Software User Guide v2 - immucor.com Software... · MIA FORA is a trademark owned by Sirona...
Transcript of Software User Guide v2 - immucor.com Software... · MIA FORA is a trademark owned by Sirona...
Copyright © 2016, Sirona Genomics, Inc. All rights reserved.
Software User Guide v2.1
Advanced NGS HLA Genotyping Software
SR-790-00017
MIA FORA Software Ver.2.1
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Copyright Notice
This documentation and the MIA FORA NGS software are the confidential information of and are
copyrighted, © 2015, by Sirona Genomics, Inc. All rights are reserved. No part of this documentation or
the software may be reproduced, copied, displayed, transmitted, modified or used without the prior written
permission of Sirona Genomics, Inc.
Trademarks
MIA FORA is a trademark owned by Sirona Genomics, Inc.
All other trademarks and registered trademarks are the property of their respective owners.
All use of the MIA FORA software and this documentation are subject to the terms of the MIA FORA
License Terms and Conditions available at www.immucor.com/miaforasoftwareterms. BY USING THE
MIA FORA SOFTWARE AND/OR THIS DOCUMENTATION, YOU ACKNOWLEDGE THAT YOU HAVE
READ, UNDERSTAND AND AGREE TO BE BOUND BY SUCH TERMS AND CONDITIONS AS THEY
MAY BE UPDATED FROM TIME TO TIME.
MIA FORA NGS Software has been CE marked in the European Union only for IVD use with MIA FORA
NGS HLA Typing Kit Part Number SR-800-10377.
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Table of Contents
1. Introduction to MIA FORA NGS ................................................................................ 4
1.1 Genotyping Strategy ................................................................................................................ 4
1.2 Competitive Alignment to Reference Alleles .......................................................................... 5
1.3 Consensus Sequence Computation by Phasing .................................................................... 6
1.4 Central Read Coverage ............................................................................................................ 7
1.5 Computed Genotypes .............................................................................................................. 8
1.6 Smart Flagging System ........................................................................................................... 8
2. Getting Started ........................................................................................................ 10
2.1 Logging In .............................................................................................................................. 10
2.2 Preference Panel .................................................................................................................... 13
2.3 User Management .................................................................................................................. 14
2.4 Disclaimer Information .......................................................................................................... 14
2.5 Laboratory Information .......................................................................................................... 15
2.6 Date and Time Format Information ....................................................................................... 15
2.7 Report Format ........................................................................................................................ 16
2.8 Review Options ...................................................................................................................... 16
2.9 Transferring Files between server and user’s machine using VNC Viewer ........................ 17
3. Projects Window ..................................................................................................... 19
3.1. Create a new project .............................................................................................................. 20
3.2. Launch data analysis ............................................................................................................. 23
4. Statistics Window .................................................................................................... 25
5. Review Window ....................................................................................................... 29
5.1. Block A: Sample Information ................................................................................................ 29
5.2. Block B. Genotype Table ....................................................................................................... 30
5.3. Block C. Variants, LD Suggestion, and Smart Guide ........................................................... 36
5.4. Block D. Allele Candidate Table ............................................................................................ 38
5.5. Block E1. Coverage Plots ...................................................................................................... 42
5.6. Block E2. Alignment Browsers ............................................................................................. 44
5.7. Block E3. Reference Alignment ............................................................................................ 48
5.8. Block F. Contig Alignment Browser...................................................................................... 55
6. Summary Window ................................................................................................... 62
7. Auxiliary Tools......................................................................................................... 63
Appendix A: Software Color Codes Description ....................................................... 72
Appendix B: Glossary ................................................................................................. 75
Appendix C: Best Practices ........................................................................................ 77
Appendix D: Clickable Functions ........................................................................... 79
Appendix E: Additional Resources ............................................................................ 81
Appendix F: Third Party Libraries’ Licenses ............................................................ 82
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1. Introduction to MIA FORA NGS
MIA FORA NGS software delivers HLA typing information for the major Class I (HLA- A,
B, and C) and Class II (DPA1, DPB1, DQA1, DQB1, DRB1 and DRB3/4/5) genes.
Genotypes are computed from massive, paired-end sequencing reads derived from the
Illumina Next Generation Sequencing (NGS) platform. The software provides accurate
and phase defined unambiguous HLA genotype information.
Figure 1-1: HLA Gene region showing relative locations of HLA Class I and Class II genes
HLA genes are one of the most complex regions of the human genome and the
accurate and complete sequence of the HLA genes is an enormously complex
endeavor. MIA FORA NGS software has been designed to take full advantage of the
NGS and provide to users a simple-to-use tool to make the right decision for an
unambiguous HLA typing call.
The software is designed to correctly identify HLA genotypes based on exon sequence.
Special consideration is given to the most highly sequenced exon regions, i.e. exons 2-
3 of Class I genes and exon 2 of Class II genes. At this time, less emphasis has been
placed on identifying intron variants.
MIA FORA NGS software includes an intuitive graphical user interface (GUI) and
complies with the requirements established by the HLA community. It provides to users:
first, the accurate and unambiguous HLA genotypes based on the latest IMGT
nomenclature and second, the complete phased sequence covered by the targeted
primers used to interrogate the HLA genes.
1.1 Genotyping Strategy
MIA FORA NGS software combines two complementary informatics strategies to
analyze each sample and then makes genotyping calls for target HLA genes using a
computed confidence score. The first strategy ranks computed allele candidates based
on mapping metrics. Coverage is calculated from competitive alignment of paired-end
NGS sequence reads with all HLA reference sequences in the latest IMGT database
and reference sequences produced by Sirona Genomics. The second strategy utilizes
Phasing by Dynamic Program to assemble reads and construct phased assembled
sequences. The approach is illustrated in Figure 1-2.
Class II Class III Class I
DP DQ DR B C A
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Figure 1-2: MIA FORA NGS genotyping strategy. Two complementary strategies are employed to compute the best fit to HLA reference alleles and resolve consensus sequences. The left side is mapping. Paired end reads are mapped using competitive alignment algorithm to rank candidate alleles. The right side is phasing. Starting with Paired end reads, these reads will mapped and go
through local assembly, then phase resolution to construct phase resolved consensus.
1.2 Competitive Alignment to Reference Alleles
Mapping is used to rank candidate alleles based on competitive alignment of paired end
sequence reads with all HLA reference sequences to make sure that we capture all
SNPs and structural variance. The reference database includes all HLA reference
sequences in the latest version of IMGT HLA database and some generated through
cloning and sequencing at Sirona Genomics. Two types of internally generated
reference sequences are used in this tool: cloned and sequenced alleles and in silico
sequences.
1) Cloned and sequenced alleles:
Cloned sequences are derived from individual long-range PCR products amplified from
IHWG cell lines and samples. Thus, these sequences represent alleles that were
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completed by filling in missing intron and exon sequences. Cloned sequences in the
database are indicated with one of three suffixes: e, v or x.
e: Sequences with the suffix e (e1, e2, etc.) are those with new intronic sequence
not represented in the IMGT database. The vast majority of cloned sequences
are of this type.
v: Sequences with the suffix v (v1, v2, etc.) are a small subset of cloned alleles
that contain new intron variants relative to existing genomic sequences in the
IMGT database.
x: Sequences with the suffix x (x1, x2, etc.) are a small subset of cloned alleles
that contain new exon variants. The suffix is added to the closest known
reference sequence but if confirmed by IMGT the allele name will change.
