Page 1 of 20 ABySS-Explorer v1.3.0: User Manual prepared by: Ka
Contents
1. 753 are also members of the paired end contig but 3 to 2534 Other members grey contig ids in the information pane are not shown Vi PLoTs la NAVIGATOR k28 o avoue EL 11 pe 1 minimize ener Weighted Percentile NXX i 100 000 200 000 Contig Length hp DADAR sae ae Aenea a nek eee eae D RETT AT AER N aetna a ae keen nN 700 000 _ 150 000 E z 100 000 50 000 Paired end contig id 2212 7212 bp 93 8 kmer cov 0 5 PE Single end contig members 550 1414 1018 1217 171 1199 1217 gba 2409513 379 907 2071 64 999 20714 8044 1356 1769 62 999 2071 804 1356 1769 336 1793 1817 1828 359 1270 2088 2055 EAL 333 1001 T ne 0 0 25 50 a5 100 K mer Coverage Figure 15 Plot view and Navigator view G Search Box There are two modes of contig search Figure 15 Modes of search can be switched by clicking and holding over the search mode button A click on the button performs a search ID 1 or IDL IBZ assembly tilename contig ID 1 ID2 etc A Find contig sequences O oS Find contig sequences O ie Find contig in Navigator a we Find contig in Navigator Figure 15 Modes of search and the corresponding format of query i Sequence Search This search finds the nucleotide sequences of the query contigs in the contigs fa format of query lt A gt lt F gt lt C gt lt A gt name of the assembly as displayed in the Assemblies panel This field is not necessary if an a2. D Statistics View gt STATISTICS Fragment Sizes k28 Libra Orientation Min Q1 Median Q3 Max Mean 5Stdew StdewiMean O factor SR00429 100 FR 186 208 215 223 246 215 52 10 03 4 66 14 33 488 36 17 92 3 67 _18 07 al Ince tater tate A AE A A AA ATA a AE TA A A A a A aTa a a aTa a a aTa ATAA a a ATAT a a ATATA A A a A A Aa a aTa Ta a a A a a a E Ta a a a aTa a a a aTa a aa ata a aTa a aTa aTa a a Ta aTa a a aTa a ata aa tatetatatetatatetatatetatetetatetetatatetatatetatetetatetetatetetatatetatetetatetetatet Scaffold Sizes Assembly W500 bp Contiguity bp Reconstruction op span bo edi me n i gt 100 bpn gt N50 lt lt Leia coer qeasor 4645027 TT 84 13 Keo __ 119060 __774386 __4647979 4o48002 52 251 B 14 Figure 7 The Statistics view The tables in Statistics view Figure 7 can be sorted by column Clicking the column header would sort the table In addition a table can be copied to the clipboard via the right click menu i Fragments Sizes file required 3 hist If multiple assemblies are selected in the Assemblies panel only the Fragment Sizes statistics from the assembly with the highest contiguity is displayed The statistics is calculated based on outliers trimmed data Outliers are outside the range Q1 1 5 Q3 Q1 Q3 1 5 Q3 Q1 where Q1 and Q3 are the 1st and 3rd quartiles in the raw data respectively Note that the Q1 and Q3 displayed are calculated after removing outli
3. The annotations in the upper plot display ABySS s threshold coverage near the local minimum and median coverage near the local maximum In the lower plot the value for Reconstruction is the integral of the upper plot to the right of each K mer Coverage value The annotations in the lower plot display the reconstruction at ABySS s threshold coverage and median coverage iii Fragment Sizes Distribution see Figure 9 file required 3 hist The fragment size distribution is a plot of the histogram in 3 hist ie Count vs Fragment Size The median is marked with an orange vertical line and the inter quartile range is shaded in blue Page 11 of 20 SRX000429 SRX000430 Weighted Percentile NXX Reconstruction 200 000 175 000 150 000 125 000 100 000 75 000 50 000 25 000 0 6 000 000 4641289 5 000 000 ee S 4 000 000 3 3 000 000 pote 2 000 000 1 000 000 50 000 100 000 150 000 200 000 250 000 50 aS Contig Length hp I mer Coverage N50 plot k mer coverage plot Figure 8 An N50 plot and a k mer coverage plot in the Plots view P PLOTS N50 Flot amp Cov Plot SRXO00429 SRXO000430 325 000 300 000 275 000 250 000 225 000 200 000 175 000 150 000 125 000 100 000 75 000 50 000 25 000 0 185 200 205 210 215 220 Fragment Size Figure 9 A fragment size distribution in the Plots view Page 12 of 20 F Navigator View files required for paired end assembly mode contigs dot 5 pa
