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FAQs SEQUENCE Pilot

General FAQs

Which hardware requirements need my computer to install SEQUENCE Pilot?
Please have a look to Products / Hardware Requirements of this website.

Why do I get the message „The application has failed to start because its side-by-side configuration is incorrect. Please see the application event log or use the command-line sxstrace.exe tool for more detail“?
Your server / client PC is missing some Microsoft C++ redistributale libraries. Please use the following link to install them:

How do I log in to SEQUENCE Pilot the first time after the installation?
Log in with user jsi and no password. Now go to Administration / Users, set up a new user and password and activate the desired user rights. After log out, the user jsi is no longer available.

You forgot your user name and / or password?
If there was only one user defined in Administration / Users, please go to SeqPilot\DB and deleted the files user.dat and user.idx. Now start SEQUENCE Pilot again and log in with user jsi and no password. Go to Administration / User, set up a new user and password and activate the desired user rights. After log out, the user jsi is no longer available.
If several users are defined in Administration / Users, please ask another user with corresponding rights to log in to SEQUENCE Pilot. Go to Administration / Users and select your user shortcut and enter a new password. Even if you don't remember your user shortcut, please set up a new user and password and activate the desired user rights.
If none of the above solutions apply, please contact our support.

Why do I get the message „connection to server lost...“ when I log in to SEQUENCE Pilot?
This message occurs when the communication between server and client PC is interrupted. Please check, that

  • in case of a client/server installation the corresponding services on server side are still running.
  • the SeqPilot folder of your installation is excluded from the control of your anti-virus software (server & client PC).
  • all ports used by SEQUENCE Pilot (7201, 7401, 7301, 7302) are released in the firewall (inbound & outbound roules, server & client PC).
  • the SeqPilot folder of your installation is shared in the network and all users have full reading and writing rights.

Why can't I edit master files / load gene files?
Please check via Administration / Users that you have all corresponding rights (edit masterfiles).

Why I get the message "... locked by user ...! Access denied." when accessing a certain master file dialogue?
Master file dialogues only can be accessed by one user at the time. This is for security reasons. The user mentioned in the message, currently is using the mentioned master file dialogue. This user needs to close the dialogue first before you can access it.

Why can't I find my patient order?
Please go to the operation Joining of the corresponding SEQUENCE Pilot module and make sure, that you select the correct date range, project (all projects) and state of the order (active or archived).

Why do I get the message „Patient locked! Readonly mode.“ when opening a patient order in the analysis view (Sequence / MLPA)?
Please make sure that no other user is accessing the same patient order. This is for security reasons.
In case of the SEQUENCE Pilot modules SEQNEXT & SEQNEXT-HLA this message also can occur in case a new analysis already is finishid, but some background processes are still accessing the order. Please go back in operation Joining then and try again in a few minutes.

How can I delete a file / run?
To delete a file / run, go to operation Joining of the corresponding SEQUENCE Pilot module, right click the file(s) / run(s) and choose item unjoin from the context menu. The file(s) / run(s) will be moved to the upper table of Joining from wich it can now be deleted (right click and choose context menu item delete).

How can I delete an order?
To delete an order (and all corresponding files / runs), go to operation LIS / Orderlist, select the order and choose context menu item delete.

Why does my sample remain in the upper joining list?
Please check column Hint for corresponding messages:

  • no DNA number (all modules): DNA number not detected in SNC (sample naming conventions); add DNA number via context menu item "settings" (single selection) or "edit DNA No..." (multi selection) and then press button [Autojoin]

  • no mix found (MLPA): mix not detected in SNC (sample naming conventions) and / or not defined in operation Mixes [master file]; if corresponding mix description exists add via context menu item settings... and then press button [Autojoin]; if mix description doesn't exist, please set up first via Mixes [master file] and then add via context menu item settings... and press button [Autojoin]

  • Order X is canceled! (all modules): corresponding order is canceled; activate corresponding order and then press button [Autojoin]

  • Order X is medically validated! (all modules): corresponding order is medically validated; remove medical valication via button [MV] (Sequence / MLPA) and press button [Autojoin]

  • Order X is archived! (all modules): corresponding order is archived; reactivate order via Sequence / MLPA and then press button [Autojoin] in Joining

  • Order X is extracted! (all modules): corresponding order is extracted; re-extract order via LIS / Orderlist, reactivate order via Sequence / MLPA and then press button [Autojoin] in Joining

  • SEQNEXT

     

    What do I need to pay attention to when defining a new panel?
    Please make sure that the columns in dialogue File Description are correctly assigned to the columns in your bed / manifest file.
    For sequence capture, enrichment and Haloplex panels please deactivate option build amplicons in dialogue File Description.
    For PCR-based panels (not Haloplex) please activate option build amplicons in dialogue File Description. Furthermore please define if the genomic coordinates still include the PCR primers or not. In case the the primers are still included please make sure that the corresponding fields / options in dialogue File Description are completed / checked.

    What do I need to pay attention to when running an analysis for data of a PCR-based panel?
    Please make sure that in the settings for your analysis the corresponding option is chosen, depending if the reads coming from the sequencer still include the primer sequences or not.

    Why did the import of the result files stop?
    Please check,

    • that the corresponding services are still running.
    • that the formate of the result files is correct (fastq, fastq.gz or bam).
    • if there are any indicating errors in SEQUENCE Pilot menu Help / Logging... or in txt files from SeqPilot\messages.
    • that enough space is left over on the harddrive, where SEQUENCE Pilot is installed.
    • that you still have enough credits in case of a chip-card version.

     

     

    Why do I see too many variants / artefacts in Sequence?

    • For big panels you can expect many variants.
    • The settings are too low for your data (Min % Coverage / Distinct/Other % Coverage).
    • You only work with mode "combined" in the settings, which is less stringent than using mode "per dir.". But please keep in mind, that using mode "per dir." is recommended only, if all base positions are covered by fwd and rev reads. If this is not the case, please use in combination with min. one of the options below setting [3] Single/double direction analysis, otherwise you risk to lose true variants.
    • Low quality score used in the settings. Therefore variants with low quality might be listed as well.
    • Possible pseudogene issues. In order to avoid these problems for small panels please make sure that a homology search has been done for all the corresponding ROIs (ROI [master file]). For big panels please activate option "Genome mapping" in the settings of your run.

    What is the quality score and how does it work?
    The quality score used by SEQNEXT is the phred score called by the sequencer for each single base position of a read.
    Based on the run settings, the user can decide, what is the minimum phred score still accepted as good quality. Bases in a reads with a quality score below this value will be ignored.
    If too many bases of one read are ignored based on a low quality score, the user can decide to ignore the whole read.

