Manual for Demo Data
SEQUENCE Pilotmodule SeqNext-HLA
Version 5.0.0(August 1st 2018)
developed by
JSI medical systems GmbHTullastr. 18
77975 EttenheimGERMANY
phone: +49-7822/440150-0fax: +49-7822/440150-20
email: [email protected]: www.jsi-medisys.com
(for research use only)
Table of Contents1 Introduction to SEQUENCE Pilot – SeqNext-HLA..........................................................................22 Installation.......................................................................................................................................2
2.1 SEQUENCE Pilot.....................................................................................................................22.2 NMDP Codes..........................................................................................................................2
3 The first login..................................................................................................................................24 SEQUENCE Pilot screen................................................................................................................35 Create your own user......................................................................................................................36 Installation of demo data.................................................................................................................47 Start a Run......................................................................................................................................68 Joining - Creation of orders.............................................................................................................79 Worklist...........................................................................................................................................710 Sequence - Analysis of demo data...............................................................................................8
10.1 Left section.............................................................................................................................810.1.1 Order/Protocol/Family table.............................................................................................810.1.2 Files, Genes, ROIs and Locations...................................................................................9
10.2 Sequences.............................................................................................................................910.3 Matching table......................................................................................................................1410.4 Result...................................................................................................................................1410.5 Results of the Demo Data....................................................................................................14
10.5.1 Genes A,B, C, DPB1, DQA1 and DRB1........................................................................1410.5.2 Gene DQB1...................................................................................................................15
10.6 Validation.............................................................................................................................2010.7 Print a report or export the result.........................................................................................2110.8 Edit bases in the electropherogram/sequences...................................................................24
11 How to analyse the order again..................................................................................................2611.1 Recalculate-Function...........................................................................................................2611.2 Delete the order and load the file again...............................................................................27
Manual for demo data SEQUENCE Pilot – module SeqNext-HLA 1
1 Introduction to SEQUENCE Pilot – SeqNext-HLA
SeqNext-HLA is a simple, easy to use desktop application for analysis of HLA sequence basedtyping data from Next Generation Sequencing platforms. It is independent of the used assay andincludes a variety of functionalities to fit the program to many different specific needs.All IMGT HLA database versions are available for download and import on the JSI website. For resultcalculation intron sequences can be regarded to exclude null alleles, moreover the phasing can bechecked. New alleles are called as well. The result can be displayed in maximum, 1 field or 2 fieldresolution and as NMPD code. After the analysis, a diagnostic report for each sample is received.
2 Installation
2.1 SEQUENCE PilotIn case you have done the Installation of SeqPilot and the HLA database already, pleaseproceed with chapter 3.
For the installation of SEQUENCE Pilot, please do the following:
Go to our website http://www.jsi-medisys.com/free-trial-license to obtain a free trial license.
Please use the link and the instructions you will receive via e-mail to download and install ourlatest version of SEQUENCE Pilot and the HLA database.
2.2 NMDP Codes
Optionally results can be shown as NMDP code. To activate the NMDP code generation deposit thethe new subdirectory NMDPFiles in the directory SeqPilot (by default C:\SeqPilot\NMDPFiles). Within this directory the current NMDP list numer.v3.txt has to be deposited:
Therefore go to
http://bioinformatics.nmdp.org/HLA/Allele_Codes/Allele_Code_Lists/Allele_Code_List_in_Numerical_Order.aspx
Download the txt-file Numeric Allele Code List (ZIP) (new nomenclature)
Extract the downloaded file to the folder C:\SeqPilot\NMDPFiles.
NMDP-codes are automatically calculated now.
3 The first loginYou can login using the Name jsi (leave the Password field empty).
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4 SEQUENCE Pilot screen
In SEQUENCE Pilot a menu is available, which consists of the items System, SeqNext-HLA andHelp. Furthermore several categories and operations are shown on the left side of the screen.Open a category or an operation by clicking on the corresponding icon.
5 Create your own user
How to get there: Category AdministrationOperation Users
After the first login, create a new user. After the new user has been saved, the user jsi is deletedautomatically.
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operations
menu
categories
Click on the category Administration on the left side of the screen and then click on theoperation Users.
Fill out the field User (max. 4 letters).
Check active and all options user is authorized to....
Press [Save].
Select the operation Logout and then login with the new user.
