BAM format file

A BAM file (.bam) is the binary version of a SAM file, which is a tab-delimited text file that contains sequence alignment data. These formats are described in details on the SAM Tools web site: http://samtools.github.io/hts-specs/.

The example input BAM files is provided in example/treated.sorted.bam and example/control.sorted.bam

Options

To show full list of available options with their description type:

probnorm bam -h

Required

-t, --treated

            Indexed BAM file for treated sample. If provided file is not indexed, probNORM will prepare it with SAMTools
            (requires SAMtools installation and increases the execution time)

-c, --control

            Indexed BAM file for control sample. If provided file is not indexed, probNORM will indexed it with SAMTools
            (requires SAMtools installation and increases the execution time)

-o, --output

            The name for the probNORM output file.

Optional

probNORM provides many additional parameters thet allow the detailed adjustment of the algorithm to the analyzed data.

-id

            probNORM can normalize only selected transcript. Quote transcripts ids in the comma separated list e.g. "RDN18,RDN25".
            Default: all transcripts are normalized.

-v, --coverage

            Coverage filtering. Only positions with coverage higher than specified are normalized.

-p, --pvalue

            P-value is the probability that a nucleotide belongs to the background distribution and is not statistically significant.
            Range [0-1]. All positions with a p-value higher than the provided one are rejected from the result.
            Default: 1 -> showing all positions.

-s, --transcript-size

            Set the percentage of transcript covered with reactive nucleotides. Those positions are further use to set normalization
            parameters. Default: 20 percent of provided transcript.

-f, --constrain-files

            The constrain files with normalized reactivities for RNAfold [ViennaRNA] and Fold [RNAStructure] will be prepared
            for each normalized transcript. Files will be saved in <output-file-name>_constrains.

Prepare input BAM files

By default the input BAM files should be sorted and indexed for the max optimization of the algorithm. User can do it by running following commands (SAMtools software is required for this steps).

samtools sort input.bam -o input_sorted.bam
samtools index input_sorted.bam

probNORM is able to sort and index provided files, however this step still requires the SAMtools and significatly increases the execution time.