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IsoEM 1.1.5: Inferring isoform and gene expression levels from RNA-Seq data
Download IsoEM 1.1.5 from github: https://github.com/mandricigor/isoem-bootstrap.
Installation:
------------
-
Create a isoem directory and download the compressed ISOEM-1.1.5
from the github repository: https://github.com/mandricigor/isoem-bootstrap -
Edit isoEMDir location in files under bin according to
your local installation - Uncompress IsoEM-1.1.5.zip into the directory.
-
Optional] On windows you might want to add the IsoEM
installation directory to the path, such that you can invoke
isoem from any location. On unix you can obtain a similar -
If you want to rebuild isoem, run the unix script "build"
provided in the compressed file.
Testing your installation:
--------------------------
To test the installation you can download the sample dataset from
http://dna.engr.uconn.edu/software/IsoEM/sample.zip
and unzip it in the installation directory of IsoEm. First see
http://dna.engr.uconn.edu/software/IsoEM/README-SAMPLE.TXT
file for more details on what the archive provides.
http://dna.engr.uconn.edu/software/IsoEM/sample.zip
and unzip it in the installation directory of IsoEm. First see
http://dna.engr.uconn.edu/software/IsoEM/README-SAMPLE.TXT
file for more details on what the archive provides.
Running IsoEM:
--------------
--------------
IsoEM takes as input a set of known isoforms in GTF format, and a
file with aligned reads in SAM format. The aligned reads MUST be
sorted by read name. If not sure, run this command to sort the
file:
file with aligned reads in SAM format. The aligned reads MUST be
sorted by read name. If not sure, run this command to sort the
file:
sort -k 1,1 aligned_reads.sam > aligned_reads_sorted.sam
The output consists of two files: one for isoform frequencies and one
for gene frequencies. Each line in these files is a pair of isoform/gene
name and isoform/gene FPKM (Fragments Per Kilobase per Million reads)
representing the frequencies inferred from the data. The output file
names are obtained from the sam file name by replacing the .sam extension
with .iso_estimates and .gene_estimates respectively.They can also be set
using the -o option.
for gene frequencies. Each line in these files is a pair of isoform/gene
name and isoform/gene FPKM (Fragments Per Kilobase per Million reads)
representing the frequencies inferred from the data. The output file
names are obtained from the sam file name by replacing the .sam extension
with .iso_estimates and .gene_estimates respectively.They can also be set
using the -o option.
You can run IsoEm from the command line as follows:
isoem [global options]* [library options]* <aligned_reads.sam>
Mandatory global options:
------------------------
-G, --GTF <GTF file> Known genes and isoforms in GTF format
------------------------
-G, --GTF <GTF file> Known genes and isoforms in GTF format
Mandatory library options: either -a or both -m and -d must be present:
-------------------------
-m, --fragment-mean <Double> Fragment length mean
-d, --fragment-std-dev <Double> Fragment length standard deviation
-a, --auto-fragment-distrib Automatically detect fragment length
distribution from uniquely mapping
paired reads (DOES NOT WORK FOR
SINGLE READS)
-------------------------
-m, --fragment-mean <Double> Fragment length mean
-d, --fragment-std-dev <Double> Fragment length standard deviation
-a, --auto-fragment-distrib Automatically detect fragment length
distribution from uniquely mapping
paired reads (DOES NOT WORK FOR
SINGLE READS)
Optional global options:
-----------------------
-c, --gene-clusters <Cluster file> Override isoform to gene mapping
defined in the GTF file with a
mapping taken from the given file.
The format of each line in the file
is "isoform gene"
-g <genome fasta file> Genome reference sequence (needed by
some library options)
-b Perform hexamer bias correction
-h, --help Show help
-r <Repeats GTF> Drop alignments falling within
annotated repeats
-----------------------
-c, --gene-clusters <Cluster file> Override isoform to gene mapping
defined in the GTF file with a
mapping taken from the given file.
