input_procheck - OligoMiner

Input - Probes Submitting

The function is the same of "Input - Single" function. However it is possible to input a FASTQ-formatted probes set previously obtained to start the filtering of probes. The OligoMiner workflow is here graphically divided into 7 different step each one with its own parameters to be set.

Please, pass mouse over the boxes or click the "?" or the "Overview" buttons to get help.

STEP 1 - File upload

Probes Upload Overview

This function allows the filtering of existing probes coming from other software, papers, or manually designed (up to 3000). Basically, it applies the entire OligoMiner filtering processes avoiding the probes mining. The required input is a FASTA-formatted file containing the probes sequences. Applying this workflow to the same probes set, using different reference genomes, will filter it for cross-species specificity. Please remind that any information contained into the header of your FASTA-formatted sequences will be lost, replaced by the "FASTA file name" of your choice.

Probes Upload Help

Upload Probes-Set File

It is possible to select a fasta file from your computer (containing all the probes you want to check in fasta format) and upload it into the application. The uploaded file will appear in the "FASTA probes sequence" text-box below, where you can check for formatting errors before lunching the analysis. Also in this case you must submit a "FASTA probes file name" to be used (see "FASTA probes file name" help box below for more details).

File Upload Help

Fiile Upload

Upload Probes-Set Name

A custom name referring to your FASTA-formatted file is required. This name identifies the new filtered probes set derived from the submitted FASTA-formatted file and it will replace its header. The following characters are not allowed: ":", ".", "/", ".", "<", ">" and the "underscore".

File Upload Help

FASTA probes file name:

Upload Porbes-Set Fasta Sequence

A FASTA-formatted file is required to let OligoMiner workflow filters the probes. It is possible to write or cut & paste a list of short FASTA-formatted sequences representing a probes set. Please remember that any information contained into the header of your FASTA-formatted probes will be lost replaced by the "FASTA probes file name" of your choice.

File Upload Help

FASTA probes sequence:

STEP 2 - Reference Genomes

Reference Genomes Overview

The list of candidate probes coming from the previous step are aligned to a specified reference genome index using the read aligner bowtie2. Alignment programs can provide a wealth of information about the relatedness of a given input sequence to other sequences present in a genome.

Reference Genome Help

Reference Genome

The reference genome index to be used for the alignement of the probes using NGS aligner "bowtie2". Take into account that using FASTA-formatted sequences coming from species too far from the selected reference genome index could not produce any suitable results.

Reference Genome Help

Genome:
Homo sapiens GRCh38 (unmasked primary-assembly)
Homo sapiens (GRCh37 Unmasked)
Mus musculus GRCm38 (unmasked primary-assembly)
Rattus norvegicus (Rnor_6.0 Unmasked)
Danio rerio (GRCz11 Unmasked)
Caenorhabditis elegans(WBcel235 Unmasked)
Drosophila melanogaster (BDGP6 Unmasked)
Arabidopsis thaliana (TAIR10 Unmasked)
Escherichia coli (ASM584v2 - Unmasked)
Saccharomyces cerevisiae (R64-1-1 - Unmasked)

STEP 3 - Linear Discriminant Analysis (LDA)

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Linear Discriminant Analysis overview

The alignment file previously produced (SAM file) is processed by a selected temperature-specific Linear Discriminant Analyisis model (LDA) such that candidate probes predicted to have more than one thermodynamically relevant target site are filtered. The LDA model identifies potentially problematic “probe”–“target site” interactions effectively as well as the much slower thermodynamic simulations.

Linear Discriminant Analysis Help

Linear Discriminant Analysis Model

Specify the temperature-specific linear discrimination model to use in LDA. Default value is 42° C. Select the temperature model closer to your experimental FISH condition (hybridization temperature).

Linear Discriminant Analysis Help

LDA model:
32°C
37°C
42°C
47°C
52°C
57°C

Linear Discriminant Analysis Threshold

The probability threshold for classifying a candidate sequence as likely to have off-target binding using the LDA model. Default value is 0.5 meaning that the LDA will filter those probes with a probability of duplexing non-target sites higher than 50%. Selecting a smaller values will improve precision (fewer false positives), but at the expense of recall (more false negatives). Selecting larger values will improve recall at the expense of precision.