4. and roll the mouse wheel to zoom The user can right click to bring up the context menu for advanced settings from JFreeChart Plot specific hot keys are available when the cursor is directly above the plots A for auto range auto range on both axes to show all data points on the plot P for previous toggles between the current and previous zoom L for legend toggles between showing and hiding the legend of the plot S for special available to k mer coverage plots only zoom to the region of the coverage threshold and median k mer coverage i N50 plot see Figure 8 file required fa or ad The file used can be selected from the file to compare drop down list from the Advanced Options tab in the Settings panel When the Navigator view is active contigs fa is used for paired end assemblies and 5 adj is used for single end assemblies This plot provides a quick way to visualize all Nxx eg N50 N80 etc of a single assemblies It also allows comparison of one or more assemblies throughout the different Nxx The contigs are arranged in descending order such that the largest contig NO is at the lower right corner while the smallest contig N100 is at the top left comer ii K mer Coverage Plot see Figure 8 file required coverage hist The k mer coverage plot consists of two subplots sharing a common K mer Coverage axis The upper plot is a plot of the histogram in coverage hist ie Count vs K mer Coverage
5. directionality of the paired end contig with light blue at the 5 end and dark blue at the 3 end See Figure 14 A click on an edge selects the edge Double clicking an edge displays its reverse complement ie from to and vice versa If the Plots view is also active a cursor green vertical line would be drawn for contig length and k mer coverage See Figure 15 Figure 12 Edge colours Default 1577 Selected 1932 Seed 1380 Paired end Partners inbound outbound Contig cl n 1053 31 3 4 10 18 oel 271 Figure 13 Graph showing the inbound and outbound partners of a selected contig Page 15 of 20 Qa NAVIGATOR k35 layout 100 ES minimize energy Paired end Contigs 1 2858 ha a Paired end Partners inbound outbound Contig g a z n Contig d a n 729 16 O 6 269 40 10 07 227 Single end contig id 2534 40605 bp 12 1 kmer cov Paired end contig id 2858 Single end contig members 2592 2704 1576 2694 66 927 211 1764 64 305 117 1346 1304 2551 729 2334 40 695 5 695 1824 25243 2636 1753 Figure 14 Graph showing single end members of a paired end contig 2858 Contig 2534 is selected and is a member of the paired end contig The light blue path 927 729 are members of the paired end contig and are 5 to 2534 The dark blue path 40
6. 4 of 20 3 User Interface and Usage The user interface is composed of seven components shown in Figure 1 projects ABySS assemblies EC SRPO0OO220 abyss 1 3 0 ABySS Explorer File View Hel A l 7 1 or D1102 G AS STATISTICS Fragment Sizes k28 Libra Orientation Min Q1 Median Q3 Max Mean Stdev Stdev Mean Q factor SRX000429 100 FR 186 208 215 223 246 215 52 10 03 4 66 14 33 Assembly NS 0 bp Contiguity bp k28 127397 778645 ls NAVIGATOR k28 layout length 2 10 100 000 200 000 labels Contig Length bp Pan e show extensions O show neighborhood number of extensions Paired end contig id 2212 7212 bp 93 8 kmer cov 1 2 3 4 5 10 20 50100 paired end assembly i Single end contig members 550 1414 1018 1217 171 1199 1217 n 379 907 2071 64 999 2071 804 1356 1769 62 999 2071 single end assembly 804 1356 1769 336 1793 1817 1828 359 1270 2088 2055 C paired end contigs 333 1001 Jaired end partner C paired end partners ze zr a TF K mer Coverage Apply Done Elapsed Time 0 581 seconds Figure 1 The user interface of ABySS Explorer v1 3 0 A Importing Assemblies Assemblies Panel Settings Panel Statistics View Plots View Navigator View Search Box m J aa q 7 Page 5 of 20 A Importing Assemblies Assemblies can be imported in two ways using the buttons shown in Figure 2 i importing an AB
7. Page 1 of 20 ABySS Explorer v1 3 0 User Manual prepared by Ka Ming Nip Cydney Nielsen Shaun Jackman Inanc Birol Canada s Michael Smith Genome Sciences Centre November 2011 ABySS Explorer is an interactive Java application that employs a novel graph based representation to display a sequence assembly and associated meta data The tool was designed with the ABySS sequence assembler in mind and was motivated by the need to examine assembly structure in particular contig connectivity and supporting paired end read relationships Table of Contents 1 Requirements 2 Running ABySS Explorer 3 User Interface and Usage A Importing Assemblies Assemblies Panel Settings Panel Statistics View Plots View Navigator View Search Box Miscellaneous Features 4 Terminology 5 External Packages 6 Technical Support TOAMmMOOW Page 2 of 20 1 Requirements System Requirements e Java SE Run Time Environment 6 or above e Smallest screen resolution supported 640 x 480 e optional internet connection and default web browser for BLAT Dataset Requirements e Assembly must be generated by ABySS 1 2 0 or above e All files from the same assembly must be in the same directory e Files used contigs fa contigs dot adj dot 3 hist coverage hist 3 dist 5 path Preferred structure of assembly directories lt name gt k lt k value gt coverage hist lt name gt 3 dist lt name gt 3 hist lt name