6 Installation of demo data
For the installation of the demo data, do the following:
Go to our websitehttp://www.jsi-medisys.de/DownLoadFiles/DemoData/SeqNext-HLA_DemoData. zip
Unzip the downloaded file, save the files DemoData_.fna and SeqNextHLA-demo.hge onyour PC.
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Go to category SeqNext-HLA / operation ROI Group [master file]
Press the [Import…] button and select the downloaded file SeqNextHLA-demo.hge.
Press [OK] in the Select Import window to import the ROI Group Demo SeqNext-HLA. Thescreen should look like above afterwards → The ROI Group “Demo SeqNext-HLA” including14 ROIs is imported.
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7 Start a Run
How to get there: Category SeqNext-HLAOperation Run
Now switch to the operation Run to start an analysis.
Press the button […] in section File and select the downloaded file DemoData_.fna.
The entry “DemoData” is created in the Patients table automatically.
Select the entry “DemoData” in this table and press [Add] behind ROI Groups:.
The window Add ROI Groups opens, select the ROI Group Demo SeqNext-HLA and press[Add→] in the window.
The ROI Group Demo SeqNext-HLA and the ROIs are then shown in section ROI. Those arethe Regions Of Interest (genes, exons, amplicons...) to be analysed. In our demo data theseare: A,B,C (exons 2, 3, 4), DQB1 (exons 2, 3) and DQA1, DPB1, DRB (exon 2).
In section Settings the Profile “Demo” is selected automatically.
Press [Start Analysis] to start the analysis. Press [Yes] in the window that opens afterwards.
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8 Joining - Creation of orders
How to get there: Category SeqNext-HLAOperation Joining
Note: Depending on your PC the analysis will take around 20 minutes.
Now switch to the operation Joining. By default this operation lists all orders loaded at the currentdate in the Lower Table. (To show orders from other dates, uncheck Date/Select Orders and press[Search]).In the Upper Table all files that are loading at the moment are listed. It is possible to follow theProgress of loading, which Step of the progress is executed and the elapsed Time.After the loading is completed (entry “completed” in column Progress), press [Update] in the dialoguepart Select Orders. Loaded files are then moved from the Upper Table to the Lower Table.
Upper Table Select Orders Lower Table
In the Lower Table, all loaded files, that were joined to an order are listed. Orders are createdautomatically. All files that have the same DNA number are joined to an order.After complete loading of the file and pressing [Update] there is one order (“P-DemoData”) in theLower Table. The Upper Table is empty. The file joined to the order is listed in the Lower Table, if youpress the “+” in the column Order.
9 Worklist
How to get there: Category SeqNext-HLAOperation Worklist
This operation lists all existing orders and their joined files. The dialogue part Select Orders and theWorklist Table are the same as Select Orders and the Lower Table in Joining (chapter 9). But incontrast to the operation Joining, there is no context menu available for the Worklist Table.To proceed with the analysis select the order “P-DemoData” in the Lower Table of Joining or in theWorklist and switch to the operation Sequence.
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10 Sequence - Analysis of demo data
How to get there: Category SeqNext-HLAOperation Sequence
Operation Sequence gives an overview about all defined regions of interest and the results. Moreoverit gives several options to do edits.
Result Sequences Show
Left section Matching table Functions
10.1 Left section
The left section gives information about the order, comments, the analysed file(s), genes, regions ofinterest and exons/locations.
10.1.1 Order/Protocol/Family table
This dialogue is divided into three sections:
Order shows all important information about the order, such as order number, DNA number,date, procedures and state.
Protocol shows a list of all edited bases for the shown Sequences (sequences of the exonselected in the Location table).
Family shows a list of all orders belonging to the same family ID.
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10.1.2 Files, Genes, ROIs and Locations
Here the sequences to be viewed and analysed can be selected:
Files: Lists all analysed files. Here the file DemoData_.fna is listed.
Genes: Lists all analysed genes of the selected file. These are A, B, C, DPB1, DQA1, DQB1,DRB1 and DRB3.
ROIs: Lists all analysed regions of interest for the selected gene. For gene A exons 2, 3 and 4are shown.
Locations: Lists all exons that are covered by the selected ROI. For the location selectedhere, sequences are shown in the electropherogram. In case introns are defined they are onlydisplayed, if the first entry (ROI) is selected.
The first gene and location (gene A and exon 2) is pre-selected and its sequences are shown. In caseyou select another file, gene or location, the sequences of the highlighted Location are displayedautomatically.
