alignmentsieve (version 3.5.4+galaxy0)

BAM file:

Output in BEDPE format?:

Instead of producing BAM files, write output in BEDPE format (as defined by MACS2). Note that only reads/fragments passing filtering criterion are written in BEDPE format.

Amount to shift fragments:

Shift the left and right end of a fragment. A positive value shift an end to the right (on the + strand) and a negative value shifts a fragment to the left. Either 2 or 4 integers can be provided. For example, '2 -3' will shift the left-most fragment end two bases to the right and the right-most end 3 bases to the left. If 4 integers are provided, then the first and last two refer to fragments whose read 1 is on the left or right, respectively. Consequently, it is possible to take strand into consideration for strand-specific protocols. Note that only properly paired reads are considered.

Shift fragment ends as appropriate for ATAC-seq:

Only include reads originating from fragments from the forward or reverse strand.:

By default (the no option), all reads are processed, regardless of the strand they originated from. For RNAseq, it can be useful to separately create bigWig files for the forward or reverse strands. Note that this tools assumes that a dUTP-based method was used, so fragments will be assigned to the reverse strand if the second read in a pair is reverse complemented.

Ignore duplicates:

If set, reads that have the same orientation and start position will be considered only once. If reads are paired, the mate position also has to coincide to ignore a read.

Minimum mapping quality:

If set, only reads with a mapping quality score at least this high are considered.

Include reads based on the SAM flag:

For example, to get only reads that are the first mate use a flag of 64. This is useful to count properly paired reads only once, otherwise the second mate will be also considered for the coverage.

Exclude reads based on the SAM flag:

For example, to get only reads that map to the forward strand, use --samFlagExclude 16, where 16 is the SAM flag for reads that map to the reverse strand.

Minimum fragment length for inclusion.:

This is primarily useful in things like ATACseq, where one would like to look specifically at mono- or di-nucleosome fragments.

Maximum fragment length for inclusion.:

A value of 0 (the default) is equivalent to no maximum.

Blacklisted regions in BED/GTF format:

One or more files containing regions to exclude from the analysis

Save the total number of reads seen and remaining after filtering to a text file?:

Save alignments NOT passing the filtering criteria?:

What it does

This tool is very much the counterpart of estimateReadFiltering, in that it can filter alignments based on a variety of desired criterion. While much of this can be done with samtools, this tool can additionally filter by fragment strand and length (e.g., for RNA-seq and ATAC-seq experiments, respectively). Finally, this program can produce BEDPE files, which can be used as input into MACS2 for peak calling, where the fragment ends have been optionally shifted.

Output

The primary output is a BAM file with all alignments passing the desired criteria. Note that all unmapped reads are removed. Additionally, an optional text file can be produced with the following entries:

Number of reads passing the filtering criteria

Total number of initial reads

Instead of producing a filtered BAM file, a BEDPE file appropriate for use with MACS2 can be used, optionally with fragment ends shifted. This is useful in cases like ATAC-seq.

The --shift option can take either 2 or 4 integers. If two integers are given, then the first value shifts the left-most end of a fragment and the second the right-most end. Positive values shift to the right and negative values to the left. See below for how setting --shift to '-5 3' would shift a single fragment:

     ----> read 1
                 read 2 <----

     ------------------------ fragment

-------------------------------- shifted fragment

The same results will be produced if read 1 and read 2 are swapped. If, instead, the protocol is strand-specific, then the first set of integers in a pair would be applied to fragments where read 1 precedes read 2, and the second set to cases where read 2 precedes read 1. In this case, the first value in each pair is applied to the end of read 1 and the second to the end of read 2. For example, suppose "-5 3 -1 4" were given as the option to --shift. The -5 3 set would produce the following:

     ----> read 1
                 read 2 <----

     ------------------------ fragment

-------------------------------- shifted fragment

and the -1 4 set would produce the following:

----> read 2
            read 1 <----

------------------------ fragment

    --------------------- shifted fragment

As can be seen, such fragments are considered to be on the - strand, so negative values then shift to the left on its frame of reference (thus, to the right relative to the + strand).

For more information on the tools, please visit our help site.

For support or questions please post to Biostars. For bug reports and feature requests please open an issue on github.

This tool is developed by the Bioinformatics and Deep-Sequencing Unit at the Max Planck Institute for Immunobiology and Epigenetics.