Compare assembled transcripts across multiple experiments
statsFile = cuffcompare(gtfFiles)gtfFiles and returns summary
statistics in the output file statsFile
[1].
cuffcompare requires the Cufflinks Support Package for the Bioinformatics Toolbox™. If the support package is not installed, then the function provides a download
link. For details, see Bioinformatics Toolbox Software Support Packages.
Note
cuffcompare is supported on the Mac and UNIX® platforms only.
statsFile = cuffcompare(gtfFiles,compareOptions)compareOptions.
statsFile = cuffcompare(gtfFiles,Name,Value)statsFile = cuffcompare(gtfFile,'OutputPrefix',"cuffComp") appends the
prefix "cuffComp" to the output file names.
[
returns the names of the output files using any of the input argument combinations in the
previous syntaxes. By default, the function saves all files to the current directory.statsFile,combinedGTF,lociFile,trackingFile] = cuffcompare(___)
Create a CufflinksOptions object to define cufflinks options, such
as the number of parallel threads and the output directory to store the results.
cflOpt = CufflinksOptions;
cflOpt.NumThreads = 8;
cflOpt.OutputDirectory = "./cufflinksOut";The SAM files provided for this example contain aligned reads for Mycoplasma
pneumoniae from two samples with three replicates each. The reads are
simulated 100bp-reads for two genes (gyrA and
gyrB) located next to each other on the genome. All the reads are
sorted by reference position, as required by cufflinks.
sams = ["Myco_1_1.sam","Myco_1_2.sam","Myco_1_3.sam",... "Myco_2_1.sam", "Myco_2_2.sam", "Myco_2_3.sam"];
Assemble the transcriptome from the aligned reads.
[gtfs,isofpkm,genes,skipped] = cufflinks(sams,cflOpt);
gtfs is a list of GTF files that contain assembled isoforms.
Compare the assembled isoforms using cuffcompare.
stats = cuffcompare(gtfs);
Merge the assembled transcripts using cuffmerge.
mergedGTF = cuffmerge(gtfs,'OutputDirectory','./cuffMergeOutput');
mergedGTF reports only one transcript. This is because the two
genes of interest are located next to each other, and cuffmerge
cannot distinguish two distinct genes. To guide cuffmerge, use a
reference GTF (gyrAB.gtf) containing information about these two
genes. If the file is not located in the same directory that you run
cuffmerge from, you must also specify the file path.
gyrAB = which('gyrAB.gtf'); mergedGTF2 = cuffmerge(gtfs,'OutputDirectory','./cuffMergeOutput2',... 'ReferenceGTF',gyrAB);
Calculate abundances (expression levels) from aligned reads for each sample.
abundances1 = cuffquant(mergedGTF2,["Myco_1_1.sam","Myco_1_2.sam","Myco_1_3.sam"],... 'OutputDirectory','./cuffquantOutput1'); abundances2 = cuffquant(mergedGTF2,["Myco_2_1.sam", "Myco_2_2.sam", "Myco_2_3.sam"],... 'OutputDirectory','./cuffquantOutput2');
Assess the significance of changes in expression for genes and transcripts between
conditions by performing the differential testing using cuffdiff.
The cuffdiff function operates in two distinct steps: the function
first estimates abundances from aligned reads, and then performs the statistical
analysis. In some cases (for example, distributing computing load across multiple
workers), performing the two steps separately is desirable. After performing the first
step with cuffquant, you can then use the binary CXB output file as
an input to cuffdiff to perform statistical analysis. Because
cuffdiff returns several files, specify the output directory is
recommended.
isoformDiff = cuffdiff(mergedGTF2,[abundances1,abundances2],... 'OutputDirectory','./cuffdiffOutput');
Display a table containing the differential expression test results for the two genes
gyrB and gyrA.
readtable(isoformDiff,'FileType','text')
ans =
2×14 table
test_id gene_id gene locus sample_1 sample_2 status value_1 value_2 log2_fold_change_ test_stat p_value q_value significant
________________ _____________ ______ _______________________ ________ ________ ______ __________ __________ _________________ _________ _______ _______ ___________
'TCONS_00000001' 'XLOC_000001' 'gyrB' 'NC_000912.1:2868-7340' 'q1' 'q2' 'OK' 1.0913e+05 4.2228e+05 1.9522 7.8886 5e-05 5e-05 'yes'
'TCONS_00000002' 'XLOC_000001' 'gyrA' 'NC_000912.1:2868-7340' 'q1' 'q2' 'OK' 3.5158e+05 1.1546e+05 -1.6064 -7.3811 5e-05 5e-05 'yes'
You can use cuffnorm to generate normalized expression tables for
further analyses. cuffnorm results are useful when you have many
samples and you want to cluster them or plot expression levels for genes that are
important in your study. Note that you cannot perform differential expression analysis
using cuffnorm.