10.2 Sequences
The sequences for the exon selected in the section Location are displayed automatically.
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combined sequence
coverageelectropherogram
count mode numberinglow abs coverage
warning lineshown location
read sequences consensussequence
perfect matchtables
location overview
haplotype 1/2 sequences
result sequences
There are different count modes possible. They can be changed with the combo box (count mode):
Exon/Intron: the first position of every exon/intron is 1.
cDNA: refers to the absolute position of the base in the gene, excluding introns.
AA: every amino acid is counted.
Gene abs.: has no function yet, count mode is the same a ROI abs.
ROI abs: first position of the ROI is 1
A location overview is available above the electropherogram:
It gives an overview of the selected location in the section Locations. You can jump to a positionwithin the electropherogram by clicking on the overview.Exons are marked blue and introns are marked yellow. Regions that are not analysed (outside theborders of the amplicon) are highlighted in a lighter color.
Heterozygous positions (differences for the called Haplotypes) are shown dark blue. Moreover belowthe overview a line is shown: The line shows how good phasing was possible depending on the readsthat are present. Green means phasing is possible, for red areas phasing is not possible.
The coverage distribution is shown graphically below the location overview (coverage graph). Youcan jump to the positions by clicking on the graph. The graph color changes from grey to pink if thecoverage is below the Expected Coverage Warning line (here the Expected Coverage Warning is setto 100, the graph color is gray because coverage is above the line).
Move the cursor tool tip over the location overview to show the following information:
ROI: analysed gene and region.
Location: Exon or intron number.
Pos.: Position in exon/intron count mode (the first position of every exon/intron is 1).
cDNA: Position in the cDNA
Amplicons: Amplicons defined for the ROI.
The following sequences are shown: consensus sequence, combined sequence, sequences forhaplotype 1 and 2 and the result sequences. The consensus sequence is a summary of allpossible alleles for that gene. The combined sequence is the combined sequence of the resultsequences for haplotype 1 and 2. The result sequence for each haplotype is the combinedsequence of all reads joined to the haplotype.Constant positions are shown as A, C, G or T. Polymorphic positions are shown in the correspondingIUB code. All intron sequences are written in small letters whereas exon sequences are written in capital letters.Moreover for intron areas the electropherogram/sequences have a grey background.
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The amino acid sequence for the sequences of haplotype 1 and 2 and of the result sequences, isshown in the current reading frame in a one letter code above the first base of the correspondingcodon. Stop codons are shown as red dots •.
The data is visualized in a coverage-electropherogram, peaks are artificial and represent thecoverage for each base position. The Expected Coverage Warning line (also shown in the locationoverview; here: 100) is also displayed.
The detected heterozygous positions are marked blue in the combined and result sequences. Theyshow the differences between haplotype 1 and 2. You can easily jump through heterozygouspositions in the sequences by using the jumper 'het pos.' in the Show window.
The perfect match tables list all alleles that show a perfect match to the haplotype for the selectedROI. For some alleles a “!” sign is listed behind the allele. This means that for those alleles thesequence for the respective exon is unknown.
Below the result sequences the reads sequences are listed. Those are the sequences of the reads,present with the highest copy numbers. Identical reads are listed only once, the copy number is listed
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in front of each read. Moreover all reads that have a perfect match to an allele can optionally bemarked with a * in front of the copy number (as shown here).The * and the copy number in front of each read can be colored. Identical colors for two or morereads mean, that the reads have a perfect match to the same allele(s) (context menu for the reads:show alleles lists identical matches).
Reverse sequences are written in italics and are highlighted in a darker colour. By default, thereverse sequence is shown as a complement reverse.
Read bases are highlighted in different colors:
darker blue: reverse sequence
lighter blue: forward sequence
orange: likely errors in base calling
..light orange: only for paired end data (not shown here): for paired end data one read pair isshown in one line. The overlapping parts of the two reads are colored light orange.
blue: insertions/deletions
red: mismatches to the shown HT
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In addition to the reads sequences, the reads view can be opened to see all detected reads for thelocation. Therefore right-click the haplotype sequence and select show reads....
In the reads view you can switch between HT1, HT2 and both HTs (Combined) using thecorresponding tab. As for the reads sequences identical reads are displayed only once, the copynumber is displayed at the right and left end of each read. Moreover all reads that have a perfectmatch to an allele can optionally be marked with a *. Bases are colored the same way as in the mainwindow.