2) In silico sequences:
Many IMGT reference sequences contain partial exon sequences. To facilitate data
analysis, the closest complete exon was copied to fill in the gaps in IMGT reference
sequences with an incomplete exon. The suffix i (i1, i2, etc.) is used to identify those
computationally filled sequences.
The extensions used in the naming of the reference sequences are to inform the user
about the reference sequence that was used to make the allele assignment. The
coverage alignment can be viewed for the IMGT and corresponding extended reference
sequences. In all cases where the sequence has either been determined by actual
sequencing or in silico extension of reference sequences, the HLA type can be reported
in the accepted IMGT format without the extensions that are used for naming these
extended sequences.
1.3 Consensus Sequence Computation by Phasing
A Dynamic Phasing algorithm generates one or two phased consensus sequences
(contigs) by de novo assembly of mapped, paired-end sequences. In the same
assembly process, polymorphic sites are identified where the minor allele frequency
exceeds a threshold of 0.2.
Once polymorphic sites are identified, Phased Resolved Consensus sequences
(phased contigs) are built based on sequence assembly and polymorphic linkage.
Following Dynamic Phasing, the Phased Resolved Consensus sequences are aligned
to the HLA allele database to determine the best fit. Consensus Alignment provides an
independent check of the genotype call. Novel alleles are identified as discrepancies
from the exon sequence of the reference alleles.
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1.4 Central Read Coverage
Central read coverage was developed to ensure base calls can be made with a high
degree of confidence. Central reads (Figure 1-3) are empirically defined as mapped
reads for which the ratio between the length of the left arm and that of the right arm
related to a particular point is between 0.5 and 2. When reads are mapped onto a
correct reference sequence, they form a continuous tiling pattern over the entire
sequenced region. However, when reads are mapped onto an incorrect reference
sequence, they form a staggered tiling pattern at some positions of the sequenced
region. To quantify this difference between the two alignment patterns, the numbers of
“central reads” are counted for any given point. Central Read Coverage ensures that
potentially mismatched reads are excluded.
Figure 1-3: Central reads of an anchor point are defined as mapped reads, where the ratio between the length of the left arm and that of the right arm related to a particular point is between 0.5 and 2. The left side of plot shows the mapping pattern of reads onto two correct references and one incorrect reference. The incorrect reference has a mosaic pattern at the two different positions between two correct reference sequences. From the plot, it can be seen that reads with an even tiling pattern are mapped to the correct reference sequence while reads mapped to the incorrect reference sequence have an uneven, staggered pattern. The coverage plot graphs on the right
side illustrate an example where central read coverage can distinguish a correct reference from an incorrect reference, while regular coverage cannot.
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1.5 Computed Genotypes
For each gene, confidence scores are used to identify the best matching alleles in the
reference database. Key coverage statistics are combined in a proprietary algorithm to
calculate confidence scores and select the top computed alleles for each gene. The
best allele candidates are then selected as the computed genotype call. Key
components of the confidence score are illustrated in Figure 1-4Error! Reference source
not found.. Coverage statistics such as number of mapped reads and minimum
coverage are calculated mapping the paired end reads to the HLA reference allele
database. Phase-resolved consensus is determined through sequence assembly and
polymorphic linkage. Candidate pairs are also evaluated and included in the calculation.
Figure 1-4: Key components of confidence scores used to rank computed alleles. Confidence
scores are calculated using a combination of mapping, phase-resolved consensus, central read coverage, and candidate pair metrics.
1.6 Smart Flagging System
A Smart Flagging System was developed to display different information about the
genotypes for all the genes in the selected sample. Above each allele is a shape
indicator. Symbols for each of the indicators are:
Triangle - confidence score
Diamond - common or well-documented (CWD) allele
Pentagon – overwritten automatic call
Hexagon - consistency with linkage disequilibrium data
Circle with a question mark or exclamation mark – Question mark means
discordant calls between the competitive alignment (EM) and the phased
resolved consensus. An exclamation mark indicates that the allele is in the
Confidence Score
Mapping
Central Coverage
Phase Resolved Consensus
Candidate Pair
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double-check list which requires careful manual review. The double-check list
includes alleles that can have issues in sample prep, sequencing or data
analysis. The circle will turn green after review and confirmed by the user.
Alleles on the double-check list include: A*02:10, DRB1*04:13, DRB1*15:01:17,
DRB1*15:11, DRB1*16:09:01.
PC - the number of phased resolved de novo contigs.
See the legend in Figure 1-5 for further explanation of each shape.
Figure 1-5: Flags (colored shapes) are used to depict each predicted genotype status. Indicators for confidence score (green triangle > blue > red > gray) green is the highest confidence score
and grey is the lowest confidence score, common or well-documented allele (green diamond) or not (amber diamond), whether a call has been edited (amber pentagon) or not (green pentagon),
whether a call is consistent with linkage disequilibrium data (green hexagon) or not (amber hexagon), and whether special review is required (amber circle with question mark), indicating a
potential novel allele in the exon sequence. PC: the number of phased contigs. If the count of contigs is different from the number of alleles of the corresponding locus, it will show amber color
in the circle. Otherwise, it is green color
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2. Getting Started
The MIA FORA NGS software is an accessory for use with Illumina next generation
sequencing (NGS) data obtained by sequencing libraries prepared by targeted
amplification of the intended genes using the MIA FORA NGS HLA typing kit. Due to the
complex nature of HLA Testing, qualified laboratory personnel must review any result to
assure accuracy.
The software can be accessed through either a VNC Viewer connection or direct access
through monitor and keyboard attached to the server. To access the server directly, log
in using KDE plasma workspace mode instead of GNOME classic mode:
1. Turn ON the server and wait till logon screen appears. Once logon screen
appears, click on User Account to log in.
2. Click on Username and enter the Password. Before clicking on the Sign-in
button, click on the Gear icon next to the Sign-In button and select “KDE plasma
workspace” from the list. Then click on the Sign In button to login to Red Hat
Linux 7.
3. Follow the instructions to log in and access MIA FORA NGS software as
described below.
2.1 Logging In
VNC viewer can be downloaded from https://www.realvnc.com/download/viewer/.
Follow the instructions on the website to install VNC Viewer on your computer.
Open the VNC Viewer application by clicking on the VNC icon. Choose the appropriate
VNC Server name or IP address and click on Connect as shown in Figure 2-1. At the next
prompt, enter your unique login name and password.
Figure 2-1: VNC login screen. Choose the appropriate VNC Server name or IP address and click on Connect button. At the next prompt enter login credentials. Each user needs appropriate access privileges and a unique username. After three failures, the login will close, and the user will have
to re-open the window and begin the login process again.
