What is Mitsucal ?

Mitsucal is a computer program designed to analyze short reads obtained from next-generation gequencing to identify mutations in various plant species.

Users are required to upload their short reads and to set several parameters for analysis.

Mitsucal was initially developed to analyze Arabidopsis thaliana mutants, and is now adapted for use with additional plant species.

Login

An e-mail address is required to log in to Mitsucal.

Please enter your e-mail address and "anonymous" as the password in the login form on the home page.

To analyze your own original data, please register your e-mail address according to the instruction in the next section.

When you are logged in, your e-mail address will be displayed at the top-right courner of the page. To log out, please click here.

Registration

Registration is only required if you want to analyze your own original data with Mitsucal.

Steps for registration

1. Open the registration page by clicking the link on the home page or this link.
2. Enter your e-mail address. Enter the letters and numbers for the CAPTCHA in the text box on the right. Click the "Registration" button.
3. After your information is verified, a confirmation URL will be sent to the provided e-mail address.
4. When registration is confirmed, a new password will be automatically generated. Please make note of your new password.

Forgotten password

If you forget your password, please open the page for Temporary password to obtain a temporary password. An e-mail will be sent to you with instructions on how to reset your password.

Start analysis

The following two steps are required to perform analysis with Mitsucal.

1. Prepare computer resources for a new mutant
2. Upload data (FASTQ files) and execute analysis

Steps to add mutant

1. Log in to Mitsucal.
2. Fill out the form for a new mutant on the home page.
   • Specify the mutant name using the following characters: a-z, A-Z, 0-9, +, -, _, and #. Do not include any spaces.
   • Select genetic background and backcrossed strains similar to your sample.
   • Select marker selection method according to your experimental design.
   • Input the Mitsucal 'Mutant ID' to be used as a control. If the ID is not known, please leave the box blank.
   All values are changeable before the program executed.
3. After filling out the form, click the "Update" button and wait for a response from the site manager.

When your click the "Update" button, an e-mail will be sent to the site manager. The site manager will prepare the computer resources for storing your files. This will typically be completed in 24 hours, but may take a few days.

Steps for data upload

1. Log in to Mitsucal.
2. Click on the desired mutant in the list at the top of the page.
3. Click the "Setup" tab.
4. Drag your FASTQ files to the upload area. After uploading all data, check the analysis settings.
5. Click the "Analyze" button in the "Anaylsis" tab. You will receive an e-mail when analysis is completed.

When you click the "Analyze" button, an e-mail will be sent to the site manager. The site manager assigns computer resources to analyze your files and starts the program. This will typically be completed in 24 hours, but may take a few days.

Results

To browse results, log in to Mitsucal.

After login, analyzed mutants appear at the top of the page. Please click on a Mutant ID to browse the results.

Sample results are shown below:

Tabs at the top of the page are links to results. We recommend viewing the results using the tabs in the following order: Chromosome Mapping, Mutation List, and Gene Viewer .

Chromosome Mapping

Chromosome mapping is required to reduce the number of candidates for mutations. Images in this tab are helpful when the sequenced genomic libraries are prepared from segregants.

Mutation List

After selecting loci by chromosome mapping, filter mismatches using the parameters below.

In a typical analysis, Mitsucal identifies hundreds of thousands of mismatches, including errors. You can narrow down the list of mismatches to find the desired mutation using the form. The table may be copied into a spreadsheet program if you would like to specify more precise filtering conditions.

Steps for filtering mismatches

Range

Select chromosome and input the region of chromosome with the corresponding mutation. This is a required fields.

Mismatches, mismatch rate

The range of mismatches and mismatch rate. If a homozygous mutation was expected, set the mismatch rate to 90-100%.

QV rate

QV represents quality value for sequence. QV are assigned to each nucleotide in a read and range from 0-40. If a homozygous mutation was expected, set the QV rate to 90-100%.

Substitution in control

If the control will not have a corresponding mutation, set to zero or a low value. If the control will have a corresponding mutation such as when the control library is originated from segregants displaying no phenotype, set to 1/3-1/2 of coverage.

