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Krait - Microsatellites Investigation and Primer Design

Du L, Zhang C, Liu Q, Zhang X, Yue B (2018) Krait: an ultrafast tool for genome-wide survey of microsatellites and primer design. Bioinformatics. 34(4):681-683. 10.1093/bioinformatics/btx665

Preface

Introduction

Krait is a robust and ultrafast tool with a user-friendly graphic interface for genome-wide investigation of microsatellites, which attempts to overcome the limitations of the currently available tools. Krait is written in Python and can be run as a standalone desktop application on Windows, Linux or Mac systems without dependencies. The microsatellite search engine is written in C and compiled as Python modules for import into Krait. Krait has many features: 1) Identification of perfect SSRs, imperfect SSRs (iSSRs), compound SSRs (cSSRs) and VNTRs from extremely large genome. 2) Locating the SSRs in gene coding region. 3) Design primer for microsatellite 4) Statistical analysis and plotting. 5) Supporting gzip compressed fasta as input file. 6) Supporting export FASTA, GFF3 or CSV. 7) Downloading DNA sequence from NCBI database.

License

Krait is distributed under a license called GNU General Public License, version 2 (GPL2).

Requirements

Krait is an operating system independent application and works on Windows, Linux and Mac systems. We recommend at least 1GB of RAM and 5GB of available hard disk space. In order to process very large genomes, a faster processor (64 bit) and larger amount of physical memory will be needed.

Installation

Windows

On windows, the preferred way to install Krait is to download the installer directly from https://github.com/lmdu/krait/releases Then double click the downloaded installer to install the program following the on-screen instructions.

MacOS

On MacOS, you can download the dmg package from the following URL: https://github.com/lmdu/krait/releases and double click the dmg file to mount it, and then drag Krait to Applcations folder to install krait.

Linux

On Linux, you don’t need to install krait, just download the package from the following URL: https://github.com/lmdu/krait/releases and then decompress the downloaded file. Just simply double click krait to run the program.

Build from source

If you can not install krait through above approaches. You can run krait from source code and build yourself binary executable. Prior to start krait, you should install python (3.6 and 3.7 recommended) required packages: PySide2, pyfastx, numpy, requests, jinja2, appdirs, primer3-py, Cython, pyinstaller.

On windows, firstly you should install mingw64 and add it to your PATH enviroment. Download the source code from github and then open the cmd.

pip3 install apsw-wheels
pip3 install -r requirements.txt
cd krait\src\libs\src
python3 setup.py build_ext --inplace -c mingw32
move *.pyd ..
cd ..\..\..
python3 main.py

On MacOS and Linux, open the terminal.

pip3 install apsw
pip3 install -r requirements.txt
cd krait/src/libs/src
python3 setup.py build_ext --inplace
mv *.so ..
cd ../../..
python3 main.py

Build binary executable

# On windows
pyinstaller win.spec

# On Linux
pyinstaller linux.spec

# On MacOS
pyinstaller mac.spec

User Interface

The general components of the Krait main user interface are shown in Figure 1.

overview

Figure 1: Krait main user interface

  1. Search for perfect microsatellites or show results if you have searched.

  2. Search for compound microsatellites or show results if you have searched.

  3. Search for imperfect microsatellites or show results if you have searched.

  4. Search for VNTRs or show results if you have searched.

  5. Mapping perfect, compound, imperfect microsatellites or VNTRs to coding region of genes.

  6. Design primers for microsatellites screened from results.

  7. Perform statistical analysis and save to file.

  8. Filter results using user inputted conditions.

  9. Display the result of analysis in table.

  10. The number of table rows.

  11. The number of table columns.

  12. The number of selected rows.

  13. The task progressing.

Import Sequences

To start a new analysis you just have to import your sequence data from files into Krait. The application accepts text or gzip compressed files containing one or more DNA sequences in FASTA format (Figure 2). These files may have the extension .fasta, .fna, .fa, .txt, .fasta.gz, .fna.gz, .fa.gz. A sequence in FASTA format begins with a single-line description or header starting with a “>” character. The rest of the header line is arbitrary but should be informative. Avoid strange characters in the sequence header, such as ’&’ or ’\’ and use ’N’ to denote in-determinations in the sequences.

fasta

Figure 2: An example for the FASTA format

Import One FASAT File

  1. Go to File Menu -> Import Fasta.

  2. Select a FASTA formatted file containing your sequences.

  3. Click Open to import FASTA file.

Import Multiple FASTA Files

  1. Put your FASTA files in a folder

  2. Go to File Menu -> Import Fastas in Folder.

  3. Select the folder to load FASTA files in that folder.

Import Fasta from NCBI

Krait provides a tool for user to download the FASTA formatted DNA sequence from NCBI nucleotide database (Figure 3).

ncbi

Figure 3: Downloading sequence from NCBI database

  1. Go to Tool Menu -> Download Sequence from NCBI to open download dialog.

  2. Input accession, version or GI number of a sequence in NCBI nucleotide database.

  3. Click Browser to select an output file path.

  4. Click OK to start download file and wait for finish.

  5. Once download finished, go to File Menu -> Import Fasta to select your downloaded file to import.

Search for Tandem Repeats

Krait allows user to identify perfect microsatellites (SSRs), compound microsatellites (cSSRs) and imperfect SSRs (iSSRs) as well as VNTRs.