After logging into VNC Viewer the remote desktop will be displayed. The VNC Viewer
may be configured for many functions, such as exporting and printing data. The latest
version of the VNC User Guide can be found at
http://www.realvnc.com/products/vnc/documentation/5.2/guides/user/VNC_User_Guide.pdf.
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To access MIA FORA NGS software, follow the steps illustrated in Figure 2-2.
Step 1: Hover over the tab at the top of the window to reveal the pull-down menu and
select Full Screen Mode.
Step 2: Click on the MIA FORA icon found on the desktop or in the Quick Start menu.
Step 3: Enter login credentials. The password must be between 4-8 characters and
contain at least one lowercase letter, one uppercase letter and one number.
When logging into MIA FORA software for the first time, labdirector is the default user to
this software. Select labdirector as the username, and type default password:
$1r0naG3n0m1c$ After that, an End User License Agreement window will show up,
which the user needs to read before clicking the Agree button to continue. The
preference panel will show up to allow labdirector to add new user, provide lab
information such as lab contacts, and disclaims. The preference panel continues to
display until all information has been entered.
Figure 2-2: Steps for logging into MIA FORA NGS software from VNC Viewer. First, maximize the
window to fit the computer display. Second, click on the MIA FORA icon either from the Quick Start menu or from the icon on the desktop. Third, enter MIA FORA NGS software login
credentials.
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Figure 2-3: End user license agreement dialog shown up when a new user log in first time.
The main window of MIA FORA NGS software, as illustrated in Figure 2-4, will be
displayed after logging in. Return to this page at any time by selecting the Projects icon
in the upper left-hand corner.
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Figure 2-4: Main MIA FORA NGS window. Projects that have been created are listed in this main page. Return to this view at any time by selecting the Projects icon. Software commands can be accessed through the side panel and dropdown menus: three drop-down menus at the top of the
home screen with eight shortcuts under Tools; an icon list along the top left side to open data views; eight icons along the bottom left side for additional project commands; two buttons on the
bottom right to create or delete projects. User name, current project, current sample, current gene, storage capacity in percentage, and last action taken will be displayed in the status bar at
the bottom of the window once project is created.
2.2 Preference Panel
A Preference window with user management and account tools will be displayed the
first time a new lab director user logs in. Bypass the Preference window by clicking on
“X” in the upper right corner to close the window. For users accessing software directly
from server, press <Esc> button on keyboard to close the window. The preferences
menu can also be accessed from the top menu under File>Preferences, shown in Figure
2-5.
Figure 2-5: Top menu toolbars. There are three drop-down menus for accessing File, Tools and
Help functions
In addition, if the laboratory information and disclaimer information are not set and the
logged in user has the director role, the preference panel will show up as in Figure 2-6.
Until all the information in the preference panel window is filled in, it will pop up every
time a user with director access opens the software.
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2.3 User Management
User management tools can be accessed through the User panel in the Preference
window, shown in Figure 2-6. When creating new accounts for users, it is important to
remember the privileges and abilities of each role. A user with the lab director role can
perform all actions with full access to the software. A lab technician can perform most of
the tasks except for confirming a sample and deleting a project. A guest has read only
access; therefore they cannot make any edits to allele calls when reviewing a project. A
guest user also cannot create, delete, export, or import projects. Detailed permission for
the three user types are show in Table 1.
Table 1. Permission for user roles: lab director, technician and guest. *Technician can report samples approved by a Lab Director.
Check
Results Comment Change
Call Approve Confirm Report Delete Add User Preferences
Lab Director
✔ ✔ ✔ ✔ ✔ ✔ ✔ ✔ ✔
Technician ✔ ✔ ✔ ✔
✔*
Guest ✔ ✔
To add a user, select the role, enter a login name and email address and create a
password. The password must contain at least 8 characters including at least one upper
and one lowercase letter and one numeral. Only a lab director may delete a user.
Figure 2-6: Preference window of setting up new accounts, delete user or change password of
current user. Note that only user with director role has the privilege to add or delete user.
2.4 Disclaimer Information
From the preferences window, the user can set up new accounts and default parameters, including laboratory information and disclaimer language to be used in the HLA typing report, shown in Figure 2-7.
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Figure 2-7: Disclaimer information panel displays the disclaimer information for the final report. A
user with lab director privileges can edit this information.
2.5 Laboratory Information
Figure 2-8: Laboratory information panel displays laboratory contact information to be shown in
final report
2.6 Date and Time Format Information
Figure 2-9: Date and Time format panel for setting date and time formats
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2.7 Report Format
Figure 2-10: Report format options.
There are three options in Report Format window: “Allele extension”,” Show comments”,
and “A4 report Page Size”. Allele extensions are those characters (e, i, v and x) added
to the names of reference sequences generated internally either through cloning-and-
sequence or computationally. “Allele Extension” option will allow users to add allele
extension characters to the allele name in the final report. For example, when “Allele
Extension “box is unchecked, A*01:01:01:01v1 will be displayed as A*01:01:01:01 in
final report. “Show comment” option allows users to display comments created during a
review process in the final report. “A4 Report Page Size” option allows users to change
the page size to A4 format (European format).
2.8 Review Options
Figure 2-11: Review Options panel
Review option tab in the preference window allows a user to set configurations in the
review window. The default order of candidate alleles can be set to either order by Call
column or by cReads column in the genotype table. To order the candidate alleles by
cReads, tick the preference and select Apply. The default order of loci can be set to
Alphabetical order or conventional order. The conventional order is A, B, C, DRB1,
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DRB3/4/5, DQA1, DQB1, DPA1, DPB1. To order the loci alphabetically, tick the
preference and select Apply.
2.9 Transferring Files between server and user’s machine
using VNC Viewer
VNC Viewer has a built-in File Transfer function. The File Transfer feature is useful for
uploading sample sheets and downloading reports. Caution is advised because this
function has the ability to transfer individual files or entire folders. Select only the
desired file to avoid transferring an entire folder instead of a single file.
Figure 2-11: VNC Viewer File Transfer window.
To fetch files from the VNC to the user’s computer:
1. Locate the VNC icon in the lower right corner of the VNC viewer window
2. Right click on the VNC icon to open a menu of options
3. Select File Transfer from the list of options
4. The file transfer dialog box will appear as shown
5. Choose the option Fetch files to: “Ask every time.”
6. Click on the button labeled Send files to open a new window and view your files
7. Select the file to transfer
8. Click on the OK button to transfer the selected file to the download location
specified above
9. A message will appear with a list of files transferred and their Download status
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Files can also be loaded directly to the server using as USB Flash Drive.
To transfer files to the VNC Server from the user’s computer:
1. Hover over the top center of the VNC viewer window, and a menu will drop down
2. Locate and press the File transfer button and the file transfer window will be
displayed
3. From the pull down menu in the lower right corner of the window, fetch files to,
select the location of the files to be transferred
4. Choose the files to be transferred and press the Send files button in the lower left
corner of the window.
A message will appear when the file transfer was completed successfully
Files can also be loaded directly to the server using as USB Flash Drive.
