Settings and Parameters
Study Setup
Project Name Enter a name for the study. Type of Study Choose between Comparison of different conditions and Comparison of different strains/species. For a cross-strain analysis an alignment file has to be provided (see below). In addition, in each genome tab an individual genomic sequence and genome annotation has to be set. When comparing different conditions no alignment file is needed and the genomic sequence and genome annotation of the organism has to be set in the first genome tab only.
Number of Genomes Set the number of different strains/conditions in the study. Press the ‘Set’ button to generate a settings tab for each strain/condition and each replicate of the study.
Number of Replicates Set the number of different replicates for each strain/condition. Press the ‘Set’ button to generate a settings tab for each strain/condition and each replicate of the study.
Output Data Path Select the folder in which all result files will be placed.
Alignment File Select the xmfa alignment file containing the aligned genomes. If the study compares different conditions, this field is inactive.
TSS prediction parameters
In the following the parameters affecting the TSS prediction procedure are described. Instead of changing the parameters manually it is also possible to select predefined parameters sets using the parameter presets drop-down menu.
step height This value relates to the minimal number of read starts at a certain genomic position to be considered as a TSS candidate. To account for different sequencing depths this is a relative value based on the 90th percentile of the expression height distribution. A lower value results in a higher sensitivity.
step height reduction When comparing different strains/conditions and the step height threshold is reached in at least one strain/condition, the threshold is reduced for the other strains/conditions by the value set here. A higher value results in a higher sensitivity. Note that this value must be smaller than the step height threshold.
step factor This is the minimal factor by which the TSS height has to exceed the local expression background. This feature makes sure that a TSS candidate has to show a higher expression in regions of locally high expression than would be necessary in regions where no expression background is detected. A lower value results in a higher sensitivity. Set this value to 1 to disable the consideration of the local expression level.
step factor reduction When comparing different strains/conditions and the step factor threshold is reached in at least one strain/condition, the threshold is reduced for the other strains/conditions by the value set here. A higher value results in a higher sensitivity. Note that this value must be smaller than the step factor threshold.
enrichment factor The minimal enrichment factor for a TSS candidate. The threshold has to be exceeded in at least one strain/condition. If the threshold is not exceeded in another condition the TSS candidate is still marked as detected but not as enriched in this strain/condition. A lower value results in a higher sensitivity. Set this value to 0 to disable the consideration of the enrichment factor.
processing site factor The maximal factor by which the untreated library may be higher than the treated library and above which the TSS candidate is considered as a processing site and not annotated as detected. A higher value results in a higher sensitivity.
step length Minimal length of the TSS related expression region (in base pairs). This value depends on the length of the reads that are stacking at the TSS position. In most cases this feature can be disabled by setting it to ‘0’. However, it can be useful if RNA-seq reads have been trimmed extensively before mapping.
base height This value relates to the minimal number of reads in the non-enriched library that start at the TSS position. This feature is disabled by default.
Normalization Settings
normalization percentile By default a percentile normalization is performed on the RNA-seq data. This value defines the percentile that is used as a normalization factor. Set this value to ‘0’ to disable normalization.
enrichment normalization percentile By default a percentile normalization is performed on the enrichment values. This value defines the percentile that is used as a normalization factor. Set this value to ‘0’ to disable normalization.
Output options
write RNA-seq graphs If this option is enabled, the normalized RNA-seq graphs are written into the output folder. Disable this option, if the normalized graphs are not needed or if they have been written before. Note that writing the graphs will increase the runtime.
TSS Clustering Settings
cluster method TSS candidates in close vicinity are clustered and only one of the candidates is kept. HIGHEST keeps the candidate with the highest expression. FIRST keeps the candidate that is located most upstream.
TSS clustering distance This value determines the maximal distance (in base pairs) between TSS candidates to be clustered together. Set this value to ‘0’ to disable clustering.
Comparative Settings
allowed cross-genome/condition shift This is the maximal positional difference (bp) for TSS candidates from different strains/conditions to be assigned to each other.
allowed cross-replicate shift This is the maximal positional difference (bp) for TSS candidates from different replicates to be assigned to each other.
matching replicates This is the minimal number of replicates in which a TSS candidate has to be detected. A lower value results in a higher sensitivity.
Classification Settings
UTR length The maximal upstream distance (in base pairs) of a TSS candidate from the start codon of a gene that is allowed to be assigned as a primary or secondary TSS for that gene.
antisense UTR length The maximal upstream or downstream distance (in base pairs) of a TSS candidate from the start or end of a gene to which the TSS candidate is in antisense orientation that is allowed to be assigned as an antisense TSS for that gene. If the TSS is located inside the coding region on the antisense strand it is also annotated as an antisense TSS.
Genome specific Settings
Name Brief unique name for this strain/condition, which can be freely chosen. As this name is also used in some filenames any special characters (including spaces) should be avoided.
Alignment ID The identifier of this genome in the alignment file. If Mauve was used to align the genomes, the identifiers are just numbers assigned to the genomes in the order as they have been chosen as input in Mauve.
The first lines of the alignment file should also contain this information:
#FormatVersion Mauve1
#Sequence1File genomeA.fa
#Sequence1Format FastA
#Sequence2File genomeB.fa
#Sequence2Format FastA
In this example `genomeA’ has ID 1 and `genomeB’ has ID 2. When loading an alignment file (xmfa) TSSpredator tries to set the alignment IDs automatically.
genome FASTA FASTA file containing the genomic sequence of this genome.
genome annotation GFF/GTF file containing genomic annotations for this genome (as can be downloaded from NCBI).
output ID The specified output ID defines which gene tag in the attributes column (in the provided gff/gtf annotation file) should be used for TSS classification. Examples are locus tag or gene id.
Graph Files
enriched plus Select the file containing the RNA-seq expression graph for the plus strand (forward) from the 5’ enrichment library.
enriched minus Select the file containing the RNA-seq expression graph for the minus strand (reverse) from the 5’ enrichment library.
normal plus Select the file containing the RNA-seq expression graph for the plus strand (forward) from the library without 5’ enrichment.
normal minus Select the file containing the RNA-seq expression graph for the minus strand (reverse) from the library without 5’ enrichment.