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1 .. image:: https://raw.githubusercontent.com/mhammell-laboratory/bamqc/master/doc/bamqc-icon.png
2 :width: 200 px
3 :alt: generated at codeology.braintreepayments.com/mhammell-laboratory/bamqc
4 :align: right
5 :target: http://codeology.braintreepayments.com/mhammell-laboratory/bamqc
6
7 =====
8 ezBAMQC
9 =====
10 *"ezBAMQC, a tool to check the quality of mapped next generation sequencing files."*
11
12 :Description:
13
14 ezBAMQC is a tool to check the quality of either one or many mapped next-generation-sequencing datasets. It conducts comprehensive evaluations of aligned sequencing data from multiple aspects including: clipping profile, mapping quality distribution, mapped read length distribution, genomic/transcriptomic mapping distribution, inner distance distribution (for paired-end reads), ribosomal RNA contamination, transcript 5’ and 3’ end bias, transcription dropout rate, sample correlations, sample reproducibility, sample variations. It outputs a set of tables and plots and one HTML page that contains a summary of the results. Many metrics are designed for RNA-seq data specifically, but ezBAMQC can be applied to any mapped sequencing dataset such as RNA-seq, CLIP-seq, GRO-seq, ChIP-seq, DNA-seq and so on. ::
15
16 :Links:
17
18 `Github Page <https://github.com/mhammell-laboratory/bamqc>`_
19
20 `Pypi Page <https://pypi.python.org/pypi/ezBAMQC>`_
21
22 `MHammell Lab <http://hammelllab.labsites.cshl.edu/software>`_
23
24 :Authors:
25 Ying Jin, David Molik, and Molly Hammell
26
27 :Version: 0.6.5
28
29 :Contact:
30 Ying Jin (yjin@cshl.edu)
31
32 Installation guide for ezBAMQC for from source installs
33 =====================================================
34
35 When installing ezBAMQC there are several options, but the main point is: since ezBAMQC uses C++ STD 11 you'll need a version of GCC that can support that, this useally means 4.8 or 4.9. beyond that, you'll need Python, R and Corrplot for interfacing with the C code.
36
37 :Intallation:
38 `Source Code <https://github.com/mhammell-laboratory/ezBAMQC/releases>`_
39
40 `Pypi <https://pypi.python.org/pypi?:action=display&name=ezBAMQC>`_
41
42 :Prerequisites:
43 * `python2.7 <https://www.python.org/download/releases/2.7/>`_
44 * `R <https://www.r-project.org/>`_
45 * `corrplot <https://cran.r-project.org/web/packages/corrplot/>`_
46 * `GCC 4.8.1 or greater <https://gcc.gnu.org/gcc-4.8/>`_ GCC 4.9.1 or greater is recomended for PyPi install
47
48 :Notes:
49 * While there are multiple methods of installing the prerequistes it may help to look at (if using a yum based linux distro):*
50 * `Devtoolset-3 <https://access.redhat.com/documentation/en-US/Red_Hat_Developer_Toolset/3/html/User_Guide/sect-Red_Hat_Developer_Toolset-Install.html>`_ for GCC compilers
51 * `IUS <https://ius.io/>`_ for Python2.7
52 * `Software Collections <https://www.softwarecollections.org/>`_ for collections of software (like devtoolset 3 or python)
53 * `rpmfinder <https://www.rpmfind.net/>`_ for searching rpms across mutliple systems
54
55 Setup
56 =====
57
58 1) Make sure that the GCC comiler is in your PATH:
59
60 ::
61
62 export PATH=/path/to/gcc:$PATH
63
64 2) Make sure that python2.7 is in your PYTHONPATH:
65
66 ::
67
68 export PYTHONPATH=/path/to/python2.7/site-packages:$PYTHONPATH
69
70 3) There are three methods of installation of ezBAMQC, from source, from setup.py, and from pypi, once prequistes are setup.