Specify a cell array, where each element is a string vector containing file names for a single sample with replicates.
alignmentFiles = {["Myco_1_1.sam","Myco_1_2.sam","Myco_1_3.sam"],...
["Myco_2_1.sam", "Myco_2_2.sam", "Myco_2_3.sam"]}
isoformNorm = cuffnorm(mergedGTF2, alignmentFiles,...
'OutputDirectory', './cuffnormOutput');
Display a table containing the normalized expression levels for each transcript.
readtable(isoformNorm,'FileType','text')
ans =
2×7 table
tracking_id q1_0 q1_2 q1_1 q2_1 q2_0 q2_2
________________ __________ __________ __________ __________ __________ __________
'TCONS_00000001' 1.0913e+05 78628 1.2132e+05 4.3639e+05 4.2228e+05 4.2814e+05
'TCONS_00000002' 3.5158e+05 3.7458e+05 3.4238e+05 1.0483e+05 1.1546e+05 1.1105e+05
Column names starting with q have the format: conditionX_N, indicating that the column contains values for replicate N of conditionX.
gtfFiles — Names of GTF filesNames of GTF files, specified as a string vector or cell array of character vectors.
Each GTF file corresponds to a sample produced by cufflinks.
Example: ["Myco_1_1.transcripts.gtf","Myco_2_1.transcripts.gtf"]
Data Types: string | cell
compareOptions — cuffcompare optionsCuffCompareOptions object | character vector | stringcuffcompare options, specified as a
CuffCompareOptions object, character vector, or string. The character
vector or string must be in the original cuffcompare option syntax
(prefixed by one or two dashes), such as '-d 100 -e 80'
[1].
Specify optional
comma-separated pairs of Name,Value arguments. Name is
the argument name and Value is the corresponding value.
Name must appear inside quotes. You can specify several name and value
pair arguments in any order as
Name1,Value1,...,NameN,ValueN.
statsFile =
cuffcompare(gtfFile,'OutputPrefix',"cuffComp",'MaxGroupingRange',90)'ConsensusPrefix' — Prefix for consensus transcript names"TCONS" (default) | string | character vectorPrefix for consensus transcript names in the output
combined.gtf file, specified as a string or character vector. This option
must be a string or character vector with a non-zero length.
Example: 'ConsensusPrefix',"consensusTs"
Data Types: char | string
'DiscardIntronRedundant' — Flag to ignore intron-redundant transfragsfalse (default) | trueFlag to ignore intron-redundant transfrags if they have the same 5' end but different 3' ends, specified as true or false.
Example: 'DiscardIntronRedundant',true
Data Types: logical
'DiscardSingleExonAll' — Flag to discard single-exon transfrags and reference transcriptsfalse (default) | trueFlag to discard single-exon transfrags and reference transcripts, specified as true or false.
Example: 'DiscardSingleExonAll',true
Data Types: logical
'DiscardSingleExonReference' — Flag to discard single-exon reference transcriptsfalse (default) | trueFlag to discard single-exon reference transcripts, specified as true or false.
Example: 'DiscardSingleExonReference',true
Data Types: logical
'ExtraCommand' — Additional commands"" (default) | character vector | stringThe commands must be in the native syntax (prefixed by one or two dashes). Use this option to apply undocumented flags and flags without corresponding MATLAB properties.
Example: 'ExtraCommand',"--library-type
fr-secondstrand"
Data Types: char | string
'GTFManifest' — Name of text file containing list of GTF files to processName of the text file containing a list of GTF files to
process, specified as a string or character vector. The file must contain one GTF file path per
line. You can use this option as an alternative to passing an array of file names to
cuffcompare.
Example: 'GTFManifest',"gtfManifestFile.txt"
Data Types: char | string
'GenericGFF' — Flag to treat input GTF files as GFFfalse (default) | trueFlag to treat input GTF files as GFF files, specified as true or false. Use this option when the input GFF or GTF files are not produced by cufflinks.
Example: 'GenericGFF',true
Data Types: logical
'IncludeAll' — Flag to include all available optionsfalse (default) | trueThe original (native) syntax is prefixed by one or two dashes.
By default, the function converts only the specified options. If the value is
true, the software converts all available options, with default values
for unspecified options, to the original syntax.
Note
If you set IncludeAll to true, the software
translates all available properties, with default values for unspecified properties. The
only exception is that when the default value of a property is NaN,
Inf, [], '', or
"", then the software does not translate the corresponding
property.
