pairedBamToBed12 converts properly paired BAM alignments to
BED12 format. Typical proper pairs will be represented by a 2 blocks BED12
entry. Additional blocks are produced when an alignment contains long deletion
(CIGAR N-op). Thickness indicates the first read of the pair. The BAM input
file must be grouped/sorted by query name (not alignment position).
Read 1: >>>>>>>>>>>> Read 2: <<<<<<<<<<<<<-----<<<<<<< The pair: >>>>>>>>>>>>------>>>>>>>>>>>>>----->>>>>>>
We developped pairedBamToBed12 in order to represent a paired alignment
on a sigle line, with splicing information. The existing tools did not provide
both features at the same time: bedtools bamtobed -split represents each
read on separate lines and, bedtools bamtobed -bedpe does not represent
spliced alignments.
In brief: download the source code, unpack it and enter the main source
directory, type make and a pairedBamToBed12 executable file will appear
the bin directory. Test it with make test. See below for an example.
wget https://github.com/Population-Transcriptomics/pairedBamToBed12/archive/pairedbamtobed12.zip unzip pairedbamtobed12.zip cd pairedBamToBed12-pairedbamtobed12 make make test ls bin/pairedBamToBed12
Usage:
pairedBamToBed12 [OPTIONS] -i <BAM>
.. tabularcolumns:: |p{4.5cm}|p{8.5cm}|
| Option | Description |
|---|---|
| -dblock | Triggers the creation of a new block when an alignment contains short deletion from reference (CIGAR D-op). |
| -color | An R,G,B string for the color used with BED12 format. Default is (255,0,0). |
| -extraG | Ignore G mismatches on first bases (experimental option for use on CAGE alignments with BWA aln). |
| -nsep | A string after which the read names are allowed to differ. Default is ___. Give an improbable value like 'nothankyou' to turn off. |
| -qual | The minimum (inclusive) mapQ sum for reporting the paired BAM into a BED12. Default is 0. |
| -x | Optional filename where unprocessed mapped pairs can be stored. |
By default it processes a properly paired pair of reads into a single BED12 line, where the start and end positions are the 5′ end of Read 1 and the 3′ end of Read 2. The BED12 blocks are used to indicate positions where the reads match, and the thick part indicates where is the contribution of Read 1. The relative orientation of the mate pairs must be forward/reverse (which is the standard in most libraries prepared for the Illumina platform).
Note
The BAM file must be sorted by read name.
Note
Reads that are not followed by their mate or not properly paired will be skipped.
$ pairedBamToBed12 -i 1proper-pair.bam
chr1 50053297 50053480 M00528:19:000000000-A88YD:1:1101:2241:12366 0 + 50053297 50053324 255,0,0 2 27,21 0,162In transcriptome analysis, the BED12 entries produced by pairedBamToBed12
represent the minimal information about a cDNA that was given by a read pair.
pairedBamToBed12 was created for the analysis of CAGEscan libraries, which
are paired-end directional libraries of random-primed 5′ cDNAs. The BED12
files are used to assemble CAGEscan clusters that combine all the pairs where
the 5′ end is in the same transcript start site peak, thus providing approximate
rudimentary transcript models for each peak. A typical analysis can be found in
Kratz et al., 2014.
This BED12 format is also supported in RIKEN's Zenbu genome browser, where one can load data in this format and visualise it either as genome intervals or as expression histograms.
Note
BWA has a bug that will set the properly paired flag for reads where one
mate is aligned very near the end of a chromosome and the other is aligned
very near the beginning of the next chromosome, when the -a option of
sampe is large. However, for CAGEscan, large numbers are necessary to
span whole gene loci. It is therefore recommended to sanitise the output
of BWA with SAMtools, using its fixmate command, that corrects the
properly paired flag since version 1.0.
Note
CAGE methods sometimes add an extra G at the beginning of the cDNAs (see
http://population-transcriptomics.org/nanoCAGE/#extra-G). This leads to
1-base shifts of some TSS peaks. From version 1.2, pairedBamToBed12
provides an experimental option, -extraG to shift the start or end
(according to the strand) of the output of one base when a G mismatch
is detected on the first base of Read1. A more detailed description of
the problem may be found in the supplemental material of the FANTOM3 paper.
The implementation here is very naive and incomplete, and was tested only on
data produced by BWA's sampe command. It relies on the MD flag and does
not understand clipping. Thus, the -extraG option available here is
not entierly satisfactory and may be removed in the future. A better
approach for instance would be to post-process the BAM file instead of
implementing a correction here.
pairedbamtobed12 only pertains to pairs mapped on the same chromosome
and is therefore unfit for representing gene fusions or interchromosomal
interactions.
At the moment, pairedbamtobed12 will output broken BED12 files if Read1
and Read2 overlap and align to the same splice junction.
pairedBamToBed12 is distrubuted under the GNU General Public License version 2.
The tool pairedBamToBed12 is copyright 2013~2015 RIKEN. It was originally
written by Nicolas Bertin as an addition to Bedtools 2.11.1. It was then
ported to Bedtools 2.21.0 by Mickaël Mendez, and then finally forked from the
Bedtools source as a stand-alone program by Charles Plessy. The documentation
was written by NB, MM and CP, and the regression tests were implemented by MM
and CP.
Bedtools is copyright Aaron Quinlan and others.