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3. Projects Window
Click on the project button to show a table of all projects created by users. Each
row displays one project with 10 columns: Project Name, Created on, Analyzed on, Lot,
Received on, Operator, Project Status (Sample, Analysis, Review, Approval, Report),
Next Step, Software Version and IMGT version. To refresh the page, click on the
Projects icon.
Figure 3-1: Projects window is displayed after clicking on the Projects button on the left panel or after successful login. When selecting any of the Statistics, Review or Summary, from the left
hand panel, a tab will display adjacent to the Project tab.
When selecting different Projects from the Project tab, the Review and
Summary tab do not refresh and must be closed to reflect the new Project
selection.
The complete project name will be generated once the project is created, which consists
of three parts: the project name provided by user, the date the project is created and a
randomly chosen English bird name. For example test_10Dec15_MURRE, where test is
provided by user through new project wizard, the second and third parts are added by
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the software. Use the complete project name generated by MIA FORA software when
setting up a MiSeq run.
The status column is shown as Figure 3-2. There are five steps to completed status:
Sample, Analysis, Review, Approval and Report. Each one is represented by one
“moving next” shape. Each status has four states: ready for next step (amber), red
(failure), blue (progressing) and green (completed).
The next step button serves two purposes. First, once a new project is created, clicking
on the next step button will launch the fastq files upload wizard. Second, if a project
analysis failed, clicking on the next step button will change the status back to ready.
Figure 3-2: Project status indicators. Amber indicates ready for next step; red indicates an error; blue indicates step in progress; green indicates step is finished.
3.1. Create a new project
To create a new project and submit the sequencing results for HLA typing, click on the
New Project button in the lower right-hand corner of the main Projects page. See Figure
3-3.
Figure 3-3: New Project initiation. Select button in lower right corner of the main application page.
After clicking on the New Project button, a dialog box will appear. Enter project name,
which is created by the user, MIA FORA kit lot number and date that kit is received.
The software will add both the date when the project is created and one of random bird
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names to create a unique, complete project name. Navigate to the Sample Barcode
sheet and confirm the sample names (Figure 3-4, to Figure 3-8).
See the MIA FORA NGS HLA Typing Kit LT24 User Guide for instructions on
how to create the Sample Barcode sheet. Sample Barcode sheet accepts
alphanumeric, dash and underscore. No special characters are accepted.
Figure 3-4: Welcome page of new project wizard
Figure 3-5: Project information dialog box of new project wizard
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Figure 3-6: Filled dialog boxes on project information page of new project wizard.
Figure 3-7: Sample barcode confirmation page of new project wizard
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Figure 3-8: Summary page of new project wizard. Note the project name is computationally generated with three parts: the part provided by user, current date and a random bird name.
3.2. Launch data analysis
To launch the data analysis, load the MiSeq fastq data files into the project by clicking
on the Next Step button. The fastq files wizard will open and navigate to load the fastq
files (Figure 3-9, Figure 3-10 and Figure 3-11). Hold down <ctrl> key to select both fastq files
together.
Figure 3-9: Welcome page of fastq files upload wizard
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Figure 3-10: Filled input box of fastq files wizard showing files selected
Figure 3-11: Confirmation page of fastq files wizard
If data analysis cannot be launched, as indicated by no change in the
state/status indicator, the user should click on Clean in the Tools menu. After
that, the data analysis pipeline should be able to pick up a project to launch data
analysis.
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4. Statistics Window
Statistics window displays sequence quality of the selected project. MiSeq quality
metrics from the selected project are shown as well as read distribution of barcoded
samples, insert distribution, and pie chart of valid reads.
Click on the Statistics button on the left side bar will bring up the statistics window
(Figure 4-1) for the selected project.
Figure 4-1: Statistics window displaying sequence quality of the selected project. MiSeq quality metrics from the selected project are shown as well as read distribution of barcoded samples,
insert distribution, and pie chart of valid reads.
Figure 4-2 shows the overall read distribution for three categories: invalid barcodes,
unused barcodes and valid barcodes. Reads with sequencing errors in the barcode or
contaminated barcode will be classified under Invalid Barcode. When a run consists of
24 samples but only a subset of samples are analyzed, any reads that are not analyzed
will be classified under Unused Barcode.
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Figure 4-2: Pie chart of sequence reads distribution among invalid barcode, unused barcode and
valid barcode reads categories
Figure 4-3 insert distribution shows the distribution of end-to-end distance between
paired-end reads after mapping onto reference sequences.
Figure 4-3: Insertion size of paired end reads distribution
Figure 4-4 and Figure 4-5 show the read count for each barcoded sample in either
histogram or plate layout. The plate layout allows the user to diagnose a failed run. By
right clicking on the barcode distribution graph, the user can save the barcode
distribution table. All other graphs on the statistics page can be saved in the same way.
Figure 4-4: Bar graph of read count for each barcoded sample
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Figure 4-5: Read count illustrated in 96-well format of plate layout. Barcode distribution within a plate is displayed in the Plate tab. Different colors represent the number of reads tagged with the index barcode for each well. The color key is displayed next to the plate diagram and ranges from
red for the lowest number of reads to blue for the highest number.
Figure 4-6 shows the distribution of nucleotides along each position of the sequencing
read. The first nucleotides of the graph represent the barcode region where the
distribution is distorted.
Figure 4-6: Distribution of nucleotides along each position of read.
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Figure 4-7 shows the distribution of quality scores at each position along the read.
Figure 4-7: Distribution of MiSeq quality scores along each position of read should above Q30.
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5. Review Window
Click on the Review button on the left side bar to display the Review window in a
tab as shown in Figure 5-1. It is important for reviewers to verify automatic calls for
flagged samples and make edits where necessary. Edits are necessary when
independent software algorithms are inconsistent in calling novel alleles, in detecting
polymorphisms, or in phasing.
Figure 5-1: Annotated review window. Block A displays sample information; Block B displays the
selected genotypes for the sample. Block C displays the Variants, Smart Guide, and LD Suggestion tables; Block D displays the table of computed allele candidates; Block E displays coverage plots and alignment browsers for mapped sequence of sample; Block F displays the
alignment browser for phase resolved de novo contigs
5.1. Block A: Sample Information
The sample information block contains Sample, Locus, and last visited date (Figure 5-2).
The Sample dropdown lists barcode ID and sample name. The Locus dropdown lists
HLA loci. Last visited field displays the date and time of the last visit. A user can view
different samples or genes through the dropdown lists, as shown in Figure 5-3 and Figure
5-4.
Figure 5-2: Sample information panel in review window
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Figure 5-3: Sample information dropdown list. The background color indicates the review status
of each sample: gray indicates the sample has not been reviewed yet (visit time = 0); yellow indicates 1 to 10 visits; blue indicates more than 10 visits; green indicates the sample was
approved. The last visited sample is indicated with red text.
Figure 5-4: Locus dropdown list sorted alphabetically
5.2. Block B. Genotype Table
The sample genotype table (Figure 5-5) displays the selected genotype for the sample.