The format of each line in the file
is "isoform gene"
-g <genome fasta file> Genome reference sequence (needed by
some library options)
-b Perform hexamer bias correction
-h, --help Show help
-r <Repeats GTF> Drop alignments falling within
annotated repeats
Optional library options:
------------------------
-s, --directed Library obtained by directed RNA-Seq
(the strand of each read is
deterministically chosen: for single
reads, the read always comes from
the coding strand; for paired reads,
the first read always comes from the
coding strand, the second from the
opposite strand)
--mate-pairs Paired reads come from the same strand
(as opposed to the default behavior
where the two reads in a pair are
assumed to come from opposite
strands)
--max-mismatches <Integer> Maximum number of mismatched allowed
for a read. This requires the genome
sequence to be specified (see -g).
Default: no limit
-q, --quality-scores Weigh the reads based on their quality
scores. This requires the genome
sequence to be specified (see -g).
--repeat-threshold <nbases> Drop all reads that have more than
this many bases inside annotated
repeats. Default: 20
--polyA <nbases> Reads have been generated from mRNAs
with polyA tails of approximately
the given number of bases
-o <file prefix> Output files prefix. It can include path.
Default: same as sam file name
------------------------
-s, --directed Library obtained by directed RNA-Seq
(the strand of each read is
deterministically chosen: for single
reads, the read always comes from
the coding strand; for paired reads,
the first read always comes from the
coding strand, the second from the
opposite strand)
--mate-pairs Paired reads come from the same strand
(as opposed to the default behavior
where the two reads in a pair are
assumed to come from opposite
strands)
--max-mismatches <Integer> Maximum number of mismatched allowed
for a read. This requires the genome
sequence to be specified (see -g).
Default: no limit
-q, --quality-scores Weigh the reads based on their quality
scores. This requires the genome
sequence to be specified (see -g).
--repeat-threshold <nbases> Drop all reads that have more than
this many bases inside annotated
repeats. Default: 20
--polyA <nbases> Reads have been generated from mRNAs
with polyA tails of approximately
the given number of bases
-o <file prefix> Output files prefix. It can include path.
Default: same as sam file name
Read Alignment:
---------------
To align the reads you can either use spliced alignment directly
on the genome (for example using tophat), or you can align on the
library of known isoforms. We recommend the second option. If you
want to do this, we provide a full step by step guide at:
http://dna.engr.uconn.edu/software/IsoEM/README-SAMPLE.TXT
on the genome (for example using tophat), or you can align on the
library of known isoforms. We recommend the second option. If you
want to do this, we provide a full step by step guide at:
http://dna.engr.uconn.edu/software/IsoEM/README-SAMPLE.TXT
Visualizing read coverage:
---------------------------------------
---------------------------------------
If you want to visualize the isoforms and their coverage by reads, you can use the isoviz
command. It produces a bedGraph file and a GTF with fpkm values file which can be uploaded
to the UCSC browser. The name of the output files are automaticaly generated from the input
file name and ends in _iso_read_coverage.bed and _isoforms_w_fpkm.gtf. They can also be set
using the -o option.
command. It produces a bedGraph file and a GTF with fpkm values file which can be uploaded
to the UCSC browser. The name of the output files are automaticaly generated from the input
file name and ends in _iso_read_coverage.bed and _isoforms_w_fpkm.gtf. They can also be set
using the -o option.
The options are almost the same as for isoem except that isoviz also needs the isoform frequency
file (e.g. obtained by running isoem). The frequency file is specified using the -f option. The
full command line synopsis is given below:
file (e.g. obtained by running isoem). The frequency file is specified using the -f option. The
full command line synopsis is given below:
isoviz [global options]* [library options]* <aligned_reads.sam>
Mandatory global options:
------------------------
-G, --GTF <GTF file> Known genes and isoforms in GTF format
-f <frequency file> Isoform FPKMs computed by IsoEM
------------------------
-G, --GTF <GTF file> Known genes and isoforms in GTF format
-f <frequency file> Isoform FPKMs computed by IsoEM
Mandatory library options:
-------------------------
-m, --fragment-mean <Double> Fragment length mean
-d, --fragment-std-dev <Double> Fragment length standard deviation
-------------------------
-m, --fragment-mean <Double> Fragment length mean
-d, --fragment-std-dev <Double> Fragment length standard deviation
Optional global options:
-----------------------
-c, --gene-clusters <Cluster file> Override isoform to gene mapping
defined in the GTF file with a
mapping taken from the given file.
The format of each line in the file
is "isoform gene"
-g <genome fasta file> Genome reference sequence (needed by
some library options)
-b Perform hexamer bias correction
-h, --help Show help
-----------------------
-c, --gene-clusters <Cluster file> Override isoform to gene mapping
defined in the GTF file with a
mapping taken from the given file.