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10.3 Matching table
In the Matching table all possible allele combinations for the highlighted gene (in dialogue partGenes) are listed, sorted by their probability. The table has the three tabs Total Result, Haplotype 1and Haplotype 2. Each tab shows the results, calculated only for the files of the selected tab.
If the entry in the column Mism. is 0, there are no mismatches to an allele combination and thecombination is listed in the Result window.
If you select an entry within this table, the sequences for the selected allele combination aredisplayed. In case there are mismatching positions, the affected locations are displayed automaticallyand the cursor is positioned to the first mismatch.
10.4 Result
This dialogue shows the total result, calculated for the selected gene in the dialogue part Genes.Here, all detected allele combinations and the HLA database version the result is based on, areshown.If there are still mismatches to an allele, there will be the entry “no results possible”. In that case youhave to check the mismatching positions in the sequences. When all mismatches are removed theresult will appear automatically.
For gene A, there are no mismatching positions, therefore the following result is listed (with HLAdatabase 3.31.0):
heterozygous result(s) - based on haplotypes: 1,2 A*02:07:01G, *24:02:01GHLA informatics group: 3.31.0 Date: 01/22/2018
The first line shows, whether it is a heterozygous or a hemizygous result and on which haplotype(s)the result calculation is based on. The second line shows the ambiguities, that is all found allelecombinations. G and P suffixes are used, moreover the result can optionally be displayed in 2-digit, 4-digit or maximum resolution or as NMDP code (see chapter 3). The last line shows, which HLAdatabase version was used for the result calculation.
10.5 Results of the Demo Data
10.5.1 Genes A,B, C, DPB1, DQA1 and DRB1
If you check genes A, B, C, DPB1, DQA1, DQB1, and DRB1 in the Genes section you can see, thatthey all have the entry compl. in the column State.
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This means for all these genes a result is found. You can check the results by clicking on a gene inthe Genes section. Moreover you can check all ROIs, by selecting them (for the selected gene) in thesection ROI - the sequences for a selected ROI are displayed.The result for the selected gene is shown in the section Result.
10.5.2 Gene DQB1
Gene DQB1 has the State complete, but a very rare allele was called. Therefore select DQB1 in theGenes table. The sequences/electropherogram change to DQB1-Exon2.
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The Matching table shows, that there is “0” mismatches present, for a very rare allele: DQB1*05:121
Moreover DQB1-E02 was called homozygous, no heterozygous positions are present:
the jumper het pos. in the dialogue Show, has the entry 0.
the sequence for the second allele is grayed out.
If you select DQB1-E03 in the section ROI you can see, that for DQB1 exon 3 the sequences areheterozygous. The jumper het pos. in the section Show, shows that 10 heterozygous positions arepresent. You can use this jumper to check the heterozygous positions. All of them are clearheterozygous positions with an almost equal coverage of two bases.
What happened here?
Select DQB1-E02 in the section ROI again, to see the sequences for exon 2. If you scroll throughexon 2 using the horizontal slider you can see, that there are two reads sequences (copy number42/35) that show several “base calling errors” (bases are marked orange). Those two reads aremarked with a “*”, showing that they have a perfect match to an allele. Moreover the copy number iscolored green. The “main” reads that were called as allele have a copy number of 465/412, the copynumber is colored red.
→ the different coloring in copy number show, that the reads belong to different alleles!
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Actually the second allele (with the copy number of 42/35) is under-represented compared to the firstallele (with a copy number of 465/412). Therefore it was not called.The matching allele(s) for a read can be viewed with the context menu 'show alleles'. Right-click theread with the *42 or *35 in front to open the context menu:
Select show alleles to open a list of alleles sorted by mismatching positions:The reads show a perfect match to the alleles listed in the first line of this dialogue (0 mismatches):
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There are two possibilities to set the reads as second allele:
1. Set the reads as HT2 manuallyRight-click on one of the reads sequences (copy number 42 or 35) and select set fragment as HT2from the context menu.
The second haplotype is now called and a result appears in the Result section automatically:
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2. Change the basecalling coverage
To test the second option, remove your first edit. Therefore right-click on DQB1-E02 in section ROIsand select editing > recalculate from the context menu.