The phasing consensus (PC) column lists the number of contigs built by the de-novo
assembly algorithm. Each Allele column lists alleles predicted to be in the same
haplotype. There are two buttons above the genotype table: Approve and Confirm.
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Figure 5-5: Genotype table for a single sample before approval. The Smart Flag is displayed above each allele. Alleles are organized by predicted haplotype; each column represents one predicted haplotype. Each cell of the table is clickable to switch to the review panel for selected gene. The highlighted alleles show automatic calls that should be manually reviewed. The legend for Smart
Flagging System can be shown by hovering cursor over the headers for allele columns.
The Approve button allows the lab director to approve the results. Once the Approve
button is clicked, a comment dialog will be shown as in Figure 5-6. The user can cancel
the action by clicking on the Cancel button. The user can add a comment in the New
Comment window. Click on the OK button to load the comment into the project log. After
the sample is approved, the Approve button will change to UnApprove.
Figure 5-6: Comment dialog box for sample approval
The Confirm button allows the lab director to confirm the results. Once the Confirm
button is clicked, a comment dialog loaded with previous comments will be displayed.
The user can cancel the action by clicking on the Cancel button. The user can add a
comment in the New Comment window. Click on the OK button to load the comment
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into the project log. After the sample is confirmed, the Confirm button will change to
UnConfirm and the Report button will appear next to the UnConfirm button (Figure 5-7).
Figure 5-7: Genotype table of alleles for a sample after approval. Three buttons are displayed
(UnApprove, UnConfirm, and Report.)
Click on the Report button to display a report dialog box (Figure 5-8). If there is ambiguity
in the two alleles from DPB1, the equivalent pairs of alleles will be displayed in the last
column “Notes”. Two options (PDF and XML) allow the user to choose a different output
format. Enter output file name in a file dialog and save the file. The PDF output file
displays all comments related to the sample displayed at the bottom if the Report
Format preference has been selected to show comment. Similarly, if the Report Format
preferences have been selected to show allele extensions, they will display on the
report. Examples for each of the files formats are shown in Figure 5-9 and Figure 5-10.
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Figure 5-8: An example of a report dialog box. Two (PDF and XML) buttons at the bottom right corner allow the user to choose either PDF or XML format. User can chose in the preference panel
whether or not to have comment and allele name extension (e, i, v, x) displayed, or A4 page format.
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Figure 5-9: Example of a report in PDF format with both comments and allele name extensions
(Preferences are set to ‘Show Comments’ and show ‘Allele Extensions’)
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Figure 5-10: Example of a report in XML format
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5.3. Block C. Variants, LD Suggestion, and Smart Guide
There are three tabs in this block- Variants, Linkage Disequilibrium suggestion (LD info),
and Smart Guide. In the Variants tab (Figure 5-11). the first column (Pos) lists
polymorphic sites of de-novo assembled contigs. The second column (Depth) provides
the coverage depth of each polymorphic site. The number outside the bracket is the
total number of reads covering that site; the number within the bracket is the number of
nucleotides not listed in either contig. The third column (Contig1) lists the nucleotide and
its occurrence in contig 1 and the fourth column (Contig2) lists the nucleotide and its
occurrence in contig 2. The fifth column (Block) indicates phasing blocks; each block is
a continuous region which can be phased with support from paired-end reads; each
phased block begins and ends with the ‘--’ symbol.
Figure 5-11: Variants table. The first column (Pos) lists the position of each polymorphic site in
the de-novo assembled contigs. The second column (Depth) lists the coverage of each polymorphic position. The number outside the bracket is the total number of reads covering that site; the number within the bracket is the number of nucleotides not listed in either contig. The third column (Contig1) lists the nucleotide and its occurrence in contig 1 and the fourth column (Contig2) lists the nucleotide and its occurrence in contig 2. The fifth column (Block) indicates
phasing blocks; each block is a continuous region which can be phased with support from paired-end reads; each phased block begins and ends with the ‘--’ symbol highlighted with amber.
The block highlighted with amber, as shown in Figure 5-12. Amber highlighted entries in
Contig1 and Contig2 indicate positions where the ratio of the nucleotide frequencies
deviates from the average ratio across the sample. These polymorphic positions are
unreliable.
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Figure 5-12: Variants table. Amber highlighted entries in Contig1 and Contig2 indicate positions where the ratio of the nucleotide frequencies deviates from the average ratio across the sample.
The LD info tab (Figure 5-13) displays LD Suggestions based information aggregated
information from the publicly available database and from analysis of internally typed
samples. The public haplotype data can be found at:
https://bioinformatics.bethematchclinical.org/HLA-Resources/Haplotype-Frequencies/
Each row represents alleles that have been observed to be associated with one
another. The LD suggestion panel is for information only; LD is not used to make
automatic allele calls.
Figure 5-13: LD Info tab displays LD Suggestion.
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The Smart Guide (Figure 5-14) provides guidelines for reviewing the HLA typing results.
Possible reasons may be provided to explain why a user should review the specified
information in the review window. The recommended actions are described to provide
instructions for review. The smart guide will only contain information when an automatic
allele call is highlighted in the genotype table on the review page.
Figure 5-14: Smart Guide lists Reasons and Actions for reviewing allele calls.
5.4. Block D. Allele Candidate Table
The allele candidate table (Figure 5-15, Figure 5-16) lists the automatic allele calls and
parameters calculated from sequence reads mapped to HLA reference sequences.
Within this table, a user is allowed to comment on a selected allele, overwrite a
automatic allele call and make a manual allele call.
The Call Column (Call) contains a check symbol to indicate the automatic or manually
selected alleles. The green check symbol indicates that there is no warning associated
with the selected allele. The amber check symbol indicates a warning associated with
the selected allele. The blue symbol in the second column indicates there is a comment
associated with the corresponding allele.
After comparing the best-matched contig with the reference allele, the software displays
the number of mismatched nucleotides between the contig and the reference allele in
the exon (MME) or intron (MMI). The alleles highlighted in gray indicate a candidate
best matched with contig 1. The alleles highlighted in amber indicate a candidate best
matched with contig 2.
A Call warning occurs in four scenarios: first, if the selected allele is not CWD; second, if
the allele is inconsistent with LD information; third, if an exon of the selected allele has a
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mismatch (MME) with the best-matched contig; fourth, if the underlying locus is
predicted to be homozygous.
There are four different sets of reference sequences: cRead (cDNA), eRead (partial
cDNA, exons 2 and 3 for class I and exon 2 for class II loci), gRead (genomic DNA),
xRead (gRead minus the eRead.) For each set of reference region, three quality metrics
are calculated: total number of reads, minimum overall coverage (Cov) and minimum
central coverage (Cen.). All 12 quality metrics are displayed for each of the candidate
allele in the table. The tooltip for metric definition can be displayed by hovering cursor
over the header for each column.