8. d end partners table lists the inbound and outbound paired end partners of the selected contig and their distance estimates for single end assembly mode only vii information pane displays information about the selected contig or vertex viii visualization viewer displays the graph and allows user interactions Page 13 of 20 There are three basic transformation operations on the graph within the visualization viewer e pan drag the background with mouse e zoom roll the mouse wheel e rotate drag the background with mouse while holding the Ctrl key The right click pop up menu within the visualization viewer has several options e Refocus on this contig available if clicked on an edge performs a Navigator search on the contig e Show nucleotide sequence available if clicked on an edge performs a sequence search on the contig e Screen capture saves a snapshot of the graph e Center graph moves the graph to the center to the visualization viewer Selected vertices can be moved by dragging with mouse Multiple vertices can be selected by creating a selection rectangle while holding the Shift key Figure 11 Vertex shapes and colours Default colour iv v All incident edges shown i ii iii iv Default shape ii iii iv One or more incident edges not shown v vi Selected iii Inferred overlap ii vi Collapsed i v vi Large overlap i All vertices have two poles and are displayed as an oval by default If all i
9. edges 1577 and 1932 Also consider this example below Q a hs Pa Ng j eA els we A far i ax N i th w gt Edges 2227 2216 1355 are extensions of 2043 Edge 2057 is not an extension of 2043 but it is an extension of 1355 However this graph is the size one neighbourhood of 2043 Neighbourhood A graph that shows the neighbours and next neighbours of a seed contig For example A neighbourhood of size one consists of all immediate neighbours of the seed contig A neighbourhood of size two consists of all immediate neighbours and next neighbours of the seed contig A neighbourhood of size three consists of all immediate neighbours next neighbours and next next neighbours of the seed contig Nxx eg N50 Nxx is the contig length where contigs of same length or longer make up xx of the reconstruction of an assembly Paired end Partner A contig that has paired end support distance estimate with another contig Quality factor Q factor The median divided by the interquartile range Reconstruction The sum of all contig lengths of an assembly Scaffold span The length of a scaffold the sum of contig length and the number of N s in the sequence Span The sum of all scaffold spans of an assembly 5 External Packages ABySS Explorer uses the following external Java packages 1 JFreeChart http www jfree org jfreechart 2 Java Universal Network Graph Framework JUNG h
10. egenerate the views C Settings Panel Page 6 of 20 The Settings panel consists of the settings for the three views Changes in settings are only applied when the Apply button is pressed The Apply button is available green when there are changes in settings i Stats Plots The Stats Plots tab Figure 4 has all the basic settings both the Statistics view and the Plots view SETTINGS Advanced Options Stats Plots min contig length bp 200 NW50 plot amp Coverage plot NW50 plot Y axis unit percentile W50 plot X axis scale Fragment Size Distribution select libraries P Figure 4 The Stats Plots tab of the Settings panel min contig length bp field sets the length in base pairs of the smallest contig used to generate the statistics and plots N50 plot amp Coverage plot check box shows the N50 plot and the k mer coverage plot when the Plots button is pressed N50 plot Y axis unit drop down box sets the Y axis unit for N50 plot as either percentile or reconstruction N50 plot X axis scale drop down box sets the X axis scale for N50 plot as either linear or logarithmic Fragment Size Distribution check box shows the Fragment Size Distribution plot when the Plots button is pressed select libraries button brings up a menu to select libraries for Fragment Sizes Statistics and the Fragment Size Distribution plot In general this button is not used if the assembly was based on one lib