     

  • SEQPATIENT

     

    Where and how can I download single reference genes?
    Single reference genes directly can be downloaded from Ensembl and NCBI. Please do the following:

    • Choose menu SEQPATIENT / Gene Admin....
    • Click on button [Extras ->] / new gene....
    • Choose the desired website (Ensembl or GenBank)
    • Enter the name of the gene (upper case letters only) in the field "Gene".
    • Press button [browse...] and then complete the field "Gene ID" and the fields in section "Web" with the corresponding information.
    • Press button [Save].
    • In the following dialoge "Change Gene" please make sure to only keep activated the desired transcript. Please check that the exon numbering is correct and adapt it if necessary by pressing button [Renumber exon names].
    • If a genome (e.g. hg19) has been installed, please map the gene file against your genome using button [Extras ->] / map to genome.

    How can I change a transcript/exon, numbering/start and/or stop codon for an existing reference gene?
    Please do the following:

    • Go to menu SEQPATIENT / Gene Admin....
    • Click on button [Extras ->] / change gene....
    • Activate / deactivate the desired transcipt(s).
    • Check the exon numbering; change manually or automatically via button [Renumber exon names] if necessary.
    • Change the positions corresponding to the start and/or stop codon if necessary.
    • Press button [Save].

    In case a genome  (e.g. hg19) was used as reference you can change the transcripts of Amp. Modules (Amp. Modules [master file]) and Seq. Primer (Seq. Primer [master file]) by right clicking and selecting the context menu item [change Transcript ID...].

    Why do I see in Gene Admin on tab Error the hint "Mismatch in Translation of cDNA (Isoform X)"?
    Each reference gene downloaded from Ensembl or NCBI comes with its own Translation. SEQUENCE Pilot calculates its own translation based on the cDNA sequence provided by these websites as well. If this translation differs to the amino acid sequence of the downloaded reference gene, you will see an error. However, SEQUENCE Pilot will always use its own calcuated translation and not the one downloaded with the reference gene.

    Why do I have to define Seq. Primers (Seq. Primer [master file])?
    Correctly defined Seq. Primers:

    • mentioned in the sample naming conventions of your result files, increase the speed of the raw data import and ensure a correct alignment of the imported result files to the corresponding reference.
    • guarantee for a correct trimming of your sequences after the alignment and therefore reduce the number of editings which have to be done by the user.
    • are necessary to build up a peak area statistics over all previously analysed sequences of a certein chemistry (gene, exon, sequencing direction, Seq. Primer). This will help to alert you to possible unrecognised heterozygous variants that are due to a very low background, such as mosaics or somatic variants.

     

     

    Why did the import of the result files stop (button Seq in the lower right of the screen stays red)?
    Please check,

    • that the corresponding services are still running.
    • that the format of the result files is correct (ab1 or scf).
    • if there are any indicating errors in SEQUENCE Pilot menu Help / Logging... or in txt files from SeqPilot\messages.
    • that enough space is left over on the harddrive, where SEQUENCE Pilot is installed.

    Why can‘t I find my patient files just loaded?
    Please check:

    • in menu Help / Logging... for the message „Result file already loaded to order, order:...“. By default the same patient file can't be loaded twice in the software using the same sample name.
    • SeqResultfiles.txt file in SeqPilot\messages for messages "Data missing number 9" which means no base calling was done by the sequencer.

    What is the difference between reanalyse and recalculate?
    Reanalyse updates the variation table.
    Recalculate removes all manual editings done by the user except ignore to the left/right.

    What is the meaning of the colour for the statistic warning in operation Sequence?
    orange (s): dissimilarity score 20-40
    red (S): dissimilarity score >40

    What is the dissimilarity score?
    Value representing the difference between the calculated relative statistic peak area over all previously analysed samples (already archived / same chemistry) of a certain base position and the current patient sample.

     

  • MLPA®

     

    Where do I find the required mix description for my kit?
    Please go to Download / MLPA® - MRC Holland Kits of this website and download the requested mix description. If the desired mix description or version is not available, please contact our SEQUENCE Pilot support team.

    Why did the import of the result files stop (button MLPA in the lower right of the screen stays red)?
    Please check,

    • that the corresponding services are still running.
    • that the formate of the result files is correct (fsa or txt).
    • if there are any indicating errors in SEQUENCE Pilot menu Help / Logging... or in txt files from SeqPilot\messages.
    • that enough space is left over on the harddrive, where SEQUENCE Pilot is installed.

    Why can't I find my controls in operation Joining?
    If the raw data files have been named correctly (sample naming conventions), the controls are listed in their own operation Controllist.

    Why can't I find my patient files just loaded?
    Please check:

    • in menu Help / Logging... for the message „Result file already loaded to order, order:...“. By default the same patient file can't be loaded twice in the software using the same sample name.
    • MlpaResultfiles.txt file in SeqPilot\messages for messages "No sizing curve found" (sizing dye missing in result file) and "No sizing file found" (no Sizing.txt deposit in SeqPilot\bin and / or Thresholds defined in operation Thresholds [master file]).

     

     

    Why can't I see any peak information?
    Please check:

    • that you use the correct Sizing.txt (SeqPilot\bin).
    • that the values in Thresholds [master file] are corresponding to the used sizing standard.

    What is the difference between reanalyse and recalculate?
    Reanalyse updates the table above the electropherogram / bar diagram.
    Recalculate resets all changes (e.g. peak assignments) done by the user.

     

  • SEQNEXT-HLA

     

    Where can I check which HLA Database is used for analysis?
    In SEQUENCE Pilot the used HLA database can be viewed and changed in menu SeqHLA / HLA DB Admin….

    How can I use and update NMDP codes?
    NMDP codes can be generated for every result. To activate the NMDP code generation create the new subdirectory NMDPFiles in directory SeqPilot of your installation.
    Within this directory the current NMDP list (numer.v3.txt) has to be deposited:

    Please note, that you have to update the NMDP list regularly. Therefore please use the procedure as descibed above.

    How are G and P suffixes calculated in SEQUENCE Pilot?
    The G and P suffixes are used to simplify the results in operation Sequence dialogue Result. By default SEQUENCE Pilot calculates the G and P suffixes automatically.

    • “G” stands for alleles which share identical nucleotide sequences for the exons encoding the peptide binding domain exon 2 and 3 for HLA class I and exon 2 only for HLA class II alleles have to be identical in nucleotide sequence, then a G Suffix is generated.
    • “P” stands for alleles, which encode for identical peptide binding domains: exon 2 and 3 for HLA class I and exon 2 only for HLA class II alleles have to be identical in protein sequence, then a P Suffix is generated.

    Alternatively files from IMGT can be downloaded where all G- and P-Groups are defined. In case you use this option, you have to update the files regularly.
    The reporting of ambiguous HLA allele typing only works, if you use maximum resolution.