Figure 5-15: Example of candidate table. This table of 17 columns lists likely allele candidates for the corresponding locus of the sample and their mapping parameters. The first column (Allele) is
the allele name of candidates. The second column (Call) with check symbols indicates the automatic or manually selected alleles. The third column (Cmt) shows whether there is comment associated with the particular allele. The fourth and fifth columns (MME, MMI) list the number of
mismatched nucleotides in the exon or intron after comparing the best-matched contig with reference allele; The alleles highlighted in gray indicate a candidate best matched with contig 1. The alleles highlighted in amber indicate a candidate best matched with contig 2. Columns 6-8
(cRead, Cov, Cen) list the mapping parameters against cDNA reference sequences. Columns 9-11 (eRead, Cov, Cen) list the mapping parameters against partial cDNA reference sequences,
specifically Exons 2 and 3 for Class I loci and Exon 2 for Class II loci. Columns 12-14 (gRead, Cov, Cen) list the mapping parameters against genomic reference sequences. Columns 15-17 (xRead, Cov, Cen) list the mapping parameters against partial genomic reference sequences that include
everything except the partial cDNA sequence listed above. The table may be sorted by clicking on any column header.
Figure 5-16: Example of candidate table where the selected alleles check symbol is amber. The amber check symbol suggests that the locus requires manual review and the reason is given in
the smart guide. The blue dialog symbol in the second column indicates there is a comment associated with the corresponding allele.
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Double-click on a header to sort on that column. Click on any cell to select the
entire row.
Technician or lab director user roles may override automatic allele calls. Common
reasons for overwriting an automatic allele call include a discrepancy in phasing,
incomplete detection of polymorphic sites, or presence of a novel allele.
Double-click on the second column (Call) to select or deselect an allele call (Figure 5-17.)
If the allele was previously checked, it will be unchecked; if the allele was unchecked, it
will be checked. Each time, the activity will be logged through the comment dialog box
(See Figure 5-18), where a user has the option to add comments about this activity.
Figure 5-17: Check or uncheck a candidate by double-clicking on the cell in the second (Call)
column
Figure 5-18: Comment dialog box when toggling the status of a selected allele
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Double-click on the cell in the third column (Cmt) to activate the comment dialog box
(Figure 5-19). Users can log comments using the comment dialog box. This action will not
change the status of the selected allele.
Figure 5-19: Comment dialog box for attaching comments to a selected allele When average
coverage is less than 40X the display shows Insufficient Data. If user wants to
show genotype of a locus without sufficient data, user could type OWI (overwrite
insufficient data) in a comment dialog to bring up genotypes masked by
insufficient data. If user wants to cancel the effect, users could type XWI in a new
comment dialog window.
Click on the tab labeled “Candidate Pair” to view the candidate pair table (Figure 5-20). In
this table, each row represents a possible combination of two alleles listed in columns
one and two. Eight parameters are displayed for each candidate pair. The cRead
column lists the number of unique reads mapped to the cDNA reference sequences of
the two alleles. The eRead column lists the number of unique reads mapped to the
partial cDNA sequence containing only Exons 2 and 3 for Class I loci and only Exon 2
for Class II loci. The gRead column lists the number of unique reads mapped to the
genomic reference sequences. The xRead column lists the number of unique reads
mapped to partial genomic sequences that exclude the exon 2/3 regions detailed above.
The mismatch numbers in the exon (MME) and intron region (MMI) for Allele1 and
Allele2. Highlight a row of possible combination of two alleles and select Coverage to
directly plot coverage for the cDNA reference sequences and the genomic reference
sequences.
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Figure 5-20: Candidate Pair table. The first and second columns (Allele1, Allele2) list the pair of
alleles examined. The third column (cRead) lists the number of unique reads mapped to the cDNA reference sequences of the two alleles; the fourth column (eRead) lists the number of unique
reads mapped to the partial cDNA sequence containing only Exons 2 and 3 for Class I loci and only Exon 2 for Class II loci. The fifth column (gRead) lists the number of unique reads mapped to
the genomic reference sequences. The sixth column (xRead) lists the number of unique reads mapped to partial genomic sequences that exclude the exon 2/3 regions detailed above. The
mismatch numbers in the exon (MME) and intron region (MMI) for Allele1 and Allele2.
5.5. Block E1. Coverage Plots
Coverage plots are generated for both cDNA and genomic DNA. The minimum
coverage across the reference should be above 20X. For each selected reference allele
in the candidate table, the coverage is plotted along either the cDNA region for the
cDNA coverage plot or the genomic region for the genomic coverage plot. Overall read
coverage is the number of individual sequence reads that were aligned across either
cDNA or genomic reference sequences (Figure 5-21).
To view coverage plots, select the alleles for review and click on the Coverage button
(first button on the left).
When the sequence reads of the sample match the selected reference sequence then
the coverage will be above the baseline across the whole region. When the sequence
reads of the sample is mismatched to the selected reference sequence then the
coverage will dip to the baseline at the mismatched position.
Positions that differ between selected alleles are highlighted with red bars (hash marks)
above the curves. Gray shaded regions display the coverage of all alleles for the gene
minus the coverage of the selected alleles. When the selected alleles map to all the
reads, then there are very low amount of gray in the coverage plot. If there is a
mismatch with the reference there is clear accumulation of reads, represented in grey,
that do not map to the reference sequence at that location.
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Figure 5-21: Coverage plots. The left panel shows coverage along the cDNA reference sequences; the right panel shows coverage along the genomic reference sequences. Red bars (hash marks) above coverage curves indicate positions that are polymorphic between the selected reference
alleles. Windows can be resized.
To view local alignment of selected reference sequences, hold down <Shift> key and
click once at the desired location within the coverage plot. Local alignment of 30 bases
of the selected reference alleles will be displayed near the clicked position (Figure 5-22).
Figure 5-22: Local alignment of sequence fragment of two selected alleles at the clicked position
(Shift-click). Nucleotides that differ between the selected alleles are shown in red text
The coverage plot will zoom in to a selected rectangular region as shown in Figure 5-23. To zoom, click and drag desired region within the plot.
Figure 5-23: Zoomed-in view of a fraction of a coverage plot
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Central coverage plots are generated for both cDNA and genomic DNA. The minimum
coverage across the reference should be above 10X. For each selected reference allele
in the candidate table, the central coverage is plotted along either the cDNA region for
the cDNA coverage plot or the genomic region for the genomic coverage plot.
To view coverage plots, select the alleles for review and click on the Central button
(second button on the left).
When the sequence reads of the sample match the selected reference sequence then
the coverage will be above the baseline across the whole region expect at exon
boundaries in cDNA central coverage plot. When the sequence reads of the sample is
mismatched to the selected reference sequence then the coverage will dip to the
baseline at the mismatched position.
Figure 5-24: Example of central read coverage plot
In the cDNA coverage plot, central reads are undefined at exon boundaries.
5.6. Block E2. Alignment Browsers
Click on the cDNA Browser to view alignment of reads against the cDNA reference
sequence, as shown in Figure 5-25. There are several buttons for navigation. Figure 5-26
and Figure 5-27 show the zoomed-in and zoomed-out view. The first reference sequence
is highlighted with gray background color and the second reference sequence is
highlighted with a tan background color. Those that differ from the first and second
reference sequences are highlighted in light blue and those bases are considered as
noise. Hovering over a read will highlight the entire read with a cyan background color
(See Figure 5-28).