11. ers If over 1 of the fragment sizes are negative separate statistics are calculated for the positive and the negative fragment sizes Outliers are also trimmed separately The fragment size is calculated as the position of the reverse read minus the position of the forward read which is positive for pairs oriented forward reverse and negative for pairs oriented reverse forward Double clicking a row displays the fragment size distribution of the corresponding library in a tab within the Plots view ii Scaffold Contig Sizes Statistics file required fa or adj The file used can be selected from the file to compare drop down box from the Advanced Options tab in the Settings panel The largest values in the Reconstruction N50 Contiguity columns have a pinkish red colour The Span column is hidden when the assembly contains no gaps of Ns In this case the table is called Contig Sizes instead of Scaffold Sizes Page 10 of 20 E Plots View N50 plot and k mer coverage plot are displayed together under the same tab They are vertically stacked when the Navigator view is shown but they are placed side by side when the Navigator view is hidden Fragment size distribution are displayed in separate tabs Tabs for N50 plot coverage plot and fragment size distribution can be shown hidden by checking unchecking the check boxes in Stats Plot Settings Please see the Settings section for more details In any plot the user can drag to pan
12. gt 5 ad lt name gt 5 path lt name gt contigs fa lt name gt contigs dot k lt another k value gt Page 3 of 20 2 Running ABySS Explorer The command for launching ABySS Explorer is where java jar lt PATH TO JAR gt argument lt PATH TO JAR gt is the path to the ABySS Explorer JAR file Make sure the JAR file and the lib directory are in the same directory argument is optional and can be any one of the following i paths to one or more ABySS assembly directories or their parent directory ii path to either an ADJ or a DOT file If no paths were specified ABySS Explorer would attempt to check whether the current directory is an ABySS assembly directory or a parent directory to one or more ABySS assembly directories iii help display information for help and exit iv version output version information and exit It is recommended that sufficient amount of heap space is requested when launching ABySS Explorer For example to request 1024 MB of maximum heap space java Xmx1024m jar AbyssExplorer jar Please consult Java s documentation for more information on java commands http java sun com javase 6 docs technotes tools windows java html Alternatively ABySS Explorer can be initially launched from the Java Web Start link on our website If prompted about unverified digital signature simply click Run to continue ABySS Explorer can then be launched from the icon created on the desktop Page
13. he current status Pressing the stop button to the left of the status bar can kill the current running process ie loading file rendering the layout of a graph in Navigator view 4 Terminology This is a list of terms used in this user manual and ABySS Explorer ABySS assembly directory This directory contains a single ABySS assembly and it must have either both i a value of k in the DOT files ii a name that starts with k followed by the value of k used to generate the assembly ie k50 and has one or more files with extensions fa or adj Contig length units bp k mer nol bp bp number of base pairs k mer number of k mers length in base pairs k 1 nol bp number of non overlapping base pairs length in base pairs if both out degree and in degree is not 1 length in base pairs k 1 if either out degree or in degree is 1 length in base pairs 2k 2 if both out degree or in degree is 1 Contiguity The square root of the sum of the squares of contig lengths of an assembly Extensions Two edges are extensions of each other when the head of one edge connect to the tail of another edge at the same pole of the shared vertex Also any edges within a path are extensions of each other Consider the example in Figure 12 where edges 1577 1932 and 1380 form a path Edge 1577 is an extension of edges 1932 and 1380 Edge 1932 is an extension of edges 1577 and 1380 Edge 1380 is an extension of