71
72 From Source
73 ~~~~~~~~~~~
74
75 1) Download source
76
77 2) Unpack tarball and go to the directory of the package:
78
79 ::
80
81 tar xvfz bamqc-0.6.6.tar.gz
82
83 cd bamqc-0.6.6
84
85 3) Run make:
86
87 ::
88
89 make
90
91 From Setup.py
92 ~~~~~~~~~~~~~
93
94 ::
95
96 python2.7 setup.py install
97
98 From Pypi
99 ~~~~~~~~~
100
101 ::
102
103 pip2.7 install BAMqc
104
105 Usage
106 =====
107
108 ::
109
110 ezBAMQC [-h] -i alignment_files [alignment_files ...] -r [refgene]
111 [-f [attrID]] [--rRNA [rRNA]] -o [dir] [--stranded [stranded]]
112 [-q [mapq]] [-l labels [labels ...]] [-t NUMTHREADS]
113
114 optional arguments:
115
116 ::
117
118 -h, --help show this help message and exit.
119 -i, --inputFile alignment files. Could be multiple SAM/BAM files separated by space. Required.
120 -r, --refgene gene annotation file in GTF format. Required
121 -f the read summation at which feature level in the GTF file. DEFAULT: gene_id.
122 --rRNA rRNA coordinates in BED format.
123 -o, --outputDir output directory. Required.
124 --stranded strandness of the library?
125 yes : sense stranded
126 reverse : reverse stranded
127 no : not stranded
128 DEFAULT: yes.
129 -q, --mapq Minimum mapping quality (phred scaled) for an alignment to be called uniquely mapped. DEFAULT:30
130 -l, --label Labels of input files. DEFAULT:smp1 smp2 ...
131 -t, --threads Number of threads to use. DEFAULT:1
132
133 Example:
134
135 ::
136
137 ezBAMQC -i test-data/exp_data/treat1.bam test-data/exp_data/treat2.bam test-data/exp_data/treat3.bam -r test-data/exp_data/hg9_refGene.gtf -q 30 --rRNA test-data/exp_data/hg19_rRNA.bed -o exp_output2
138
139 Please find the example output from folder test-data.
140
141 FAQ
142 ====
143 Q: Why use ezBAMQC?
144
145 A: ezBAMQC is efficient and easy to use. With one command line, it reports a comprehensive evaluation of the data with a set of plots and tables.The ability to assess multiple samples together with high efficiency make it especially useful in cases where there are a large number of samples from the same condition, genotype, or treatment. ezBAMQC was written in C++ and supports multithreading. A mouse RNA-seq sample with 120M alignments can be done in 8 minutes with 5 threads.
146
147 Q: Why the total number of reads reported by ezBAMQC does not match with samtools flagstat?
148
149 A: The difference is because of non-uniquely mapped reads or multiply aligned reads (multi-reads). Samtools flagstat counts each multiple aligment as a different reads, but ezBAMQC counts reads accoriding to the read ID, i.e., each individual read will be counted once no matter that it is a uniquely mapped read or multi-read.
150
151 Q: What is "Low Quality Reads" ?
152
153 A: Reads marked as qc fail accoriding to SAM format or reads with mapping quality lower than the value set by the option -q will be considered as "Low Quality Reads".
154
155 Q: How the setting of option -q alter the results?
156
157 A: Reads with low quality, i.e., did not pass -q cutoff, are only counted in Total Reads, Mapped Reads, and Mappability by mapping quality plot. The rest of the report does not include low quality reads.
158
159 Q: Do multi-reads (non-uniquely mapped reads) have been considered in Read distribution and gene quantification?
160
161 A: No. Only uniquely mapped reads were counted.
162
163
164 Acknowledgements
165 ================
166
167 #) Samtools contributors
168 #) Users' valuable feedback
169
170 Copying & Distribution
171 ======================
172
173 ezBAMQC is free software: you can redistribute it and/or modify
174 it under the terms of the GNU General Public License as published by
175 the Free Software Foundation, either version 3 of the License, or
176 (at your option) any later version.
177
178 This program is distributed in the hope that it will be useful,
179 but *WITHOUT ANY WARRANTY*; without even the implied warranty of
180 *MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE*. See the
181 GNU General Public License for more details.
182
183 You should have received a copy of the GNU General Public License
184 along with ezBAMQC. If not, see `this website <http://www.gnu.org/licenses/>`_