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Figure 5-25: Alignment browser against cDNA reference sequence. Four buttons on the top left corner allow easy navigation of the view. Prev and Next buttons jump to the previous or next
polymorphic sites. Zoom In and Zoom Out buttons will zoom the display in or out. Five tracks on the top of the alignment display coverage, length, poly site, the first and second selected alleles (if
two alleles are selected) and annotation of the reference sequence showing exon and intron locations. The red rectangle marks the cursor position where the position and the number of
nucleotides are displayed in blue text.
Figure 5-26: Zoomed-in view of cDNA alignment browser
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Figure 5-27: Zoomed-out view of cDNA alignment browser. The yellow arrows are reverse reads
and the gray arrows are forward reads
Figure 5-28: Highlight of read under cursor with cyan color in cDNA browser
Click on Genomic Browser to view alignment of the sequence reads against the
genomic reference sequence, as shown in Figure 5-29 to Figure 5-32. Genomic browser
functions the same as those in the cDNA browser.
Due to large number of actual reads, only a partial set of mapped reads are
plotted in the cDNA and genomic browser, while the numbers next to the red
rectangle represent the total number of all mapped reads.
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Figure 5-29: Alignment browser against genomic reference sequence. Four buttons on the top left
corner allow easy navigation of the view. Prev and Next buttons jump to the previous or next polymorphic sites. Zoom In and Zoom Out buttons will zoom the display in or out. Five tracks on top of the alignment display coverage, length, poly site, the first and second selected alleles (if
two alleles are selected) and annotation of the reference sequence showing exon and intron locations. The red rectangle marks the cursor position where the position and number of each
nucleotide are displayed in blue text.
Figure 5-30: Zoomed-in view of the genomic alignment browser
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Figure 5-31: Zoomed-out view of the genomic alignment browser
Figure 5-32: Individual reads under the cursor are highlighted in cyan in the genomic browser. The
deletion is indicated by missing bases in the reference sequence.
5.7. Block E3. Reference Alignment
Click on the Reference Alignment button to view the reference sequence alignment of
selected alleles as shown in Figure 5-33 (for cDNA reference sequences) and in Figure
5-34 (for genomic reference sequences).
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Figure 5-33: cDNA reference alignment of selected references in the candidate table. Pink bars
delineate the exon boundaries. Positions different among selected alleles are highlighted
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Figure 5-34: Genomic reference alignment of selected references in the candidate table. Pink bars delineate the intron-exon boundaries. Positions that differ among the selected alleles are
highlighted
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Click on the Consensus Alignment button to display multiple sequence alignment
between selected alleles and their best-matched de novo assembled contig (Figure 5-35
to Figure 5-38). The Phased Resolved Consensus sequence can be aligned with any
selected candidate alleles by clicking on the Consensus Alignment browser. The
alignment between selected alleles from the candidate table and the best-matched
contig will display. The best-matched contig is defined as the least number of exon
mismatches.
Figure 5-35: Multiple alignment of selected cDNA reference sequences with de novo contig1 sequence. Positions that differ between contig1 and the reference sequence are highlighted.
When no sequence is available the position is denoted with N.
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Figure 5-36: Multiple sequence alignment of selected cDNA reference sequences with de novo
contig2 sequence. Positions that differ between contig2 and the reference sequence are highlighted
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Figure 5-37: Multiple alignment of selected genomic reference sequences with de novo contig1 sequence. Positions different between contig1 sequence and reference sequences are
highlighted. Exon regions are shaded in gray
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Figure 5-38: Multiple alignment of selected genomic reference sequences with de novo contig2
sequence. Positions different between contig2 sequence and reference sequences are highlighted. Exon regions are shaded in gray
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Figure 5-39. Display amino acid codon in the contig sequence alignment window. The amino acid codon can be displayed by double clicking on any nucleotide.
Figure 5-40. Contig Alignment Browser centered at the select base position from contig sequence alignment. To align a specific position in consensus alignment browser with the contig alignment
browser, select and hold shift and double-click on the nucleotide in the consensus alignment browser. A red vertical box will highlight the selected position in the contig alignment browser.
Click on the Clear button to de-select any selected alleles in the candidate table.
5.8. Block F. Contig Alignment Browser
The contig alignment browser (Figure 5-41 to Figure 5-44) displays the de novo assembled
contigs for the selected sample. The functions in the browser are similar to the genomic
alignment browser (Figure 5-29) shown above. In addition, the contig alignment browser
has an “Export Consensus” button allowing the user to export the contigs in fasta
format.
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Figure 5-41: Contig browser showing reads mapped to de novo contigs. Five buttons on the top left corner allow easy navigation of the view. Prev and Next buttons jump to the previous or next
polymorphic sites. Zoom In and Zoom Out buttons will zoom the display in or out. The Export Consensus button allows exporting the contig (consensus) sequences. Five tracks on top of the alignment display coverage, length, poly site, the first and second selected alleles (if two alleles are selected) and annotation of the reference sequence showing exon and intron locations. The
red rectangle marks the cursor position where the position and the number of each nucleotide are displayed in blue text.
Figure 5-42: Zoomed-in view of contig browser showing reads mapped onto de novo contigs
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Figure 5-43: Zoomed-out view of contig browser showing reads mapped onto de novo contigs.
Gray rectangles denote paired-end reads; yellow arrows denote non-paired-end reads (singletons)
Figure 5-44: Contig Browser showing a single read highlighted in cyan; highlighting is visible by
hovering the mouse over the display
The unsequenced region between the paired-end reads is indicated with N.
5.8.1 Interaction between Variants table and contig alignment browser
Click on any position in the first column (Pos) of the Variants table to highlight the
polymorphic site in the contig alignment browser as shown in Figure 5-45.
Figure 5-45: Interaction between Variants table and contig alignment browser; clicking on a position in the first column (Pos) of the Variants table will center the Variants in the contig
alignment browser
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5.8.2 Interaction between cDNA browser and contig alignment browser
Click on a location in the cDNA browser to center it in the contig alignment browser as
shown in Figure 5-46.
Figure 5-46: Interaction between cDNA alignment browser and contig alignment browser. Click on
a position in cDNA browser to center the corresponding position in the contig alignment browser.
The opposite action is also possible (click on contig browser to center the cDNA browser).
5.8.3 Interaction between genomic browser and contig alignment browser
Click on a location in the genomic browser to center the contig alignment browser at the
corresponding position as shown in Figure 5-47.
Figure 5-47: Interaction between genomic alignment browser and contig alignment browser. Click
on a position in genomic browser to center the corresponding position in the contig alignment browser. The opposite action is also possible (click on contig browser to center the genomic
browser)
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5.8.4 Interaction between genomic coverage plot and contig alignment browser
Hold down <Ctrl> key and double click on a position in the genomic coverage plot to
center the contig alignment browser at the corresponding position (Figure 5-48).
