The RF Count Genome module is an extension of the RF Count module, introduced in version 2.8.0 to process genome-level alignments. It can process any number of genome-level SAM/BAM files to calculate per-base RT-stops/mutations and read coverage. For any information on the RC file format, or on the RF Count algorithm, please refer to the manual page of rf-count.

Usage

To list the required parameters, simply type:

$ rf-count-genome -h
Parameter Type Description
-p or --processors int Number of processors (threads) to use (Default: 1)
-wt or --working-threads int Number of working threads to use for each instance of SAMTools/Bowtie (Default: 1).
Note: RT Counter executes 1 instance of SAMTools for each processor specified by -p. At least -p <processors> * -wt <threads> processors are required.
-o or --output-dir string Output directory for writing counts in RC (RNA Count) format (Default: rf_count_genome/)
-ow or --overwrite Overwrites the output directory if already exists
-t or --tmp-dir string Path to a directory for temporary files creation (Default: /tmp)
Note: If the provided directory does not exist, it will be created
-s or --samtools string Path to samtools executable (Default: assumes samtools is in PATH)
-r or --sorted In case SAM/BAM files are passed, assumes that they are already sorted lexicographically by transcript ID, and numerically by position
-t5 or --trim-5prime int[,int] Comma separated list (no spaces) of values indicating the number of bases trimmed from the 5'-end of reads in the respective sample SAM/BAM files (Default: 0)
Note #1: Values must be provided in the same order as the input files (e.g. rf-count -t5 0,5 file1.bam file2.bam, will consider 0 bases trimmed from file1 reads, and 5 bases trimmed from file2 reads)
Note #2: If a single value is specified along with multiple SAM/BAM files, it will be used for all files
-fh or --from-header Instead of providing the number of bases trimmed from 5'-end of reads through the -t5 (or --trim-5prime) parameter, RF Count will try to guess it automatically from the header of the provided SAM/BAM files
-f or --fasta string Path to a FASTA file containing the reference transcripts
Note: Transcripts in this file must match transcripts in SAM/BAM file headers
-ndd or --no-discard-duplicates Reads marked as PCR/optical duplicates, discarded by default, will be also considered
-pn or --primary-only Considers only primary alignments (SAM bitwise flag != 256)
-po or --paired-only When processing SAM/BAM files from paired-end experiments, only those reads for which both mates are mapped will be considered
-pp or --properly-paired When processing SAM/BAM files from paired-end experiments, only those reads mapped in a proper pair will be considered
-mq or --map-quality int Minimum mapping quality to consider a read (Default: 0)
-ls or --library-strandedness string Defines which genomic strand alignment-derived counts must be assigned to (check "Strandedness of genome-level alignments" below for possible values, Default: unstranded (with -m or -co), second-strand otherwise)
Note: strandedness specified via -ls can be overridden for individual samples by appending a colon followed by the library type to the sample name (check "Strandedness of genome-level alignments" below for additional details).
-co or --coverage-only Only calculates per-base coverage (disables RT-stops/mutations count)
-m or --count-mutations Enables mutations count instead of RT-stops count (for SHAPE-MaP/DMS-MaPseq)
Mutation count mode options
-om or --only-mut string Only the specified mutations will be counted
Note #1: mutations must be provided in the form [original]2[mutated]. For example, "A2T" (or "A>T", or "A:T") will only count mutation events in which a reference A base has been sequenced as a T. IUPAC codes are also accepted. Multiple mutations must be provided as a comma (or semi-colon) separated list (e.g. A2T;C:N,G>A)
Note #2: when specified, this parameter automatically disables insertion and deletion count
Note #3: when specified, an extra ouput folder frequencies/ will be generated, with a text file for each sample, containing the overall base substitution frequencies
-ds or --discard-shorter int Discards reads shorter than this length (excluding clipped bases, Default: 1)
-q or --min-quality int Minimum quality score value to consider a mutation (Phred+33, requires -m, Default: 20)
-es or --eval-surrounding When considering a mutation/indel, also evaluates the quality of surrounding bases (±1 nt)
Note: the quality score threshold set by -q (or --min-quality) also applies to these bases
-nd or --no-deletions Ignores deletions
-ni or --no-insertions Ignores insertions
-na or --no-ambiguous Ignores ambiguously mapped deletions
Note: the default behavior is to re-align them to their right-most valid position (or to their left-most valid position if -la has been specified)
-la or --left-align Re-aligns ambiguously mapped deletions to their left-most valid position
-rd or --right-deletion Only the right-most base in a deletion is marked as mutated
-ld or --left-deletion Only the left-most base in a deletion is marked as mutated
-md or --max-deletion-len int Ignores deletions longer than this number of nucleotides (Default: 10)
-me or --max-edit-distance float Discards reads with editing distance frequency higher than this threshold (0<m≤1, Default: 0.15 [15%])
-eq or --median-quality int Median quality score threshold for discarding low-quality reads (Phred+33, Default: 20)
-dc or --discard-consecutive int Discards consecutive mutations within this distance from eachothers
-cc or --collapse-consecutive Collapses consecutive mutations/indels toward the 3'-most one (recommended for SHAPE-MaP experiments)
-mc or --max-collapse-distance int Maximum distance between consecutive mutations/indels to allow collapsing (requires -cc, ≥0, Default_ 2)


Strandedness of genome-level alignments

An important difference with transcriptome-level analyses is that, at the level of the genome, the structure signal can be originated by either of the two DNA strands, dependening on where the gene resides. The strandedness depends on how the library has been generated. Specifying the proper strandedness of the library is crucial as it determines the way rf-count-genome will assign the RT-stop/mutation counts to the two genomic strands.
The strandedness of each sample can be specified in two ways:

  1. by specifying a single library type for all samples being analyzed via the -ls (or --library-strandedness)
  2. by appending a colon followed by the library type to each individual SAM/BAM file being passed to rf-count-genome (see below)
Value Strandedness
0 or u or unstranded The information on the genomic strand that originated the transcript is not preserved
1 or f or first or first-strand R1 aligns to the strand complementary to the one that originated the transcript
2 or s or second or second-strand R1 aligns to the same strand that originated the transcript


Second-strand is the default (and only accepted) mode for the analysis of RT-stop-based RNA structure mapping experiments. When mutation count (-m) or coverage-only (-co) modes are enabled, if the strandedness of the samples is not specified, samples are assumed to be unstranded.
For instance, in the examples below:

$ rf-count-genome -m -f reference.fasta -r sample1.bam:s sample2.bam:f sample3.bam
$ rf-count-genome -m -f reference.fasta -r -ls second-strand sample1.bam:s sample2.bam:f sample3.bam

sample1.bam is generated using a second-strand directional library prep strategy, whìle sample1.bam is generated using a first-strand directional library prep strategy. In the first example sample3.bam is assumed to have been generated using a non-directional library prep strategy (unstranded), while in the second example sample3.bam is assumed to have been generated using a second-strand directional library prep strategy (-ls second-strand).

When processing unstranded experiments, rf-count-genome will generate a single output RC file, named after the sample, having the .plus.rc suffix. When processing stranded experiments, two output RC files will be generated, named after the sample, respectively having the .plus.rc and .minus.rc suffixes and corresponding to the plus and minus strands of the genome.
Transcriptome-level RC files can be generated starting from genome-level RC files using the extract tool of the rf-rctools module. For additional information, please refer to the manual page.