KGBassembler Manual. A Karyotype-based Genome Assembler for Brassicaceae Species. Version 1.2. August 16 th, 2012

Transcription

1 KGBassembler Manual A Karyotype-based Genome Assembler for Brassicaceae Species Version 1.2 August 16 th, 2012 Authors: Chuang Ma, Hao Chen, Mingming Xin, Ruolin Yang and Xiangfeng Wang Contact: Dr. Xiangfeng Wang, xwang1@clas.arizona.edu Dr. Chuang Ma, chuangma2006@gmail.com

2 1 Introduction The Brassicaceae family contains about 3,700 species, including the most important model plant Arabidopsis thaliana and many agronomically important vegetable crops. Due to the lack of genetic or physical maps for many of non-model plants in Brassicaceae species, the sequence reads from next-generation sequencing (NGS) can be only assembled to contigs or scaffolds. Here we presented a Brassicaceae genome assembler, named KGBassembler, to assemble contigs and/or scaffolds to full chromosomes based on karyotype maps of Brassicaceae species and without the need of genetic and physical maps. KGBassembler is an easy-to-use tool featured with a graphical user interface (GUI), allowing users to use automatic assembling of chromosomes based on the karyotype maps obtained from comparative chromsome painting (CCP) experiments and/or to manually edit the layouts of contigs according to the in silico generated karyotypes. KGBassembler has been applied to assemble the genome of Arabidopsis lyrata, Thellungiella parvula and Eutrema salsugineum on a laptop. 2 Download The latest version of KGBassembler can be available from 3 Installation No installation is required to implement KGBassembler. Note: the Linux users need to add the execute permission to KGBassembler with the command: $chmod u+x KGBassembler_Linux_X_X.

3 4 Run 4.1 Launch KGBassembler Double click the KGBassembler, a graphical user interface (GUI) will be presented for loading project data, and visualizing the in silico generated karyotype. The following is a screenshot of KGBassembler window. Details about this figure are described in the following table. No. Description 1 File menu Four file menus: ("File", "Contigs2Blocks", "Blocks2Chromosomes", "View digital karyotypes"). The first three are for the assembly of Brassicaceae genomes. The last one can be used to view the digital karyotypes. 2 Progress bar Display the status of running progress. 3 Main window Display the full file paths customized in the configure file and visualize the insilico karyotypes.

4 4.2 Load project To start the genome assembly,please select "File->Load project" (or use "Ctrl+L") to load project data specified in the configure file for the NAMES of the karyotype file, the contig sequences file and the BLAT alignment file. If the configure file is setup correctly, all the path parameters will be displayed in the main window of KGBassembler, and the "Setting parameters" button on "Contigs2Blocks" menu will be checkable. Please double check the paths of project data before running the next step. An example of the configure file is shown in the following figure. Note that the annotation lines are started with the "#" character. The names of contig sequence file, the BLAT alignments file, the karyotype file are specified at the "CONTIG_FASTA:", "BLAT_PSL:" and "CCP_KARYOTYPE:" lines, respectively. The name of the result directory is given at the "OUTPUT_DIR: " line. Note: Make sure you have read and write permissions for "OUTPUT_DIR".

5 The following figure shows the format of karyotype file. The letter-labels and orientations of conserved blocks for each chromosome are listed in one line and separated by a comma. Two adjacent blocks are separated by the tab character. Note: 1) The configure file, the karyotype file, the contig sequences file and the BLAT alignment file should be in the SAME directory of the KGBassembler. 2) The names of the karyotype file, the contig sequences file, the BLAT alignment file and the result directory should be specified in the configure file before loading project data. 3) The contig sequences should be in FASTA format. 4) The BLAT alignments between Arabidopsis genes (protein sequences) and contig sequences should be in PSL format. The alignment tool BLAT can be downloaded fromhttp:// Users who cannot handle the BLAT mapping can send the request to us for help.