    How can I use the G and P suffixes from IMGT files instead of the calculated ones from SEQUENCE Pilot?
    Files from IMGT can be downloaded where all G- and P-Groups are defined. In case you use this option, you have to update the files regularly (with every database update).
    To use the nomenclature from a file, please do the following:

    • Go to http://hla.alleles.org/wmda/.
    • Download the files hla nom g.txt and hla nom p.txt.
    • Save the files in the folder of the respective database in folder Settings\HLANom of your SEQUENCE Pilot installation (e.g. the files for HLA DB 3.31 must be saved in folder Settings\HLANom\3.24.0).
    • Open the lis.ini file, located in SeqPilot\bin your SEQUENCE Pilot installation with a text editor.
    • Go to section [SeqPilot] and deposit the entry HLANomFileAllele=yes in a new line.
    • After restarting SEQUENCE Pilot the G and P suffixes for all newly started runs will be used from this file.

    How can I inactivate G and P suffixes?
    In case you do not want to use G and P suffixes at all, enter the new line Enter ReportAmbigousAllele=no in the section [SeqNextHLA] of your in the lis.ini file, located in the SeqPilot\bin of your SEQUENCE Pilot installation.

    Where can I find the result?
    In operation Sequence, section Result. Here the result, calculated for the selected gene in the dialogue part Genes is listed.
    The first line shows, whether it is a heterozygous or a hemizygous result and on which haplotypes the result calculation is based on. The second line shows the ambiguities, that is all found allele combinations. G and P suffixes are used automatically. The last line shows, which HLA database version was used for the result calculation. Optionally the result can be listed as NMDP code.
    If there are still mismatches to an allele there will be the entry “no results possible”. In that case you have to check the mismatching positions in the electropherogram. When all mismatches are removed the result will appear automatically.

    What does the Matching table display?
    This table shows all possible hemizygous results/allele combinations, sorted by their probability.
    If you select an entry within this table it is highlighted green. This allele or allele combination is displayed in the electropherogram then. If there are mismatching positions for that allele or allele combination, the affected result files are displayed automatically and the cursor is positioned to the first mismatch.
    All allele combinations with no mismatches are listed as result in section Result.

    Is it possible to set a preferred result in case ambiguities are present?
    Yes, this is possible in the Matching table, using the context menu item prefer alleles.

    Is it possible to compare the sequences of my resulting allele sequences?
    Yes, this is possible in the Matching table using the context menu item show differences….

    Can new alleles be called as well?
    Yes, if a new allele is present one mismatch remains to all known allele combinations from the IMGT HLA database. The new allele can be set manually in operation Sequence section Genes using the context menu new allele…. Afterwards the result “new allele most similar to…” is shown in section Result.

    How can I export the results?
    The results shown in the Results window in operation Sequence can be exported as txt file. Therefore, please use the context menu export / result... in section Genes.
    Moreover a report can be printed or saved as pdf. Therefore use the button [Print] in dialogue part Functions.
    Results can also be exported automatically to any LIMS systems after analysis, with our TALKMASTER interface.

     

     

    Which files can be analysed?
    Fastq and fastq.gz files from all common sequencing platforms from Illumina, IonTorrent, MinION and PacBio can be analysed.

    Which one time procedures need to be set up before I can start analysis?
    The HLA database needs to be installed and ROIs need to be defined in operation ROI [master file].

    How can I deposit the DNA number in the file name?
    By default all signs in the file name before the first "_" are used as DNA number. In case this is not the DNA number of your file name, please contact our SEQUENCE Pilot support team. We can adapt our software, so that the DNA number is read correctly.

    Is it possible to automatically start a Run?
    Yes, with the Autorun option. For further details, please contact our SEQUENCE Pilot support team.

    How can I make analysis faster?

    • Switch off Second Alignment: The analysis will be faster, by adding the entry SecondLevelCalcAlignmentScore=no in the file lis.ini (SeqPilot\bin of your SEQUENCE Pilot installation) in section [SeqNextHLA]. When this entry is set a second alignment for reads which can be mapped to more than one locus is not done any more. But please note, that this might lead to single reads getting mapped to the wrong locus (especially for loci B/C or DRB pseudogenes) - depending on the allele combinations.
    • Activate muliple processing cores: To make analysis faster multiple processing cores can be used to compute the result files of a run. Several worker processes can be started, which process several files in parallel (in case the Run is started for several files, each worker processes one file). The number of worker processes should be related to the number of cores available on the server / client PC. For further details please contact our SEQUENCE Pilot support team.
    • Analysis is slow because the Coverage is very high: The following settings in operation Run, utton [Settings], tab Expert Settings can be adapted:
      • Load file: For very big files, only part of the reads can be loaded and analysed. This makes analysis much quicker. Here the percentage of reads that should be loaded and used for analysis can be entered. 100% means, that all reads are loaded.
      • Load reads: Here an absolute minimum number of reads that must be loaded can be entered. This setting can overrule the setting Load file mentioned above. In case the percentage value is too low to load the number of  reads defined here, more reads are loaded until the number of reads defined.

    Analysis is slowing down my PC. What can I do?
    By default the importer process uses the maximum number of cores. Therefore the computer might be very slow during data analysis, which might be a problem in case other programs are used as well.
    The number of cores used during import can be restricted with the entry MaxImporterThreadCores=1 in section [SeqNextHLA] the lis.ini file (SeqPilot\bin of your SEQUENCE Pilot installation). The number behind this entry is the number of cores used for import.

    Can I load data for the same patient several times?
    This should only be done in case the fastq files contain data for different loci. In case you want to load replicates for one patient of the same loci, please contact our SEQUENCE Pilot support team to discuss all possible options.

    Are introns analysed as well?
    Intron sequences are displayed but not used for result calculation. They are only used to exclude known null alleles.

    I have two possible results listed. Is it possible to exclude one of them?
    Yes, phasing can optionally be checked. If possible, allele combinations are then removed from the result based on the phasing. To use this option, please activate the setting "Check phasing" on tab Expert Settings.

    Can I exclude null alleles?
    For result calculation null alleles which show mismatches/insertions/deletion in the introns are called or excluded as result automatically. This function is active by default, but it can be switched off. To switch it off the setting "Check null allele intron" on tab Expert Settings has to be deactivated.

    How can I change the resolution?
    By default 3 field resolution (maximum resolution) is used.
    In case another resolution should always be used (2 field = 4 digits or 1 field= 2 digits), this can be set in the lis.ini file, located in folder SeqPilot\bin of your SEQUENCE Pilot installation. To change to 2 digits or 4 digits, please make the entry DefaultResolution=2 digits or DefaultResolution=4 digits in section [SeqNextHLA].
    For already analysed files open the patient sample in operation Sequence, right click a locus in section Genes and select item change resolution and positions… from the context menu.