14. ncident edges displayed connect to the same pole then the two poles of the vertex would be collapsed together in which the vertex becomes a circle Vertices are gray and filled by default They are dark gray when selected They are unfilled when one or more incident edges are not shown Double clicking an unfilled vertex would center the graph on one of the edges that are not shown The Navigator options show extensions size of neighbourhood and number of extensions can limit the number of edges displayed A vertex is said to have an inferred overlap if the overlap between some incident contigs is not found In this case the vertex is yellow A vertex is said to have an large overlap when e the overlap is larger more negative than the threshold distance and e the overlap is larger than half the length of the longer contig of the overlap Page 14 of 20 The threshold overlap is calculated as Q1 3 Q3 Q1 where Q1 and Q3 are the 1st and 3rd quartile of the overlap in the assembly In this case the vertex is orange Edges are gray by default The seed edge of a neighbourhood is light green A selected edge is green and is thicker than a regular edge See Figure 12 The inbound and outbound contigs with paired end read support to the currently selected contig are coloured light and dark purple respectively See Figure 13 Single end contigs that are members of a paired end contig are coloured blue A blue gradient indicates the
15. ners available when the single end assembly radio button is selected iii Advanced Options Page 8 of 20 Th Advanced Options tab Figure 6 has the advanced settings for the Statistics view and the Plots view SETTINGS Advanced Options __ Stats Plots file to compare SRPOOO 20 contigs fa ngth unit of ler Figure 6 The Advanced Options tab of the Settings panel file to compare drop down box sets the file for Scaffold Contig Sizes statistics and N50 plot This drop down box lists the names of all fa and adj files in the assembly directories added However there are special cases where the file selected is ignored for N50 plot When the single end assembly radio button from Navigator settings is selected either 5 adj or 5 fa instead of contigs fa would be used to generate the N50 plot When the paired end assembly radio button is selected contigs fa would be used to generate the N50 plot Note that contigs fa contains the paired end assembly whereas 5 fa and 5 adj contain the single end assembly unit of length drop down box sets the unit of length for contigs in Scaffold Contig Sizes Statistics and N50 plot as one of bp number of base pairs k mer number of k mers available to fa files only nol bp number of non overlapping base pairs available to adj files only When the Navigator view is active the unit of length will always be bp for N50 plot Page 9 of 20
16. rary ii Navigator Page 7 of 20 The Navigator tab Figure 5 has all the settings for Navigator view SETTINGS Advanced Options Stats Plots Navigator length 10 10 labels show extensions G O show neighborhood number of extensions 1 2 3 4 5 10 20 50100 paired end assembly single end assembly Figure 5 The Navigator tab of the Settings panel length check box enables rendering of length of contigs as a wave packet length slider sets the length represented as half period of the wave packet available when length check box is checked show extensions radio button constructs a neighborhood such that only extensions to the seed contig are shown edges shown are limited to the number of extensions show neighborhood radio button constructs a neighborhood such that all neighbor edges are shown edges shown are limited to the size of neighborhood size of neighborhood number of extensions slider sets the size of the neighborhood of the seed contig labels check box shows the identifier labels of the contigs paired end assembly radio button displays the graph of the paired end assembly single end assembly radio button displays the graph of the single end assembly paired end contigs check box highlights the single end contigs that make up a paired end contig available when the single end assembly radio button is selected paired end partners check box highlights the paired end part