6 4.3 Determine regions of conserved blocks on contigs The next step of running KGBassembler is to determine the regions of conserved blocks on contigs with the local and global search strategies. All the related parameters in this step are presented on acontrol panel, which is opened by clicking the button "Contigs2Blocks->Setting parameters". In the group box (a), the "minimal identity" and "minimal coverage" are used to eliminate lowquality BLAT alignments. In the group box (b), the "Gene Number" and "Fault-Tolerant Ratio (FTR)" are two parameters for the sliding window approach. When the "Gene Number" is 10 and the "FTR" is 0.30, the KGBassembler will scan the contigs with the window length of 10 aligned genes and assign a block ID to the window region in which more than 7(i.e., (1-FTR) *GeneNumber) Arabidopsis genes belonging to the same conserved block. Note: a lower "FTR" applies a strict rule to determine the genome blocks in contigs and yields the assembled chromosomes with higher quality. A less stringent "FTR" (e.g., 0.30) is recommended for retaining more contigs with block information. In the group box (c), the "Chromosome FTR" is a parameter to determine the most possible chromosome where the contigs should be belonged to. The "Minimal Gene Number" is a parameter used to control whether the letter-labels of identified blocks are displayed on the assembled digital karyotype. The "Chromosome FTR" should be smaller than The latter-

7 labels will not be displayed if the gene numbers of blocks are less than the "Minimal Gene Number". In the group box (d), two parameters "Minimal Contig Size (Kb)" and "Minimal Gene Number" are provided to ignore short contigs, if the information is not sufficient to assign the blocks to contigs. Two logical operations including "AND" and "OR" are available for different criterions of contig filtering. Here "Minimal Gene Number" indicates the minimal number of aligned genes on the contig. Once all the parameters are set up, please press "OK" button for recording these settings and changing the status of the button "Contigs2Blocks->Run" to be checkable. Click the "Run" button to complete the determination of conserved blocks on contigs. The results will be graphical visualized in the main window of KGBassembler. the color and latter-labels of 25 (A- X, 0) blocks are shown at the top of the image. Here we add another block "0" to represent the Arabidopsis genes whose block lable-letters have not been determined. The character in the bracket indicates the orientation of contigs (+: plus strand; -: minus strand; 0: undertmined strand). The label of contigs with short size (less than 0.02*chromosome size) is not displayed, but can be retrieved from the file "Blocks2Chromosomes.txt" in the "other" subdirectory of "OUTPUT_DIR".

8 4.4 Assemble contigs into large-scaffolds or whole-chromosomes KGBassemble provides the option of manually adjusting the layout of contigs obtained from Section 3.3. By clicking the "Manual adjustment" button on the "Blocks2Chromosomes" menu, an editable form containing the order and orientation information of contigs will be presented (shown in the following figure). Please do remember to press "OK" button to record the modification. Note: users can also directly modify layout of contigs in the "tmp_block2chromosomes_adusted.txt" file, which is located in the same directory of KGBassembler. Note: For the guide of adusting contig layouts, KGBassemble has been updated to outputs two html files describing the information of aligned Arabidopsis genes in each contig and the synteny maps between Arabidopsis and the assembled genome, respectively. The per-contig view page (percontigview.html) provides the information, including the contigs assigned for each assembled chromosome, the contig length, the orientation on assembled chromosome, number of aligned Arabidopsis genes. In the svg file (located in "percontigview" subdirectory), KGBassembler visulizes the location, block-label and block-color of these aligned genes, and displays the statistical results for each probable contig about the number of aligned Arabidopsis genes belonging to. The detailed information about the aligned genes was also output in the textual files in the same subdirectory.

9 The synteny map page (syntenymaps.html) provides the hyperlink of synteny maps between each chromosome of Arabidopsis and the assembled genome in svg format (located in the syntenymaps subdirectory). The detailed information of synteny regions are shown in the textual files in this subdirectory, in which each line describing the Arabidopsis gene ID, locations of this gene on Arabidopsis and the assembled genomes.

10 After record the modification, the "Assemble chromosomes" button on the "Blocks2Chromosomes" menu will be checkable. Click this button to re-assemble the wholechromosomes defined by the mapping information of contigs. Once finished, the assembled in silico karyotype will be plotted in the main window of KGBassembler and saved in the SVG (Scalable Vector Graphics) file ("DigitalKaryotype.svg") in the result directory. The assembled karyotype can be also reloaded for further visualization by clicking the "Open SVG file" on the "View digital karyotypes" menu. Note that KGBassembler does not estimate the gap size between two adjacement contigs, and thus directly connectes them together to form the final chromosomes.