    What can I check in case a result is not called automatically?
    Please contact our SEQUENCE Pilot support team.

    What can I do in case background is present for one or several loci?
    It needs to be checked to which locus the background belongs to. In case it belongs to a locus which is not set up as ROI (e.g. locus E, G, Y…), ROIs for this locus need to be set up in ROI [master file] and included in the corresponding ROI group (ROI Group [master file]). In case you do not want to call results for these ROIs, you can set them to analysis mode “only mapping” in ROI [master file].

    A result is called but mismatches are shown for the locus. What does this mean?
    A result is always called as soon as there are no mismatches in exon areas. In intron areas mismatches are not regarded for result calculation.

    In operation Sequence allele sequences from the IMGT HLA database are marked blue instead of green. What does this mean?
    In case an intron sequence is unknown for a haplotype, it is checked if the intron sequence of another group member is known.
    If yes, the sequence of the group member is listed. The haplotype sequence for the intron is then marked blue instead of green. You can see which allele the intron sequence belongs to when you move your mouse to the blue intron sequence (tool tip). This feature can be switched off using the entry Show2DigitsIntronAlleles=no in section [SeqPilot] of the lis.ini file (SeqPilot\bin of your SEQUENCE Pilot installation).
    If no, no intron sequence is shown.

    In operation Sequence the number in front of the read sequences can be highlighted in different colors. Moreover a * can be in front. What does this mean?
    Identical reads are listed only once. The copy number is listed in front of each read. Moreover all reads that have a perfect match to an allele can optionally be marked with a * in front of the number.
    The * and the copy number in front of each read can be colored. Identical colors for two or more reads mean, that the reads have a perfect match to the same allele(s) (context menu item "show alleles lists identical matches").
    Please note, that black is not regarded as a color. Black */copy numbers mean that no other reads with the same perfect matches are present.

    Can I get a quick overview about coverages?
    Yes, therefore please press the button [Summary] in operation Sequence. The table can be exported as txt-file using the corresponding context menu item.

     

  • SEQHLA

     

    Where can I check which HLA Database is used for analysis?
    In SEQUENCE Pilot the used HLA database can be viewed and changed in menu SeqHLA / HLA DB Admin….

    How can I use and update NMDP codes?
    NMDP codes can be generated for every result. To activate the NMDP code generation create the new subdirectory NMDPFiles in directory SeqPilot of your installation.
    Within this directory the current NMDP list (numer.v3.txt) has to be deposited:

    Please note, that you have to update the NMDP list regularly. Therefore please use the procedure as descibed above.

    How are G and P suffixes calculated in SEQUENCE Pilot?
    The G and P suffixes are used to simplify the results in operation Sequence dialogue Result. By default SEQUENCE Pilot calculates the G and P suffixes automatically.

    • “G” stands for alleles which share identical nucleotide sequences for the exons encoding the peptide binding domain exon 2 and 3 for HLA class I and exon 2 only for HLA class II alleles have to be identical in nucleotide sequence, then a G Suffix is generated.
    • “P” stands for alleles, which encode for identical peptide binding domains: exon 2 and 3 for HLA class I and exon 2 only for HLA class II alleles have to be identical in protein sequence, then a P Suffix is generated.

    Alternatively files from IMGT can be downloaded where all G- and P-Groups are defined. In case you use this option, you have to update the files regularly.
    The reporting of ambiguous HLA allele typing only works, if you use maximum resolution.

    How can I use the G and P suffixes from IMGT files instead of the calculated ones from SEQUENCE Pilot?
    Files from IMGT can be downloaded where all G- and P-Groups are defined. In case you use this option, you have to update the files regularly (with every database update).
    To use the nomenclature from a file, please do the following:

    • Go to http://hla.alleles.org/wmda/.
    • Download the files hla nom g.txt and hla nom p.txt.
    • Save the files in the folder of the respective database in folder Settings\HLANom of your SEQUENCE Pilot installation (e.g. the files for HLA DB 3.31 must be saved in folder Settings\HLANom\3.24.0).
    • Open the lis.ini file, located in SeqPilot\bin your SEQUENCE Pilot installation with a text editor.
    • Go to section [SeqPilot] and deposit the entry HLANomFileAllele=yes in a new line.
    • After restarting SEQUENCE Pilot the G and P suffixes for all newly started runs will be used from this file.

    How can I inactivate G and P suffixes?
    In case you do not want to use G and P suffixes at all, enter the new line Enter ReportAmbigousAllele=no in the section [SeqPilot] of your in the lis.ini file, located in the SeqPilot\bin of your SEQUENCE Pilot installation.

    Where can I find the result?
    In operation Sequence, section Result. Here the result, calculated for the selected gene in the dialogue part Genes is listed.
    The first line shows, whether it is a heterozygous or a hemizygous result and on which haplotypes the result calculation is based on. The second line shows the ambiguities, that is all found allele combinations. G and P suffixes are used automatically. The last line shows, which HLA database version was used for the result calculation. Optionally the result can be listed as NMDP code.
    If there are still mismatches to an allele there will be the entry “no results possible”. In that case you have to check the mismatching positions in the electropherogram. When all mismatches are removed the result will appear automatically.

    What does the Matching table display?
    This table shows all possible hemizygous results/allele combinations, sorted by their probability.
    If you select an entry within this table it is highlighted green. This allele or allele combination is displayed in the electropherogram then. If there are mismatching positions for that allele or allele combination, the affected result files are displayed automatically and the cursor is positioned to the first mismatch.
    All allele combinations with no mismatches are listed as result in section Result.

    Is it possible to set a preferred result in case ambiguities are present?
    Yes, this is possible in the Matching table, using the context menu item prefer alleles.

    Is it possible to compare the sequences of my resulting allele sequences?
    Yes, this is possible in the Matching table using the context menu item show differences….

    Can new alleles be called as well?
    Yes, if a new allele is present one mismatch remains to all known allele combinations from the IMGT HLA database. The new allele can be set manually in operation Sequence section Genes using the context menu new allele…. Afterwards the result “new allele most similar to…” is shown in section Result.

    How can I export the results?
    The results shown in the Results window in operation Sequence can be exported as txt file. Therefore, please use the context menu export / result... in section Genes.
    Moreover a report can be printed or saved as pdf. Therefore use the button [Print] in dialogue part Functions.
    Results can also be exported automatically to any LIMS systems after analysis, with our TALKMASTER interface.

     

     

    How can I load ProtransKits?
    Under Download/SEQHLA - ProtransKits of this website you can find an installation file including pre-defined amplification modules and sequencing primers for ProtransKits.

    Which files can be analysed?
    Files from ABI sequencers should have the extension .ab1 and files from Beckman sequencers .scf. If you use sequencers from other manufacturers like MegaBase, please contact our SEQUENCE Pilot support team.

