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== Introduction ==
== Introduction ==
'''NOTE: The current version works only for single end reads. If the input bam file contains paired end sequences, reads from the same fragment will be counted independently'''
* This tool was initially developed for RNA-seq data to quantify gene/exon expression but can also be used for other sequences as well.
* The tools calculates the read count for each region in the input list of regions from a BAM file, and also outputs the normalized read count as Read Per Million Mapped Reads per Kilobases (RPKM). To correct for the bias of the read count due to GC bias, it will also output the GC content of each region along with the total reads mapped to the corresponding GC content bins. By providing this statistic, the extent of GC bias can be investigated and if excessive bias is observed this statistic can be used to correct the global GC bias.
* This tool was initially developed for RNA-seq data to quantify gene/exon expression but can also be used for other purposes as well.
== Usage ==
== Usage ==
./readCount --reference hg19.fa --regions refFalt.exon.hg19 --min_overlap 5 --uniq --exon_out exon.readcount --gene_out gene.readcount input.bam
./readCount --reference hg19.fa --regions refFalt.exon.hg19 --min_overlap 5 --uniq --exon_out exon.readcount --gene_out gene.readcount input.bam
The following example generate normalized read counts using the number of reads that are only mapped to targets:
./readCount --reference hg19.fa --regions refFalt.exon.hg19 --min_overlap 5 --uniq --norm_by_mapped2target --exon_out exon.readcount --gene_out gene.readcount input.bam
== Input files ==
== Input files ==
Required input files are --reference, --regions and input.bam
Required input files are --reference, --regions and input.bam
* An example --regions file is a BEF file and looks like the following:
* An example --regions file is a BED file and looks like the following:
1 1000 2000 GENE1
1 1000 2000 GENE1
1 5000 6000 GENE1
1 5000 6000 GENE1
--min_overlap : minimum number of bases overlapping an input region to consider that the read is in the region.
--min_overlap : minimum number of bases overlapping an input region to consider that the read is in the region.
--min_map_quality : reads with map quality below this number will be ignored
--uniq : If a read is mapped to multiple regions (e.g. exons) this read will be counted only once toward the read count in the gene
--uniq : If a read is mapped to multiple regions (e.g. exons) this read will be counted only once toward the read count in the gene
--norm_by_mapped2target : The RPM and RPKM will be normalized by reads mapped only to target regions. Default is to use all mapped reads in a bam file.
--exon_cout : read count for each exon. A read may be counted to multiple exons if this read is mapped to multiple exons.
--exon_cout : read count for each exon. A read may be counted to multiple exons if this read is mapped to multiple exons.
--gene_cout : read count for each gene.
--gene_cout : read count for each gene.
== Output files ==
== Output files ==
AACSL:5 2171 15 0.780 0.359 49.65 443653
AACSL:5 2171 15 0.780 0.359 49.65 443653
AADAC:3 1607 172 8.949 5.569 38.39 468603
AADAC:3 1607 172 8.949 5.569 38.39 468603
== Download ==
The latest version of source code v0.01 can be [[Media:readcount.0.01.tar.gz | downloaded]] here. To compile, cd readcount.0.01/ and type make. The executable is under bin/ directory.
== Contact ==
== Contact ==