17. ssembly is selected or a DOT ADJ file is loaded lt F gt file name This field is not necessary unless the file of interest is not contigs fa lt C gt one contig id or a comma space separated list of contig ids This field is always required The sequences found are displayed in another window The user can perform a web BLAT search on the sequences found Please note that there are restrictions on the usage of web BLAT Example queries 123 k32 123 k32 ecoli 4 fa 123 ii Navigator Search Navigator search applies the settings when the Apply button is enabled green query one contig id ie 100 This search displays the neighbourhood of the query contig in the Navigator view query two contig ids separated by a comma ie 100 105 This search displays contigs that form the shortest path between the two contigs of interest in the Navigator The path found ignores the direction of edges and so may not be a valid sequence path If the query has more than two contig ids either separated by spaces or commas a sequence search is performed instead H Miscellaneous Features i Double clicking a view title maximizes the corresponding view by hiding other views shown The hidden views can be shown by pressing the toggle buttons in Assemblies panel ii Assemblies panel and Settings panel can be hidden or shown by pressing the triangle button on the divider iii The status bar at the bottom of the interface displays t
18. th files required for single end assembly mode 5 adj 5 path 3 dist The Navigator view Figure 10 displays the graph of an assembly In general edges represent contigs and vertices represent the overlap between contigs ii Ul IV V minimize energy Paired end Contigs 3 2191 7 a NAVIGATOR k28 layout 100 Single end contig id 416 28 bp 253 kmer cow Paired end Partners 5 inbound outbound Paired end contig id 2191 1 onti d Single end contig members 784 1288 388 2159 EREE E 4 2 112794 339 2159 1107 1266 1999 1377 297 1848 1407 35 1881 629 502 695 71 1173 1889 638 1482 72 34 31 685 1514 1531 1225 878 493 45 1794 525 1864 1983 414 134 784 745 901 1263 960 2120 926 1251 1429 225 447 947 1861 1271 1296 625 261 752 1462 421 vi Figure 10 The Navigator view in single end assembly mode i layout progress bar shows the progress of the layout of the graph ii pause button pauses the layout and displays the current state of the graph iii resume button resumes the layout iv minimize energy check box layout stops when the energy of the graph is minimized or 2000 iterations has run otherwise the edges and vertices are not spaced out v paired end contigs drop down box shows the selected contig s corresponding paired end contig s for single end assembly mode only vi paire
19. ttp jung sourceforge net 3 Batik SVG Toolkit http xmlgraphics apache org batik All external packages required by ABySS Explorer are included in the download 6 Technical Support Please direct your bug reports questions and suggestions to the ABySS Google Group abyss users googlegroups com You can also read and search existing discussions on the Google Group at http groups google com group abyss users End of User Manual
20. ySS assembly directory ii importing an ADJ or DOT file an Figure 2 The buttons for importing assemblies These commands are also available in the File menu The button adds one or more ABySS assemblies to the Assemblies panel When the file chooser is launched one or more ABySS assembly directories or their parent directories can be selected The button removes one or more selected assemblies from the Assemblies panel The ADJ DOT button loads one file either an ADJ file or a DOT file The ADJ files must reside in a directory whose name starts with k followed by the value of k When an ABySS assembly directory is loaded all three visualization views Statistics Plots Navigator can be launched When either an ADJ file or DOT file is loaded only the Navigator view is available B Assemblies Panel ASSEMBLIES The Assemblies panel Figure 3 lists the ABySS assemblies loaded The colored squares denote the colors that will be used in the Plots view Multiple assemblies can be selected for Statistics view and Plots view but Navigator view can only view one assembly at a time The views can be launched from the three toggle buttons When there are no changes in the selection of assemblies or in the settings described in the next section pressing these buttons would only toggle the visibility of the views Otherwise pressing the green buttons would apply any new Figure 3 The Assemblies panel settings and r
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