Search for SSRs

SSR Search Results

After SSR search finished, a table containing results will be displayed. An example was shown in Figure 4. The result table contains 9 columns.

ssrres

Figure 4: An example for SSR search result

column

description

id

unique identifier generated by Krait

sequence

the name of sequence where SSR was found

standard

the standardized motif

motif

repeat unit of SSR

type

SSR type, mononucleotide, dinucleotide etc corresponding to motif length

repeat

number of repeats

start

start position of SSR in original sequence, 1-based

end

end position of SSR in original sequence, 1-based

length

the length of SSR (bp)

Show SSR Results

If you have searched SSRs, you can click SSR search button ssr or go to SSRs (toolbar) -> Show Perfect SSRs or go to View Menu -> Show Perfect SSRs to display SSR results in table.

Remove SSR Results

You can go to SSRs (toolbar) -> Remove Perfect SSRs or View Menu -> Remove Perfect SSRs to remove searched SSR results.

Search for cSSRs

cSSR Seach Results

After cSSR search finished, a table containing results will be displayed. An example was shown in Figure 5. The result table contains 10 columns.

cssrres

Figure 5: An example for cSSR search result

column

description

id

unique identifier generated by Krait

sequence

the name of sequence where cSSR was found

start

start position of cSSR in original sequence

end

end position of cSSR in original sequence

motif

the motifs of individual SSR in a cSSR

complexity

the number of individual SSR in a cSSR

length

the length of cSSR

gap

total bases of distance between two SSR

component

the SSR id of individual SSRs comprised cSSR

structure

the detail of SSRs comprised cSSR

Show cSSR Results

If you have searched cSSRs, you can click cSSR search button cssr or go to cSSRs (toolbar) -> Show Compound SSRs or View Menu -> Show Compound SSRs to dispay cSSR result in table.

Remove cSSR Results

You can go to cSSRs (toolbar) -> Remove Compound SSRs or View Menu -> Remove Compound SSRs to remove searched cSSR results.

Search for iSSRs

iSSR Search Results

After iSSR search finished, a table containing results will be displayed. An example was shown in Figure 6. The result table contains 13 columns.

issrres

Figure 6: An example for iSSR search result

column

description

id

unique identifier generated by Krait

sequence

the name of sequence where SSR was found

standard

the standardized motif

motif

repeat unit of SSR

type

SSR type, mononucleotide, dinucleotide etc. corresponding to motif length

start

start position of SSR in original sequence

end

end position of SSR in original sequence

length

the length of SSR (bp)

match

the number of matches

substitution

the number of substitutions

insertion

the number of insertions

deletion

the number of deletions

score

the score of iSSR

Show iSSR Results

If you have searched iSSRs, you can click iSSR search button issr or go to iSSRs (toolbar) -> Show Imperfect SSRs or View Menu -> Show Imperfect SSRs to dispay iSSR result in table.

Remove iSSR Results

You can go to iSSRs (toolbar) -> Remove Imperfect SSRs or View Menu -> Remove Imperfect SSRs to remove searched cSSR results.

Search for VNTRs

VNTR Search Results

After VNTR search finished, a table containing results will be displayed. An example was shown in Figure 7. The result table contains 8 columns.

vntrres

Figure 7: An example for VNTR search result

column

description

id

unique identifier generated by Krait

sequence

the name of sequence where SSR was found

motif

repeat unit of SSR

type

VNTR type, motif length

repeat

number of repeats

start

start position of SSR in original sequence

end

end position of SSR in original sequence

length

the length of SSR (bp)

Show VNTR Results

If you have searched VNTRs, you can click VNTR search button vntr or go to VNTRs (toolbar) -> Show VNTRs or go to View Menu -> Show VNTRs to display VNTR results in table.

Remove VNTR Results

You can go to VNTRs (toolbar) -> Remove VNTRs or View Menu -> Remove VNTRs to remove searched VNTR results.

Mapping Repeats in Genes

Start Mapping Repeats

  1. Search SSRs, cSSRs, iSSRs or VNTRs (See 3 Search for Repeats )

  2. Go to Mapping (toolbar) -> Import Annotation File to select a GTF or GFF file corresponding to the imported sequence file.