    Why did the import of the result files stopp (button Seq in the lower right of the screen stays red)?
    Please check,

    • that the corresponding services are still running.
    • that the formate of the result files is correct (ab1 or scf).
    • if there are any indicating errors in SEQUENCE Pilot menu Help / Logging... or in txt files from SeqPilot\messages.
    • that enough space is left over on the harddrive, where SEQUENCE Pilot is installed.

    Why can‘t I find my patient files just loaded?
    Please check:

    • in menu Help / Logging... for the message „Result file already loaded to order, order:...“. By default the same patient file can't be loaded twice in the software using the same sample name.
    • SeqResultfiles.txt file in SeqPilot\messages for messages "Data missing number 9" which means no base calling was done by the sequencer.

    Are introns analysed as well?
    Intron sequences are displayed but not used for result calculation.

    Can I exclude Null alleles?
    Null alleles showing base changes in intron areas are not called or excluded automatically. But the intron areas are displayed in SEQHLA. Moreover in case a splice site mutation is present, there will be the warning X present in column Warnings, in table Positions / Resultfiles in operation Sequence. Therefore the user can check the intronic positions and set or exclude certain null alleles in the Matching table manually using context menu item "exclude alleles...".

    How can I change the resolution?
    By default 3 field resolution (maximum resolution) is used.
    In case another resolution should always be used (2 field = 4 digits or 1 field= 2 digits), this can be set in the lis.ini file, located in folder SeqPilot\bin of your SEQUENCE Pilot installation. To change to 2 digits or 4 digits, please make the entry DefaultResolution=2 digits or DefaultResolution=4 digits in section [SEQPILOT].
    For already analysed files open the patient sample in operation Sequence, right click a locus in section Genes and select item change resolution and positions… from the context menu.
    For future analysis, it needs to be set in LIS / Procedures. Procedures are generated automatically by SEQUENCE Pilot when loading a result file and no corresponding procedure exists. To change the resolution for a locus:

    • Select the procedure in field Name.
    • Select the desired resolution in dialogue part HLA. The following resolutions are available:
      • max resolution = 3 field (default option)
      • 4 digits = 2 field (shortens the results to 4 digits)
      • 2 digits = 1 field (shortens the results to 2 digits)

    Furthermore all constant positions, that is all positions in the consensus sequence, which show an A, C, G or T, can be ignored here as well, by checking the corresponding setting. In operation Sequence they are highlighted grey then. These bases can be edited, but done changes don't effect the entries of the Matching table.

     

What do I need to pay attention to when defining a new panel?
Please make sure that the columns in dialogue File Description are correctly assigned to the columns in your bed / manifest file.
For sequence capture, enrichment and Haloplex panels please deactivate option build amplicons in dialogue File Description.
For PCR-based panels (not Haloplex) please activate option build amplicons in dialogue File Description. Furthermore please define if the genomic coordinates still include the PCR primers or not. In case the the primers are still included please make sure that the corresponding fields / options in dialogue File Description are completed / checked.

What do I need to pay attention to when running an analysis for data of a PCR-based panel?
Please make sure that in the settings for your analysis the corresponding option is chosen, depending if the reads coming from the sequencer still include the primer sequences or not.

Why did the import of the result files stop?
Please check,

  • that the corresponding services are still running.
  • that the formate of the result files is correct (fastq, fastq.gz or bam).
  • if there are any indicating errors in SEQUENCE Pilot menu Help / Logging... or in txt files from SeqPilot\messages.
  • that enough space is left over on the harddrive, where SEQUENCE Pilot is installed.
  • that you still have enough credits in case of a chip-card version.

Why do I see too many variants / artefacts in Sequence?

  • For big panels you can expect many variants.
  • The settings are too low for your data (Min % Coverage / Distinct/Other % Coverage).
  • You only work with mode "combined" in the settings, which is less stringent than using mode "per dir.". But please keep in mind, that using mode "per dir." is recommended only, if all base positions are covered by fwd and rev reads. If this is not the case, please use in combination with min. one of the options below setting [3] Single/double direction analysis, otherwise you risk to lose true variants.
  • Low quality score used in the settings. Therefore variants with low quality might be listed as well.
  • Possible pseudogene issues. In order to avoid these problems for small panels please make sure that a homology search has been done for all the corresponding ROIs (ROI [master file]). For big panels please activate option "Genome mapping" in the settings of your run.

What is the quality score and how does it work?
The quality score used by SEQNEXT is the phred score called by the sequencer for each single base position of a read.
Based on the run settings, the user can decide, what is the minimum phred score still accepted as good quality. Bases in a reads with a quality score below this value will be ignored.
If too many bases of one read are ignored based on a low quality score, the user can decide to ignore the whole read.

Where and how can I download single reference genes?
Single reference genes directly can be downloaded from Ensembl and NCBI. Please do the following:

  • Choose menu SEQPATIENT / Gene Admin....
  • Click on button [Extras ->] / new gene....
  • Choose the desired website (Ensembl or GenBank)
  • Enter the name of the gene (upper case letters only) in the field "Gene".
  • Press button [browse...] and then complete the field "Gene ID" and the fields in section "Web" with the corresponding information.
  • Press button [Save].
  • In the following dialoge "Change Gene" please make sure to only keep activated the desired transcript. Please check that the exon numbering is correct and adapt it if necessary by pressing button [Renumber exon names].
  • If a genome (e.g. hg19) has been installed, please map the gene file against your genome using button [Extras ->] / map to genome.

How can I change a transcript/exon, numbering/start and/or stop codon for an existing reference gene?
Please do the following:

  • Go to menu SEQPATIENT / Gene Admin....
  • Click on button [Extras ->] / change gene....
  • Activate / deactivate the desired transcipt(s).
  • Check the exon numbering; change manually or automatically via button [Renumber exon names] if necessary.
  • Change the positions corresponding to the start and/or stop codon if necessary.
  • Press button [Save].

In case a genome  (e.g. hg19) was used as reference you can change the transcripts of Amp. Modules (Amp. Modules [master file]) and Seq. Primer (Seq. Primer [master file]) by right clicking and selecting the context menu item [change Transcript ID...].

Why do I see in Gene Admin on tab Error the hint "Mismatch in Translation of cDNA (Isoform X)"?
Each reference gene downloaded from Ensembl or NCBI comes with its own Translation. SEQUENCE Pilot calculates its own translation based on the cDNA sequence provided by these websites as well. If this translation differs to the amino acid sequence of the downloaded reference gene, you will see an error. However, SEQUENCE Pilot will always use its own calcuated translation and not the one downloaded with the reference gene.