Example: 'IncludeAll',true
Data Types: logical
'IncludeContained' — Flag to include transfrags contained by other transfragsfalse (default) | trueFlag to include transfrags contained by other transfrags in the
same locus in the output combined.gtf, specified as true
or false. By default, cuffcompare does not include these
contained transfrags. If the value is true, the contained transfrags include
a contained_in attribute indicating the first container transfrag
found.
Example:
'IncludeContained',true
Data Types: logical
'MaxAccuracyRange' — Number of bases from terminal exons to use when assessing exon accuracy100 (default) | positive integerNumber of bases from the free ends of terminal exons to use when assessing exon accuracy, specified as a positive integer.
Example:
'MaxAccuracyRange',80
Data Types: double
'MaxGroupingRange' — Number of bases to use for grouping transcript start sites100 (default) | positive integerNumber of bases to use for grouping transcript start sites, specified as a positive integer.
Example:
'MaxGroupingRange',90
Data Types: double
'OutputPrefix' — Prefix for cuffcompare output files"cuffcmp" (default) | string | character vectorPrefix for cuffcompare output files,
specified as a string or character vector. This option must be a string or character vector with
a non-zero length.
Example:
'OutputPrefix',"cuffcompareOut"
Data Types: char | string
'ReferenceGTF' — Name of GTF or GFF file containing reference transcriptsName of the GTF or GFF file containing reference transcripts to
compare to each sample, specified as a string or character vector. If you provide a file, the
function compares each sample to the references in the file and marks isoforms as
overlapping, matching, or novel.
The function stores these tags in the output files .refmap and
.tmap files.
Example:
'ReferenceGTF',"references.gtf"
Data Types: char | string
'SequenceDirectory' — Name of directory containing FASTA sequences to classify input transcripts as repeatsName of directory containing FASTA sequences to classify input transcripts as repeats, specified as a string or character vector. The directory must contain FASTA-format files with the underlying genomic sequences and contain one FASTA file per reference. Name each FASTA file after the chromosome with the extension .fa or .fasta.
Example: 'SequenceDirectory',"./SequenceDirectory/"
Data Types: char | string
'SnCorrection' — Flag to consider only reference transcripts that overlap with input transfragsfalse (default) | trueFlag to consider only reference transcripts that overlap with
any of the input transfrags, specified as true or false.
If the value is true:
The function ignores any reference transcripts that do not overlap with any of the input transfrags.
You must also specify the ReferenceGTF option.
Example:
'SnCorrection',true
Data Types: logical
'SpCorrection' — Flag to consider only input transcripts that overlap with reference transcriptsfalse (default) | trueFlag to consider only input transcripts that overlap with any
of the reference transcripts, specified as true or false.
If the value is true:
The function ignores any input transcripts that do not overlap with any of the reference transcripts and reports no novel loci.
You must also specify the ReferenceGTF option.
Example:
'SpCorrection',true
Data Types: logical
'SuppressMapFiles' — Flag to prevent creation of .tmap and .refmap filesfalse (default) | trueFlag to prevent the creation of .tmap and
.refmap files, specified as true or
false. Set the value to true to prevent the function
from generating the files.
Example:
'SuppressMapFiles',true
Data Types: logical
statsFile — Name of text file containing statistics"cuffcmp.stats"Name of the text file containing statistics related to the accuracy of the transcripts in each sample, returned as string. The function performs the tests for sensitivity (Sn) and specificity (Sp) at various levels, including the nucleotide, exon, and intron levels, and reports the results in this file.
The default file name is "cuffcmp.stats". If you specify
OutputPrefix, the function uses it instead of
"cuffcmp".
combinedGTF — Name of file containing union of all transfrags in each sample"cuffcmp.combined.gtf"Name of the file containing the union of all transfrags in each sample, returned as a string.
The default file name is "cuffcmp.combined.gtf". If you specify
OutputPrefix, the function uses it instead of
"cuffcmp".
lociFile — Name of file with all processed loci"cuffcmp.loci"Name of file with all processed loci across all transcripts, returned as a string.
The default file name is "cuffcmp.loci". If you specify
OutputPrefix, the function uses it instead of
"cuffcmp".
trackingFile — Name of file containing transcripts with identical coordinates"cuffcmp.tracking"Name of the file containing transcripts with identical coordinates, introns, and strands, returned as a string.
The default file name is "cuffcmp.tracking". If you specify
OutputPrefix, the function uses it instead of
"cuffcmp".
[1] Trapnell, Cole, Brian A Williams, Geo Pertea, Ali Mortazavi, Gordon Kwan, Marijke J van Baren, Steven L Salzberg, Barbara J Wold, and Lior Pachter. “Transcript Assembly and Quantification by RNA-Seq Reveals Unannotated Transcripts and Isoform Switching during Cell Differentiation.” Nature Biotechnology 28, no. 5 (May 2010): 511–15.