  3. Click the mapping button mapping to start mapping.

Locating Results

If mapping task finished, the repeats mapped in gene exon, intron, CDS or UTRs will be marked as different colors in table. An example was shown in Figure 9.

mappingres

Figure 9: An example for mapping result

Select by Color

This function allows selection of repeats on the basis of their color i.e. the region in where the repeat located. You can go to Mapping (toolbar) -> Show SSRs in CDS, Exon, UTR or Intron to screen as shown in Figure 10.

color

Figure 10: Selection by Colors

Remove Mapping Results

You can go to Mapping (toolbar) -> Remove mapping result to remove mapping results.

Primer Design

Start Primer Design

  1. Select tandem repeats in table that you want to design primer.

  2. Go to Primer (toolbar) -> Specify Primer3 Setttings to set parameters for primer3.

  3. Click design primer button primer to start design primer for selected tandem repeats.

Primer Design Results

After primer design, a table contains primer details will be display. The result table contains 12 columns. An example for primer design was shown in Figure 11.

primerres

Figure 11: An example for primer design

id

unique identifier generated by Krait

category

the type of repeats, ssr, issr, cssr or vntr

target

the id of the tandem repeat

entry

the primer number for tandem repeats. A tandem repeats can contains multigroup primers

product

the product size of primer

forward

the sequence of forward primer

reverse

the sequence of reverse primer

tm1,tm2

the temperature of the forward primer and reverse primer

gc1,gc2

the GC content of the forward primer and reverse primer

stability1,2

the end stability of the forward primer and reverse primer

Show or Remove Primer Design Results

If you have designed primers, you can go to Primer (toolbar) -> Show Designed Primer to display primer results. The primer results can be removed by Primer (toolbar) -> Remove Designed Primer.

Settings for Primer Design

The settings for primer design shown in Figure 12 are the same with Primer3 tags. Click the setting name will redirect to the corresponding tags in Primer3 manual.

primersettings

Figure 12: Setting panel for primer design

Filter Data in Table

Start Filter Data

  1. Verify that the current display is a table with rows.

  2. Input the conditions into input box as shown in Figure 13.

  3. Press Enter to filter rows in table.

filter

Figure 13: Filter condition input box.

Construct Filter Conditions

Krait provides comparison operators and logical operators that allow you to construct the condition. The comparison operators include =, >, <, >=, <=; the logical operators include AND, OR. There is a special operator IN can be used. The condition is case-insensitive. Thus you can use column name and operators to construct a condition to filter table rows. The numeric column can be allowed to use all comparison operators and the text column only be allowed to use = and IN. Examples:

  1. Suppose you want to find SSRs whose motif is ATG, you can construct a condition:

motif=ATG

  1. If you want to find SSRs whose motif is ATG and repeats greater than 10, you can construct a condition:

motif=ATG and repeat>10

  1. If you want to find SSRs whose motifs are ATG, AT, AAAG and repeats between 10 and 15, you can construct a condition:

motif in (ATG,AT,AAAG) and repeats>=10 and repeats<=15

  1. If you want to find SSRs whose length less than 12 or length greater than 20, you can construct a condition:

length<12 or length>20

Statistical Analysis

Perform statistical Analysis

If you have searched SSRs, cSSRs, iSSRs or VNTRs, you can just click the statistics button stat to start statistical analysis.

If you have performed statistical analysis, click the statistics button will display the statistical results. If you want to perform statistical analysis again, you can go to Statistics (toobar) -> Statistical Analysis.

Statistical Analysis Results

Once statistical analysis finished, a statistical report contains several tables and graphs will be generated and save to a html file. You can use browser (Chrome or firefox) to view html report.

report

Figure 14: A part of statistical report

You can also go to File Menu -> Export Statistical Report to perform statistics and export html formatted statistical report.

Refresh Statistical Results

If you have performed statistical analysis, you can go to Statistics (toolbar) -> Refresh Statistical Analysis to regenerate statistical results.

Export Output Files

Krait allows user to export Table (CSV, GTF, GFF) and FASTA formatted files.

Export Table

  1. Select rows in table that you want to export.

  2. Go to File Menu -> Export Selected as Table and select a format (CSV, GTF, GFF or TEXT) and provide an output file name.

  3. Click Save to export to output file.

Export FASTA

  1. Select rows in table that you want to export.

  2. Go to File Menu -> Export Selected as Fasta and provide an output file name.

  3. Click Save to export to output file

Feedback

Report issues: https://github.com/lmdu/krait/issues.

Contact email: adullb@qq.com

Indices and tables