The format of each line in the file
is "isoform gene"
-g <genome fasta file> Genome reference sequence (needed by
some library options)
-b Perform hexamer bias correction
-h, --help Show help
Optional library options:
------------------------
-s, --directed Dataset obtained by directed RNA-Seq
(the strand of each read is
deterministically chosen: for single
reads, the read always comes from
the coding strand; for paired reads,
the first read always comes from the
coding strand, the second from the
opposite strand)
--mate-pairs Paired reads come from the same strand
(as opposed to the default behavior
where the two reads in a pair are
assumed to come from opposite
strands)
--max-mismatches <Integer> Maximum number of mismatched allowed
for a read. This requires the genome
sequence to be specified (see -g).
Default: no limit.
-q, --quality-scores Weigh the reads based on their quality
scores. This requires the genome
sequence to be specified (see -g).
-o <file prefix> Output files prefix. It can include path.
Default: same as sam file name
-O <dir prefix> Output directory prefix
-B <nr bootstraps> Number of bootstraps to perform
Default: 200
-C <confidence intervals> Confidence Intervals (CI).
Default 95 % (200 bootstrap iterations).
------------------------
-s, --directed Dataset obtained by directed RNA-Seq
(the strand of each read is
deterministically chosen: for single
reads, the read always comes from
the coding strand; for paired reads,
the first read always comes from the
coding strand, the second from the
opposite strand)
--mate-pairs Paired reads come from the same strand
(as opposed to the default behavior
where the two reads in a pair are
assumed to come from opposite
strands)
--max-mismatches <Integer> Maximum number of mismatched allowed
for a read. This requires the genome
sequence to be specified (see -g).
Default: no limit.
-q, --quality-scores Weigh the reads based on their quality
scores. This requires the genome
sequence to be specified (see -g).
-o <file prefix> Output files prefix. It can include path.
Default: same as sam file name
-O <dir prefix> Output directory prefix
-B <nr bootstraps> Number of bootstraps to perform
Default: 200
-C <confidence intervals> Confidence Intervals (CI).
Default 95 % (200 bootstrap iterations).
--endseq Reads have been generated using an end-sequencing protocol
Source Code:
------------
The source code can be found in the src directory under the
installation path.
installation path.
Revision history
----------------
-
Version 1.1.5 (1/20/16) - added TPM and ECPM estimates for genes and isoforms - added option to compute confidence intervals (bootstrapping) - added option for reading alignments from standard input -
Version 1.1.4 (12/31/15) - added `endseq` option
-
Version 1.1.3 (10/11/15) - bug fix in handling CIGAR with indels in convert-iso-to-genome-coords - bug fix related to hisat/hisat2 alignments
-
Version 1.1.1 (11/5/12) - bug fix related to clipped read alignments (CIGAR with S field)
-
Version 1.1.0 (4/24/12) - added support for alignments with insertions and deletions
-
Version 1.0.6 (8/12/11) - extract-isoform-sequences-from-genome (see http://dna.engr.uconn.edu/software/IsoEM/README-SAMPLE.TXT) generates transcripts in a randomized order - isoviz generates a gtf with fpkm values - added output file name option
-
Version 1.0.5 (5/08/11) - bugfix related to paired read data
-
Version 1.0.4 (2/22/11) - added polyATail option - further memory and speed improvements -
Version 1.0.3 (8/30/10) - correct for annotated repeats
-
Version 1.0.2 (8/05/10) - improved memory requirements for storing genome sequence - added hexamer bias correction option - added isoviz visualization tool -
Version 1.0.1 (6/25/10) - added support for mate pairs - added support for max number of mismatches - performance improvements -
Version 1.0.0 (6/16/10) - first public release
Contact
-------
For questions or suggestions regarding IsoEM you can contact:
-
Igor Mandric (imandric1@student.gsu.edu)
-
Sahar Al Seesi (sahar@engr.uconn.edu)
-
Ion Mandoiu (ion@engr.uconn.edu)
-
Marius Nicolae (man09004@engr.uconn.edu)
-
Serghei Mangul (serghei@cs.gsu.edu)
-
Alex Zelikovsky (alexz@cs.gsu.edu)