11 Besides the in silico karyotype, KGBassembler also generates other information,including the sequences of assembled chromosomes ("ChromosomeSeq.fasta"), the statistical results of Arabidopsis genes retained for block inferrence and genome assembly ("stat_repalignment.txt"). the statistical results of contigs on the assembled chromosomes ("stat_contigsonchrom.txt"), the regions of identified blocks in the contigs ("Contigs2Blocks.txt"), the predicted layouts of contigs on chromosomes ("Blocks2Chromosomes.txt"), and the support information of karyotype ("KaryotypeSupportInfo.txt"). There are 3 columns in the file "Contigs2Blocks.txt", which respectively represent the contig ID, contig size, and the label-letters of conserved chromosomal blocks and their regions in the contigs. Example: #ContigID ContigSize BlockLabel:ContigStart-ContigEnd c A: ,B: c C: c D: c D: There are 6 columns in the file "Blocks2Chromosomes.txt", providing information including the chromosome ID, the contig ID, the contig size, the label-letter of conserved chromosomal blocks, the number of aligned Arabidopsis genes in the contig, and the strand of contigs. Example: #ChromID ContigID ContigSize BlockLabel GeneNum ContigStrand Chr1 c A 11 - Chr1 c A 14 - Chr1 c A 14 - Chr1 c A 7 - Chr1 c A 8 - In the file "KaryotypeSupportInfo.txt", there are totally 9 columns.

12 1. GeneID - the identifiers of Arabidopsis genes. 2. chromid - the chromosome ID. 3. chromstart- the start position of aligned Arabidopsis genes on the assembled chromosome. 4. chromend - the end position of aligned Arabidopsis genes on the assembled chromosome. 5. contigid - the contig ID. 6. genecol - the color of Arabidopsis genes shown in the assembled karyotype. 7. gblocklabel - the corresponding block label-letters of Arabidopsis genes in Arabidopsis karyotype. 8. genestatus - the check status of Arabidopsis genes indicating the confidence in the genome assembly (confidence level from high to low is "1st", "2nd", "3rd" and "4th"). 9. BlockLabel - the label-letter of predicted blocks. Example: #GeneID chromid chromstart chromend contigid genecol gblocklabel genestatus BlockLabel AT1G02380 Chr c154 #f4ea00 A 1st A AT1G02370 Chr c154 #f4ea00 A 1st A AT1G02340 Chr c154 #f4ea00 A 1st A AT1G02335 Chr c154 #f4ea00 A 1st A 5 Release Note August 16th, 2012, KBGassembler (version 1.2) were released. (1). A bug was fixed to filter low-quality BLAT alignments between contigs and Arabidopsis genes.

13 (2). A function was added to place the contigs accoording to the location of orthologous genes on the Arabidopsis genome. July 5th, 2012, the executables of KBGassembler (version 1.1) were released for Windows and Linux platforms. (1). A function was added to output the synteny regions between each pair of chromosomes in Arabidopsis and the assembled genome (textual files in the subdirectory syntenymaps of the result directory). (2). A function was added to generate svg files for visualzing the linear genomic synteny as dot-plot graphs ((svg files in the subdirectory syntenymaps of the result directory)). (3). A function was added to organize the synteny maps in a html file (syntenymaps.html). (4). The karyotype files were updated for more species with the avaiable CCP-based karyotype maps. (5). A function was added to display the information of orthologous genes on each contig in SVG format (svg files in the subdirectory percontigview of the result directory). (6). A function was added to output the support information of each contig in textual format (txt files in the subdirectory percontigview of the result directory). (7). A function was added to generate a html file (percontigview.html) for organizing the information in each contig used in the genome assembly. (8). Updated KGBassembler to generate a intermediate file (BestPSL2Block) for recording the retained Arabidopsis gene hits after the filtration with the parameters in Section 3.3. (9). Updated KGBassembler to generate a textual file (stat_repalignment.txt) for the statistical restuls of Arabidopsis gene hits used in the Phase I (Congigs2Blocks). (10). A function was added to summerize the contigs used for the genome assembley and output in a textual file (stat_contigsonchrom.txt). April 26, 2012, the executables of KBGassembler (version 1.0) were released for Windows and Linux platforms.