Why do I have to define Seq. Primers (Seq. Primer [master file])?
Correctly defined Seq. Primers:

  • mentioned in the sample naming conventions of your result files, increase the speed of the raw data import and ensure a correct alignment of the imported result files to the corresponding reference.
  • guarantee for a correct trimming of your sequences after the alignment and therefore reduce the number of editings which have to be done by the user.
  • are necessary to build up a peak area statistics over all previously analysed sequences of a certein chemistry (gene, exon, sequencing direction, Seq. Primer). This will help to alert you to possible unrecognised heterozygous variants that are due to a very low background, such as mosaics or somatic variants.

Why did the import of the result files stop (button Seq in the lower right of the screen stays red)?
Please check,

  • that the corresponding services are still running.
  • that the format of the result files is correct (ab1 or scf).
  • if there are any indicating errors in SEQUENCE Pilot menu Help / Logging... or in txt files from SeqPilot\messages.
  • that enough space is left over on the harddrive, where SEQUENCE Pilot is installed.

Why can‘t I find my patient files just loaded?
Please check:

  • in menu Help / Logging... for the message „Result file already loaded to order, order:...“. By default the same patient file can't be loaded twice in the software using the same sample name.
  • SeqResultfiles.txt file in SeqPilot\messages for messages "Data missing number 9" which means no base calling was done by the sequencer.

What is the difference between reanalyse and recalculate?
Reanalyse updates the variation table.
Recalculate removes all manual editings done by the user except ignore to the left/right.

What is the meaning of the colour for the statistic warning in operation Sequence?
orange (s): dissimilarity score 20-40
red (S): dissimilarity score >40

What is the dissimilarity score?
Value representing the difference between the calculated relative statistic peak area over all previously analysed samples (already archived / same chemistry) of a certain base position and the current patient sample.

Where do I find the required mix description for my kit?
Please go to Download / MLPA® - MRC Holland Kits of this website and download the requested mix description. If the desired mix description or version is not available, please contact our SEQUENCE Pilot support team.

Why did the import of the result files stop (button MLPA in the lower right of the screen stays red)?
Please check,

  • that the corresponding services are still running.
  • that the formate of the result files is correct (fsa or txt).
  • if there are any indicating errors in SEQUENCE Pilot menu Help / Logging... or in txt files from SeqPilot\messages.
  • that enough space is left over on the harddrive, where SEQUENCE Pilot is installed.

Why can't I find my controls in operation Joining?
If the raw data files have been named correctly (sample naming conventions), the controls are listed in their own operation Controllist.

Why can't I find my patient files just loaded?
Please check:

  • in menu Help / Logging... for the message „Result file already loaded to order, order:...“. By default the same patient file can't be loaded twice in the software using the same sample name.
  • MlpaResultfiles.txt file in SeqPilot\messages for messages "No sizing curve found" (sizing dye missing in result file) and "No sizing file found" (no Sizing.txt deposit in SeqPilot\bin and / or Thresholds defined in operation Thresholds [master file]).

Why can't I see any peak information?
Please check:

  • that you use the correct Sizing.txt (SeqPilot\bin).
  • that the values in Thresholds [master file] are corresponding to the used sizing standard.

What is the difference between reanalyse and recalculate?
Reanalyse updates the table above the electropherogram / bar diagram.
Recalculate resets all changes (e.g. peak assignments) done by the user.

Where can I check which HLA Database is used for analysis?
In SEQUENCE Pilot the used HLA database can be viewed and changed in menu SeqHLA / HLA DB Admin….

How can I use and update NMDP codes?
NMDP codes can be generated for every result. To activate the NMDP code generation create the new subdirectory NMDPFiles in directory SeqPilot of your installation.
Within this directory the current NMDP list (numer.v3.txt) has to be deposited:

Please note, that you have to update the NMDP list regularly. Therefore please use the procedure as descibed above.

How are G and P suffixes calculated in SEQUENCE Pilot?
The G and P suffixes are used to simplify the results in operation Sequence dialogue Result. By default SEQUENCE Pilot calculates the G and P suffixes automatically.

  • “G” stands for alleles which share identical nucleotide sequences for the exons encoding the peptide binding domain exon 2 and 3 for HLA class I and exon 2 only for HLA class II alleles have to be identical in nucleotide sequence, then a G Suffix is generated.
  • “P” stands for alleles, which encode for identical peptide binding domains: exon 2 and 3 for HLA class I and exon 2 only for HLA class II alleles have to be identical in protein sequence, then a P Suffix is generated.

Alternatively files from IMGT can be downloaded where all G- and P-Groups are defined. In case you use this option, you have to update the files regularly.
The reporting of ambiguous HLA allele typing only works, if you use maximum resolution.

How can I use the G and P suffixes from IMGT files instead of the calculated ones from SEQUENCE Pilot?
Files from IMGT can be downloaded where all G- and P-Groups are defined. In case you use this option, you have to update the files regularly (with every database update).
To use the nomenclature from a file, please do the following:

  • Go to http://hla.alleles.org/wmda/.
  • Download the files hla nom g.txt and hla nom p.txt.
  • Save the files in the folder of the respective database in folder Settings\HLANom of your SEQUENCE Pilot installation (e.g. the files for HLA DB 3.31 must be saved in folder Settings\HLANom\3.24.0).
  • Open the lis.ini file, located in SeqPilot\bin your SEQUENCE Pilot installation with a text editor.
  • Go to section [SeqPilot] and deposit the entry HLANomFileAllele=yes in a new line.
  • After restarting SEQUENCE Pilot the G and P suffixes for all newly started runs will be used from this file.

How can I inactivate G and P suffixes?
In case you do not want to use G and P suffixes at all, enter the new line Enter ReportAmbigousAllele=no in the section [SeqNextHLA] of your in the lis.ini file, located in the SeqPilot\bin of your SEQUENCE Pilot installation.

Where can I find the result?
In operation Sequence, section Result. Here the result, calculated for the selected gene in the dialogue part Genes is listed.
The first line shows, whether it is a heterozygous or a hemizygous result and on which haplotypes the result calculation is based on. The second line shows the ambiguities, that is all found allele combinations. G and P suffixes are used automatically. The last line shows, which HLA database version was used for the result calculation. Optionally the result can be listed as NMDP code.
If there are still mismatches to an allele there will be the entry “no results possible”. In that case you have to check the mismatching positions in the electropherogram. When all mismatches are removed the result will appear automatically.

What does the Matching table display?
This table shows all possible hemizygous results/allele combinations, sorted by their probability.
If you select an entry within this table it is highlighted green. This allele or allele combination is displayed in the electropherogram then. If there are mismatching positions for that allele or allele combination, the affected result files are displayed automatically and the cursor is positioned to the first mismatch.
All allele combinations with no mismatches are listed as result in section Result.

Is it possible to set a preferred result in case ambiguities are present?
Yes, this is possible in the Matching table, using the context menu item prefer alleles.

