pyReadCounters
pyReadCounters is part of the pyCRAC package. Produces a gene hittable file, two GTF output files showing to which genomic features the reads overlap. Finally the tool produces a read statistics file that provides information about the complexity of your dataset.
Output file examples
A hittable file:
# generated by pyReadCounters version 1.1.0, Mon Apr 16 20:34:22 2012 # /usr/local/bin/pyReadCounters.py -f RNAseq_data.novo -c 1 --unique # total number of reads 12534556 # total number of paired reads 10947376 # total number of single reads 483095 # total number of mapped reads: 11430471 # total number of overlapping genomic features 7019550 # sense 5960669 # anti-sense 1058881 # feature sense_overlap anti-sense_overlap number of reads YEF3 49930 3629 24221 PMA1 32621 2650 21776 COX1 24559 1037 15174 TFP1 21539 1689 13506 HSC82 21177 1458 12729 ADH1 20245 1467 11351 AI5_ALPHA 20022 918 13101 AI4 19390 886 12638 AI3 17823 798 11473 AI2 17590 790 11297 RPL10 16822 1113 8797 ENO2 16336 1125 8913 TEF1 15578 1333 5450
An example of a GTF 'count_output' file:
# generated by Counters version 1.2.0, Tue Jan 8 22:47:29 2013 # pyReadCounters.py -f PAR_CLIP_unique.novo --mutations=TC -v # total number of reads: 2455251 # total number of paired reads: 0 # total number of single reads: 2455251 # total number of mapped reads: 2455251 # total number of overlapping genomic features: 5153943 # sense: 2640600 # anti-sense: 2513343 chrXIV reads exon 661572 661605 2 + . gene_id "INT_0_6716,YNR016C"; gene_name "INT_0_6716,ACC1"; # 661596S; chrXIV reads exon 661720 661738 1 + . gene_id "INT_0_6716,YNR016C"; gene_name "INT_0_6716,ACC1"; # 661726S; chrXIV reads exon 661839 661878 4 + . gene_id "INT_0_6716,YNR016C"; gene_name "INT_0_6716,ACC1"; # 661875S;
This output file also reports whether a read contains a mutation.
For example:
# 661596S
Indicates that the read had a nucleotide substitution ("S") at genomic coordinate 661596. The chromosome name can be found in the first column.
Parameter list
File input options:
-f FILE, --input_file=FILE
provide the path to your novo, SAM/BAM or gtf data
file. Default is standard input. Make sure to specify
the file type of the file you want to have analyzed
using the --file_type option!
-o OUTPUT_FILE, --output_file=OUTPUT_FILE
Use this flag to override the standard file names. Do
NOT add an extension.
--file_type=FILE_TYPE
use this option to specify the file type (i.e.
'novo','sam' or 'gtf'). This will tell the program
which parsers to use for processing the files. Default
= 'novo'
--gtf=annotation_file.gtf
type the path to the gtf annotation file that you want
to use
Common pyCRAC options:
--ignorestrand
To ignore strand information and all reads overlapping
with genomic features will be considered sense reads.
Useful for analysing ChIP or RIP data
--overlap=1
sets the number of nucleotides a read has to overlap
with a gene before it is considered a hit. Default =
1 nucleotide
-r 100, --range=100
allows you to add regions flanking the genomic
feature. If you set '-r 50' or '--range=50', then the
program will add 50 nucleotides to each feature on
each side regardless of whether the GTF file has genes
with annotated UTRs
Options for SAM/BAM and Novo files:
--mutations=delsonly
Use this option to only track mutations that are of
interest. For CRAC data this is usually deletions
(--mutations=delsonly). For PAR-CLIP data this is
usually T-C mutations (--mutations=TC). Other options
are\: do not report any mutations: --mutations=nomuts.
Only report specific base mutations, for example only
in T's, C's and G's :--mutations=[TCG]. The brackets
are essential. Other nucleotide combinations are also
possible
--align_quality=100, --mapping_quality=100
with these options you can set the alignment quality
(Novoalign) or mapping quality (SAM) threshold. Reads
with qualities lower than the threshold will be
ignored. Default = 0
--align_score=100
with this option you can set the alignment score
threshold. Reads with alignment scores lower than the
threshold will be ignored. Default = 0
--unique
with this option reads with multiple alignment
locations will be removed. Default = Off
--blocks
with this option reads with the same start and end
coordinates on a chromosome will be counted as one
cDNA. Default = Off
-m 100000, --max=100000
maximum number of mapped reads that will be analyzed.
Default = All
-d 1000, --distance=1000
this option allows you to set the maximum number of
base-pairs allowed between two non-overlapping paired
reads. Default = 1000
--discarded=FILE
prints the lines from the alignments file that were
discarded by the parsers. This file contains reads
that were unmapped (NM), of poor quality (i.e. QC) or
paired reads that were mapped to different chromosomal
locations or were too far apart on the same
chromosome. Useful for debugging purposes
-l 100, --length=1000
to set read length threshold. Default = 1000