Linear Discriminant Analysis Help

LDA threshold (0-1):

Experimental Salt Concentration

The mM Na+ concentration to be used for Tm calculation, the default value is 390 mM. This value will affect the Tm of probes displayed in the final output of the analysis. It is suggested to use the same value from the probe design step.

Linear Discriminant Analysis Help

[Na+] (mM):

Experimental Formamide Percent

The percent formamide to be used for probe Tm calculation, the default is 50%. This value will affect the Tm of probes displayed in the final output of the analyisis. It is suggested to use the same value from the probe design step.

Linear Discriminant Analysis Help

[formamide] (%):

STEP 4 - Substrings off-tagets searching

Substring Off-target Searching Overview

To ensure probe specificity, candidate sequences are screened for the presence of high-abundance substrings (kmers) that may be missed by alignment programs because of their short lengths and could lead to off-target binding. This is done using an external utility called Jellyfish, which builds specific kmer indices starting from reference genomes and use them to check the probability that a kmer within the probe will bind an off-target sequence. It is possible to choose 9 different kmer lengths for each reference genome and decide under which condition a candidate probe will be discarded.

Substrings Off Target searching Help

Substrings Length

The length, in base pairs, of the kmers to be searched inside the probes sequence. Choose the correct kmer length basing also on the probes mean length. Default value is 18 bp.

Substrings Off Target searching Help

Substrings length (bp):
16 bp
18 bp
20 bp
22 bp
24 bp
26 bp
28 bp
30 bp
32 bp

Substrings Maximum Number Of Off Target

Filter probes with off-target kmers occurring at least the specified number of time. In default mode if a probes has 5 or more kmer that binds an off target sequence then the entire probe will be discarded.

Substrings Off Target searching Help

Substrings MAX off-targets:

STEP 5 - Secondary structure prediction

Secondary Structure Prediction Overview

This step identifies and filters probe sequences predicted to adopt secondary structures in a given set of experimental conditions. This is done using an external utility called NUPACK that discards probes predicted to have more thermodynamically stable secondary structures than the desired probe-target duplex. It is possible to set the experimental condition in which the probes would form unwanted secondary structures/homodimers together with the stringency of the filtering.

Secondary Structure Prediction Help

Experimental Temperature

This is the temperature at which the probes will be hybridized. The default value is 47 °C.

Secondary Structure Prediction Help

Th (°C):

Secondary Structure Probability Threshold

The probability threshold for a probe having a linear structure to use for discarding. Default value is 0.1. With default setting all the probes that have a 10% probability or more to have a linear structure will be saved.

Secondary Structure Prediction Help

Linear probability (0-1):

Experimental Salt Concentration

The mM Na+ concentration at which probes will be hybridized, default value is 390 mM. Take into accounts that NUPACK's allowable range of Na+ concentration is 50-1100 mM

Secondary Structure Prediction Help

[Na+] (mM):

Experimental Formamide Percent

The percent formamide at which probes will be hybridized, default value is 50%.

Secondary Structure Prediction Help

[formamide] (%):

STEP 6 - "Stripping" melting temperature calculation

Stripping Melting Temperature Calculation

This step calculates the melting temperature of probes as an additional value to put beside the main one. This allow users to compare the expected melting temperatures of probes during the hybridization step with those expected during the stripping phase (different salt and formamide concentration).

Stripping Melting Temperature Help

Experimental Salt Concentration

The mM Na+ concentration of the new experimental condition, default value is 390 mM.

Stripping Melting Temperature Help

[Na+] (mM):

Experimental Formamide Percent

The percent formamide of the new experimental condition, default value is 50%.

Stripping Melting Temperature Help

[formamide] (%):

STEP 7 - Output probes strand

Output Probes Strand

The final probes orientation will be identical to the selected genomic strand. Choosing plus(+) will give you probes with sequence identical to the plus(+) strand of your submitted FASTA-formatted sequence. On the contrary, selecting minus(-), will give you probes with sequence identical to the minus(-) strand of your submitted FASTA-formatted sequence.

Output Probes Strand Help

Strand:
plus(+)
minus(-)