The ROI is recalculated, all manual edits are removed. Therefore DQB1-E02 is set homozygousagain.Now right click on DQB1-E02 in section ROIs again, and select editing > settings... from the contextmenu. The following dialogue opens:
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This dialogue shows Settings, that can be adapted for the complete file (in Operation Run or Joining)or for the selected ROI (in operation Sequence, as shown). The first setting – Basecalling coverage –decides if a second allele is called or not. With the value 25 %, a second allele is called, in case thecoverage of a second base reaches at least 25 % of the coverage of the highest base (is set as 100%).In our example the second base reaches only around 15 % of the first base. Therefore the secondallele is not called. Change the Basecalling coverage to 15 % in the dialogue Settings and press[OK]. The result is recalculated automatically. The second allele is now called and we get the sameresult as before.
10.6 Validation
After checking all results the order can be technically and medically validated. Technical and medicalvalidation show, that all results have been confirmed. After medical validation all files are locked andno further edits can be done.
To validate the complete order at one time, press the buttons [T.V.] and [M.V.] shown in the dialoguepart Functions for technical and medical validation respectively:
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After technical validation there is the entry V in the column Condition of Files, Genes and Locations.After medical validation there is the entry S in the column Condition.
Moreover single files, genes, ROIs and location can be validated separately. Therefore select theentry to validate and press [T.V.] or [M.V.] in the corresponding section:
Technical and medical validation can be removed by clicking [T.V.] and [M.V.] again!
10.7 Print a report or export the result
Now a sequencing report can be printed. This lists the results for all analysed genes. Moreover thesequenced region, average coverage, perfect match coverage and positions with low coverage aredisplayed for each exon.
Press [print...] in the dialogue part Functions to open the Print/Preview.
Here you can choose what to print:
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Mark the genes to print. The options select all, select none and select all not printed helpschecking the genes.
In the field report select the report form SeqNext-HLA Report short.
Open a print preview using [Preview...] and use [Print] to print your results.
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Optionally the result can also be exported as a txt-file. Therefore go to section Files and selectexport > result in the context menu:
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10.8 Edit bases in the electropherogram/sequences
For gene DQB1 we already showed:
How to set a read as haplotype
How to change the Basecalling coverage
Sequences can also be edited manually in the electropherogram/sequences. Remove the validationfirst, otherwise you will not be able to do edits. Therefore click [TV] and [MV] in the section Functionsagain.
In case there are mismatching positions to a proposed allele combination, you can change the basein the sequences manually. Therefore you can jump to the position in the sequences using thejumper mism. in the dialogue part Show (there are no mismatching positions present in our demodata).
Right-click a base in the result file sequence or in the combined sequence to open a context menu.
Just select the desired base. In case the context menu of the combined sequence is used, the editsare done for both haplotypes simultaneously.Moreover a sequence can be ignored to left or to right using this context menu.
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If the base of the consensus sequence (first line, highlighted green) should be set in the filesequences or combined sequence, just “left-click” the base. This is only possible for constant basepositions (A,T,G,C)!
Edited base positions show a black mark below the edited base.
You can jump to edited positions in the sequences using the jumper edited in the Show window.
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11 How to analyse the order againYou have two possibilities to repeat your analysis. You can either use the Recalculate-Function toremove all manual edits or delete the order and load the file again.
11.1 Recalculate-Function
Using this function already loaded orders are recalculated. This means manual edits are removedand changed settings (such as an updated software version, updated HLA database or a modifiedlis.ini file) are automatically used to recalculate the result.
To recalculate do the following:
Medical validated orders are locked and can not be recalculated. In case the order ismedically validated, remove the validation by pressing [MV] in the dialogue Functions (in theoperation Sequence).
Then switch to the operation Joining. List the file by pressing “+” in the column Order. Right-click the file and select unjoin from the context menu.
The file is moved to the Upper Table. Right-click it and select recalculate from the contextmenu.
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After the analysis is finished press [Autojoin] in the dialogue part Select Orders. The file will bejoined to the order in the Lower Table automatically.
11.2 Delete the order and load the file again
With this option the order is deleted.
To delete the order select the category LIS and the operation Orderlist. In case no order islisted, remove the mark in Date and press [Search].
Then right-click the order and select the context menu item delete order.
Now you can load the file again using the operation Run as described in chapter 8.
For a detailed description have a look at the Manual SEQUENCE Pilot – module SeqNext-HLA.
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