: Load kmers for 23S detection. Also accepts load28skmers or loadlsukmers.
load5skmers: Load kmers for 5S detection.
loadtrnakmers: Load kmers for tRNA detection.
longkmers: Enable all long kmer loading for rRNA and tRNA detection.
klong16s: Kmer length for 16S detection. Also accepts klong18s or klongssu.
klong23s: Kmer length for 23S detection. Also accepts klong28s or klonglsu.
klong5s: Kmer length for 5S detection.
klongtrna: Kmer length for tRNA detection.

Advanced scoring parameters

e1: ORF scoring parameter e1 (frame-specific penalty).
e2: ORF scoring parameter e2 (frame-specific penalty).
e3: ORF scoring parameter e3 (frame-specific penalty).
f1: ORF scoring parameter f1 (frame-specific bonus).
f2: ORF scoring parameter f2 (frame-specific bonus).
f3: ORF scoring parameter f3 (frame-specific bonus).
p0: Gene calling parameter p0 (probability adjustment).
p1: Gene calling parameter p1 (probability adjustment).
p2: Gene calling parameter p2 (probability adjustment).
p3: Gene calling parameter p3 (probability adjustment).
p4: Gene calling parameter p4 (probability adjustment).
p5: Gene calling parameter p5 (probability adjustment).
p6: Gene calling parameter p6 (probability adjustment).
q1: Gene calling parameter q1 (quality adjustment).
q2: Gene calling parameter q2 (quality adjustment).
q3: Gene calling parameter q3 (quality adjustment).
q4: Gene calling parameter q4 (quality adjustment).
q5: Gene calling parameter q5 (quality adjustment).
lookback: Lookback distance for both directions during gene calling.
lookbackplus: Lookback distance in the plus direction.
lookbackminus: Lookback distance in the minus direction.

Statistics parameters

verbose=false: Print detailed progress information.
extended=false: Print extended statistics. Also accepts extendedstats or verbosestats.

Examples

Basic Gene Calling

callgenes.sh in=genome.fasta out=genes.gff

Calls genes in a bacterial genome and outputs GFF annotations.

Full Analysis

callgenes.sh in=contigs.fa out=calls.gff outa=proteins.faa out16S=16S.fa stats=stats.txt

Calls genes, extracts proteins, identifies 16S rRNA, and saves statistics.

High Sensitivity Settings

callgenes.sh in=metagenome.fa out=genes.gff minlen=30 minOrfScore=30 clearcutoffs=true

Uses more permissive settings for calling genes in metagenomic data.

Multi-pass Analysis

callgenes.sh in=genome.fa out=genes.gff passes=2 extended=true

Uses iterative refinement with 2 passes and detailed statistics output.

rRNA and tRNA Detection

callgenes.sh in=genome.fa out=all.gff out16S=16S.fa out18S=18S.fa longkmers=true

Detection of protein-coding genes and RNA genes with enhanced kmer matching.

Algorithm Details

Gene Calling Strategy

CallGenes implements a probabilistic gene calling algorithm using the GeneCaller.callGenes() method with GeneModel for k-mer frequency scoring. The algorithm combines multiple evidence sources through six reading frame analysis:

K-mer-based scoring: Uses probabilistic gene models (PGM files) with StatsContainer and FrameStats classes to score potential ORFs based on k-mer frequency patterns
Start/stop codon analysis: Evaluates start and stop signals using minStartScore and minStopScore thresholds in context-dependent scoring
Frame-specific penalties: Applies different scoring based on reading frame to account for codon usage bias
Overlap resolution: Intelligently handles overlapping ORFs with configurable overlap limits

Dual Strategy for Sequence Processing

The algorithm employs different strategies based on sequence type:

Single sequences: Direct ORF calling with strand-specific processing
Paired reads: Optional merging with BBMerge or error correction with ECCO before gene calling

RNA Gene Detection

CallGenes includes specialized modules for non-coding RNA detection:

rRNA detection: Supports 16S, 18S, 23S, and 5S rRNA identification using kmer matching and sequence alignment
tRNA detection: Identifies transfer RNA genes with adjustable sensitivity parameters
Configurable thresholds: Identity and bias parameters allow fine-tuning for different organisms

Multi-pass Refinement

The multi-pass feature enables iterative model improvement:

Initial gene calling with default or provided model
Model training on initial predictions
Refined gene calling with improved model
Optional additional passes for further refinement

Performance Characteristics

Memory usage: Scales with genome size and model complexity; typically 6GB default heap
Threading: Automatic parallelization across available CPU cores
I/O optimization: Supports compressed input files and ordered/unordered output modes
Scalability: Handles single genomes to large metagenomic assemblies

Output Formats

CallGenes produces multiple output formats:

GFF3: Standard genome annotation format with detailed gene features
FASTA: Amino acid sequences for protein-coding genes
Statistics: Performance metrics in text or JSON format
Histograms: Gene length distributions for quality assessment

Support

For questions and support:

Email: bbushnell@lbl.gov
Documentation: bbmap.org