Is it possible to compare the sequences of my resulting allele sequences?
Yes, this is possible in the Matching table using the context menu item show differences….

Can new alleles be called as well?
Yes, if a new allele is present one mismatch remains to all known allele combinations from the IMGT HLA database. The new allele can be set manually in operation Sequence section Genes using the context menu new allele…. Afterwards the result “new allele most similar to…” is shown in section Result.

How can I export the results?
The results shown in the Results window in operation Sequence can be exported as txt file. Therefore, please use the context menu export / result... in section Genes.
Moreover a report can be printed or saved as pdf. Therefore use the button [Print] in dialogue part Functions.
Results can also be exported automatically to any LIMS systems after analysis, with our TALKMASTER interface.

Which files can be analysed?
Fastq and fastq.gz files from all common sequencing platforms from Illumina, IonTorrent, MinION and PacBio can be analysed.

Which one time procedures need to be set up before I can start analysis?
The HLA database needs to be installed and ROIs need to be defined in operation ROI [master file].

How can I deposit the DNA number in the file name?
By default all signs in the file name before the first "_" are used as DNA number. In case this is not the DNA number of your file name, please contact our SEQUENCE Pilot support team. We can adapt our software, so that the DNA number is read correctly.

Is it possible to automatically start a Run?
Yes, with the Autorun option. For further details, please contact our SEQUENCE Pilot support team.

How can I make analysis faster?

  • Switch off Second Alignment: The analysis will be faster, by adding the entry SecondLevelCalcAlignmentScore=no in the file lis.ini (SeqPilot\bin of your SEQUENCE Pilot installation) in section [SeqNextHLA]. When this entry is set a second alignment for reads which can be mapped to more than one locus is not done any more. But please note, that this might lead to single reads getting mapped to the wrong locus (especially for loci B/C or DRB pseudogenes) - depending on the allele combinations.
  • Activate muliple processing cores: To make analysis faster multiple processing cores can be used to compute the result files of a run. Several worker processes can be started, which process several files in parallel (in case the Run is started for several files, each worker processes one file). The number of worker processes should be related to the number of cores available on the server / client PC. For further details please contact our SEQUENCE Pilot support team.
  • Analysis is slow because the Coverage is very high: The following settings in operation Run, utton [Settings], tab Expert Settings can be adapted:
    • Load file: For very big files, only part of the reads can be loaded and analysed. This makes analysis much quicker. Here the percentage of reads that should be loaded and used for analysis can be entered. 100% means, that all reads are loaded.
    • Load reads: Here an absolute minimum number of reads that must be loaded can be entered. This setting can overrule the setting Load file mentioned above. In case the percentage value is too low to load the number of  reads defined here, more reads are loaded until the number of reads defined.

Analysis is slowing down my PC. What can I do?
By default the importer process uses the maximum number of cores. Therefore the computer might be very slow during data analysis, which might be a problem in case other programs are used as well.
The number of cores used during import can be restricted with the entry MaxImporterThreadCores=1 in section [SeqNextHLA] the lis.ini file (SeqPilot\bin of your SEQUENCE Pilot installation). The number behind this entry is the number of cores used for import.

Can I load data for the same patient several times?
This should only be done in case the fastq files contain data for different loci. In case you want to load replicates for one patient of the same loci, please contact our SEQUENCE Pilot support team to discuss all possible options.

Are introns analysed as well?
Intron sequences are displayed but not used for result calculation. They are only used to exclude known null alleles.

I have two possible results listed. Is it possible to exclude one of them?
Yes, phasing can optionally be checked. If possible, allele combinations are then removed from the result based on the phasing. To use this option, please activate the setting "Check phasing" on tab Expert Settings.

Can I exclude null alleles?
For result calculation null alleles which show mismatches/insertions/deletion in the introns are called or excluded as result automatically. This function is active by default, but it can be switched off. To switch it off the setting "Check null allele intron" on tab Expert Settings has to be deactivated.

How can I change the resolution?
By default 3 field resolution (maximum resolution) is used.
In case another resolution should always be used (2 field = 4 digits or 1 field= 2 digits), this can be set in the lis.ini file, located in folder SeqPilot\bin of your SEQUENCE Pilot installation. To change to 2 digits or 4 digits, please make the entry DefaultResolution=2 digits or DefaultResolution=4 digits in section [SeqNextHLA].
For already analysed files open the patient sample in operation Sequence, right click a locus in section Genes and select item change resolution and positions… from the context menu.

What can I check in case a result is not called automatically?
Please contact our SEQUENCE Pilot support team.

What can I do in case background is present for one or several loci?
It needs to be checked to which locus the background belongs to. In case it belongs to a locus which is not set up as ROI (e.g. locus E, G, Y…), ROIs for this locus need to be set up in ROI [master file] and included in the corresponding ROI group (ROI Group [master file]). In case you do not want to call results for these ROIs, you can set them to analysis mode “only mapping” in ROI [master file].

A result is called but mismatches are shown for the locus. What does this mean?
A result is always called as soon as there are no mismatches in exon areas. In intron areas mismatches are not regarded for result calculation.

In operation Sequence allele sequences from the IMGT HLA database are marked blue instead of green. What does this mean?
In case an intron sequence is unknown for a haplotype, it is checked if the intron sequence of another group member is known.
If yes, the sequence of the group member is listed. The haplotype sequence for the intron is then marked blue instead of green. You can see which allele the intron sequence belongs to when you move your mouse to the blue intron sequence (tool tip). This feature can be switched off using the entry Show2DigitsIntronAlleles=no in section [SeqPilot] of the lis.ini file (SeqPilot\bin of your SEQUENCE Pilot installation).
If no, no intron sequence is shown.

In operation Sequence the number in front of the read sequences can be highlighted in different colors. Moreover a * can be in front. What does this mean?
Identical reads are listed only once. The copy number is listed in front of each read. Moreover all reads that have a perfect match to an allele can optionally be marked with a * in front of the number.
The * and the copy number in front of each read can be colored. Identical colors for two or more reads mean, that the reads have a perfect match to the same allele(s) (context menu item "show alleles lists identical matches").
Please note, that black is not regarded as a color. Black */copy numbers mean that no other reads with the same perfect matches are present.

Can I get a quick overview about coverages?
Yes, therefore please press the button [Summary] in operation Sequence. The table can be exported as txt-file using the corresponding context menu item.

Where can I check which HLA Database is used for analysis?
In SEQUENCE Pilot the used HLA database can be viewed and changed in menu SeqHLA / HLA DB Admin….

How can I use and update NMDP codes?
NMDP codes can be generated for every result. To activate the NMDP code generation create the new subdirectory NMDPFiles in directory SeqPilot of your installation.
Within this directory the current NMDP list (numer.v3.txt) has to be deposited:

Please note, that you have to update the NMDP list regularly. Therefore please use the procedure as descibed above.