Limit to SNPs affecting translation

Select this option to limit mutations that cause missense (amino acid substitution), nonsense (stop codon), and splicing defects (mismatches in 2bp end of intron).

Limit to typical substitution pattern (G -> A)

It is known that ethylmethane sulfonate (EMS) mutagenesis frequently cause G -> A substitution. Select this option to limit results to this type of mutation.

Update table

After completing the form, click the "Update table" button. It will take a few seconds or minutes for the changes to be reflected.

Mutation List

The Mutation List table shows all mutations corresponding to the specified conditions.

Clicking the gene number in the leftmost column will open a new Gene Viewer tab for that gene number.

Gene

Gene number containing mismatches. Clicking this link will open the Gene Viewer to show the aligned reads. Gene name (annotation) is in the rightmost column.

bp

The coordinates of nucleotides in the gene having mismatches. Please note that there are margins upstream and downstream of the gene.

Reads:Sum

Reads covering the nucleotide.

Reads:Mismatch

Mismatches at the nucleotide.

Reads:Ratio

Reads:Mismatch / Reads:Sum.

QV:Sum

Sum of QV (Quality Value) of nucleotides at the position.

QV:Mismatch

Sum of QV of mismatched nucleotides at the coordinate.

QV:Ratio

QV:Mismatch / QV:Sum.

Control

This column is displayed only when the control is set. Please note that only the same mutations observed in the control are shown. For example, when the sample has a G -> A mismatch and the control has G -> T mismatch, no mismatch is displayed.

Control:Sum, Mismatch, Ratio

Reads of control covering the nucleotide.

Location

Location of the mutation in the gene: OutOfGene, UTR, Intron, 3Intron, 5Intron, or coding is shown. 3Intron and 5Intron indicate that the mismatch is in the conserved 2bp at the end of an intron, which will cause a splicing error.

Nucleotide substitution

There is a mismatch between the reference sequence and the read, e.g. reference (G) -> read (A).

Amino acid substitution

When a mutation is in the translated region, the substituted amino acid is shown. If a mutation does not change the translation, "synonymous" is shown.

Gene Viewer

Gene Viewer visualizes the results of mapping and mutation. One example is displayed below.

The horizontal axis indicates the nucleotide position in base pairs (bp). The brown, gray, and orange boxes indicate the coding sequence, intron and UTR, respectively. The multicolored bars represent nucleotide sequence, with A, T, C and G indicated by magenta, orange, cyan and blue, respectively.

The vertical axis shows the coverage of each nucleotide. The red lines indicate mismatches.

PCR primers can be designed by dragging the region of interest in the image,

Setup

Name of mutant

Enter your mutant name. This parameter does not have any effect on results. To reflect this change, please reload the page.

Data type

Select file format.

Background strain of mutant

Select the strain from which the mutant originates.

Backcrossed strain

Select the strain used to backcross.

Marker selection

Select chromosome markers (SNPs) for chromosome mapping.

• Unique SNPs not in control (the mutant was backcrossed to parental strain)

  • When a mutant originating from an Arabidopsis transgenic line is backcrossed to the parental line.
  • When a mutant originating from the Oryza sativa Nipponbare strain is backcrossed to Nipponbare.

• Shared SNPs with control (the mutant was crossed to a non-parental strain)

  • When a mutant originating from an Arabidopsis Col strain is crossed to a Ler strain.
  • When a mutant originating from the Oryza sativa Nipponbare strain is crossed to Kasalath.

Mutagen

Select the mutagen used to create the mutant.

• EMS

  Select this option when a DNA alkylating agent such as EMS, which typically causes G -> A substitution, was used.

• Radiation

  Select this option when mutants will have deletions and insertions in addition to base substitutions.

Control

Input the mutant ID to be used as a control.

It is recommended to select, as a control, a mutant isolated from the same screening but having a different phenotype. When the non-mutagenized parental line is also sequenced, please input its mutant ID.

Search database

Some tools are provided to search the Mitsucal database.

BLAST

Search for a gene or chromosome in Mitsucal using BLAST.

Keyword search

You can do a simple keyword search for gene number and annotation.