How are G and P suffixes calculated in SEQUENCE Pilot?
The G and P suffixes are used to simplify the results in operation Sequence dialogue Result. By default SEQUENCE Pilot calculates the G and P suffixes automatically.

  • “G” stands for alleles which share identical nucleotide sequences for the exons encoding the peptide binding domain exon 2 and 3 for HLA class I and exon 2 only for HLA class II alleles have to be identical in nucleotide sequence, then a G Suffix is generated.
  • “P” stands for alleles, which encode for identical peptide binding domains: exon 2 and 3 for HLA class I and exon 2 only for HLA class II alleles have to be identical in protein sequence, then a P Suffix is generated.

Alternatively files from IMGT can be downloaded where all G- and P-Groups are defined. In case you use this option, you have to update the files regularly.
The reporting of ambiguous HLA allele typing only works, if you use maximum resolution.

How can I use the G and P suffixes from IMGT files instead of the calculated ones from SEQUENCE Pilot?
Files from IMGT can be downloaded where all G- and P-Groups are defined. In case you use this option, you have to update the files regularly (with every database update).
To use the nomenclature from a file, please do the following:

  • Go to http://hla.alleles.org/wmda/.
  • Download the files hla nom g.txt and hla nom p.txt.
  • Save the files in the folder of the respective database in folder Settings\HLANom of your SEQUENCE Pilot installation (e.g. the files for HLA DB 3.31 must be saved in folder Settings\HLANom\3.24.0).
  • Open the lis.ini file, located in SeqPilot\bin your SEQUENCE Pilot installation with a text editor.
  • Go to section [SeqPilot] and deposit the entry HLANomFileAllele=yes in a new line.
  • After restarting SEQUENCE Pilot the G and P suffixes for all newly started runs will be used from this file.

How can I inactivate G and P suffixes?
In case you do not want to use G and P suffixes at all, enter the new line Enter ReportAmbigousAllele=no in the section [SeqPilot] of your in the lis.ini file, located in the SeqPilot\bin of your SEQUENCE Pilot installation.

Where can I find the result?
In operation Sequence, section Result. Here the result, calculated for the selected gene in the dialogue part Genes is listed.
The first line shows, whether it is a heterozygous or a hemizygous result and on which haplotypes the result calculation is based on. The second line shows the ambiguities, that is all found allele combinations. G and P suffixes are used automatically. The last line shows, which HLA database version was used for the result calculation. Optionally the result can be listed as NMDP code.
If there are still mismatches to an allele there will be the entry “no results possible”. In that case you have to check the mismatching positions in the electropherogram. When all mismatches are removed the result will appear automatically.

What does the Matching table display?
This table shows all possible hemizygous results/allele combinations, sorted by their probability.
If you select an entry within this table it is highlighted green. This allele or allele combination is displayed in the electropherogram then. If there are mismatching positions for that allele or allele combination, the affected result files are displayed automatically and the cursor is positioned to the first mismatch.
All allele combinations with no mismatches are listed as result in section Result.

Is it possible to set a preferred result in case ambiguities are present?
Yes, this is possible in the Matching table, using the context menu item prefer alleles.

Is it possible to compare the sequences of my resulting allele sequences?
Yes, this is possible in the Matching table using the context menu item show differences….

Can new alleles be called as well?
Yes, if a new allele is present one mismatch remains to all known allele combinations from the IMGT HLA database. The new allele can be set manually in operation Sequence section Genes using the context menu new allele…. Afterwards the result “new allele most similar to…” is shown in section Result.

How can I export the results?
The results shown in the Results window in operation Sequence can be exported as txt file. Therefore, please use the context menu export / result... in section Genes.
Moreover a report can be printed or saved as pdf. Therefore use the button [Print] in dialogue part Functions.
Results can also be exported automatically to any LIMS systems after analysis, with our TALKMASTER interface.

How can I load ProtransKits?
Under Download/SEQHLA - ProtransKits of this website you can find an installation file including pre-defined amplification modules and sequencing primers for ProtransKits.

Which files can be analysed?
Files from ABI sequencers should have the extension .ab1 and files from Beckman sequencers .scf. If you use sequencers from other manufacturers like MegaBase, please contact our SEQUENCE Pilot support team.

Why did the import of the result files stopp (button Seq in the lower right of the screen stays red)?
Please check,

  • that the corresponding services are still running.
  • that the formate of the result files is correct (ab1 or scf).
  • if there are any indicating errors in SEQUENCE Pilot menu Help / Logging... or in txt files from SeqPilot\messages.
  • that enough space is left over on the harddrive, where SEQUENCE Pilot is installed.

Why can‘t I find my patient files just loaded?
Please check:

  • in menu Help / Logging... for the message „Result file already loaded to order, order:...“. By default the same patient file can't be loaded twice in the software using the same sample name.
  • SeqResultfiles.txt file in SeqPilot\messages for messages "Data missing number 9" which means no base calling was done by the sequencer.

Are introns analysed as well?
Intron sequences are displayed but not used for result calculation.

Can I exclude Null alleles?
Null alleles showing base changes in intron areas are not called or excluded automatically. But the intron areas are displayed in SEQHLA. Moreover in case a splice site mutation is present, there will be the warning X present in column Warnings, in table Positions / Resultfiles in operation Sequence. Therefore the user can check the intronic positions and set or exclude certain null alleles in the Matching table manually using context menu item "exclude alleles...".

How can I change the resolution?
By default 3 field resolution (maximum resolution) is used.
In case another resolution should always be used (2 field = 4 digits or 1 field= 2 digits), this can be set in the lis.ini file, located in folder SeqPilot\bin of your SEQUENCE Pilot installation. To change to 2 digits or 4 digits, please make the entry DefaultResolution=2 digits or DefaultResolution=4 digits in section [SEQPILOT].
For already analysed files open the patient sample in operation Sequence, right click a locus in section Genes and select item change resolution and positions… from the context menu.
For future analysis, it needs to be set in LIS / Procedures. Procedures are generated automatically by SEQUENCE Pilot when loading a result file and no corresponding procedure exists. To change the resolution for a locus:

  • Select the procedure in field Name.
  • Select the desired resolution in dialogue part HLA. The following resolutions are available:
    • max resolution = 3 field (default option)
    • 4 digits = 2 field (shortens the results to 4 digits)
    • 2 digits = 1 field (shortens the results to 2 digits)

Furthermore all constant positions, that is all positions in the consensus sequence, which show an A, C, G or T, can be ignored here as well, by checking the corresponding setting. In operation Sequence they are highlighted grey then. These bases can be edited, but done changes don't effect the entries of the Matching table.