Figure 5-48: Interaction between genomic coverage plot and contig alignment browser. Hold down
<Ctrl> key and double-click on a position in genomic coverage plot to center the corresponding position in the contig browser
5.8.5 Interaction between cDNA coverage plot and contig alignment browser
Hold down <Ctrl> key and double-click on a position in cDNA coverage plot to center
the contig browser at the corresponding position (Figure 5-49).
Figure 5-49: Interaction between cDNA coverage plot and contig alignment browser. Hold down <Ctrl> key and double-click on a position in cDNA coverage plot to center the corresponding
position in the contig browser
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5.8.6 Interaction between cDNA coverage plot and cDNA alignment browser
Hold down <Shift> key and double-click on a position in the cDNA coverage plot to open
a new tab displaying the cDNA Alignment Browser and centered at the corresponding
position (Figure 5-50).
Figure 5-50: Interaction between the cDNA coverage plot and the cDNA browser. Hold down
<Shift> key and double-click on a position in the cDNA coverage plot to center the corresponding position in the cDNA browser
5.8.7 Interaction between genomic coverage plot and genomic alignment browser
Hold down <Shift> key and double-click on a position (shown as a blue bar) in the
genomic coverage plot to open a new tab displaying the Genomic Alignment Browser
centered at the corresponding position (Figure 5-51).
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Figure 5-51: Interaction between genomic coverage plot and genomic browser. Hold down <Shift>
key and double-click on a position in the genomic coverage plot to center the corresponding position in the genomic browser
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6. Summary Window
Summary table shows genotype calls for HLA genes for each sample in the project.
HLA genes are listed as columns. Alleles that require manual review is highlighted pink
in the summary table. A Smart Guide tooltip window will show when a user hovers the
mouse cursor over the highlighted allele.
Click on the summary button to bring up the summary window as shown in Figure
6-1. Click on any allele name to open the review window for that sample. Click on a
sample name to open the dialog box to approve that sample.
Figure 6-1: Summary table of genotypes for a project. The Smart Flagging System is displayed
above each allele, see Section 1 for details. Alleles that requires manual review is high-lighted in the summary table. A Smart Guide tooltip window will show when users hover the mouse cursor over the highlighted allele call. Green filled circles next to the sample names indicate approved samples. Right click to open the context menu (show at the right.) The summary table may be
exported in two different formats. The long CSV form shows alleles for one sample in a single row while the short CSV form shows two haplotypes (rows) for one sample. If ambiguity for DPB1 is found, then the equivalent allele pairs will be reported in an extra column in exported CSV file
together with user comments.
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7. Auxiliary Tools
Click on Reference Alignment on the left side bar to open the reference sequence
alignment window as shown in Figure 7-1. Users can use this to check alignment of any
selected alleles of the same locus.
Figure 7-1: Reference Alignment Tool. The top left panel allows the user to switch between cDNA and genomic as well as switch between different genes; the left panel allows the user to choose
alleles and the right panel displays alignments for the selected alleles
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Click on the Codon button on the left side bar to display a DNA codon table as
shown in Figure 7-2.
Figure 7-2: DNA Codon Table
Click on the Backup button on the left side bar to export a project into external
storage. After the user selects the location to store the backup data through a file dialog,
a status bar will appear in the lower right hand of the software window to indicate the
backup status.
Click on the Restore button on the left side bar to import a project from external
storage. After the user selects the backed up folder through a file dialog, a loading bar
will appear in the lower right hand of the software window to indicate the restore status.
Click on the Export log button on the left side bar to export the project log to a file.
Click on the Search button to activate the search features available to query for
detailed criteria on samples or alleles. The search feature can also be accessed from
the top menu: Open Tools and select Search, and the search window will appear. The
search has several criteria that can be used to find specific samples or alleles for the 9
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HLA genes (A, B, C, DPA1, DPB1, DQA1, DQB1, DRB1, DRBo which includes DRB3,
4, and 5).
The options in the search window are explained in Figure 7-3 (Boxes A–H). After setting
up the options in the search window, click on the Find button to find projects in the
specified date range that contain the samples or alleles consistent with the search term.
To search for a specific allele, select “Allele” from the pull down menu in Box A. To
search specific samples, select “Sample” from the pull down menu in Box A. Enter a
search term for the sample or allele name in Box B. Format restriction is applied when
searching for an Allele.
The search term for allele must follow the following format: Gene*allele name. For
example, to find samples and projects that contain the allele DRB1*01:01:01 of gene
DRB1, the search term should be DRB1*01:01:01.
The user can search projects that contain linked alleles of the 9 HLA genes by entering
alleles separated by commas. For example, to find projects that have alleles A*01:01:01
and DRB1*01:01:01, the search terms for the two alleles should be entered in Box B as
A*01:01:01, DRB1*01:01:01. The search terms for alleles can be in any order.
Each gene should be entered only once in the search box.
To gain more search flexibility, the percent “%” and the underscore “_” wildcard
characters can be used in the search terms. Also, the prefix can be used to search for
the desired allele or sample. For example, if the search term in Box B is DRB1*01 then
it would be considered to be DRB1*01%. In this case, all alleles that start with DRB1*01
will be displayed. If the search term in Box B is DRB1*0_ then both DRB1*01, DRB1*02,
until DRB1*09 will be displayed.
To search the date range, select created after date in Box D and created before date in
Box E. The default values of Box D and Box E are respectively set to be the creation
dates of the earliest and latest projects currently available in the project table.
Box F has two states: checked or unchecked. When Box F is checked the allele search
term is case sensitive. If this box is unchecked then the matching is not case sensitive.
When Box G is checked, the search term must be exactly match with the allele name or
sample name in the projects. In this setting, “%” and “_” are not considered to be
wildcard characters.
Note that “%” and “_” are wildcard characters only if Box G is unchecked. In this case
the search term will be considered a match with the name of an allele if the search term
is a prefix of the allele name. For example, if the search term in Box B is DRB1*01 then
it would be considered to be DRB1*01%. In this case, all alleles that start with DRB1*01
are considered to be matching.
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The output of the search will be displayed in Box H. Each row displays the sample
name, if there is genotype data, barcode ID, project name, the date of project creation,
and the name of the user who created the project. Double-clicking on a row will display
the review panel of that sample and project.
Figure 7-3: Search window. A, Select Allele or Sample for search criteria; B, Box to enter search
term; C, Find button; D and E, Select the desired date range; F, when this box is checked the search term is case sensitive; G, when this box is checked the search term is exactly matched to
the names of alleles or samples and % and _ are not considered wildcard characters; H, the output of the search
The content in the search results table is clickable to directly open the appropriate review panel.
Click on the NMDP button to open a wizard and view instructions for updating the
NMDP ambiguity code (Figure 7-4, Figure 7-5, Figure 7-6, Figure 7-7)
Figure 7-4: Welcome page for NMDP ambiguity code wizard
Figure 7-5: Filled file input page of NMDP ambiguity code wizard. Users need to download the file
from the HLA resource page: https://bioinformatics.bethematchclinical.org/HLA-Resources/Allele-Codes/Allele-Code-Lists/Allele-Code-List-in-Numerical-Order
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Figure 7-6: Sample of input file for the NMDP ambiguity code wizard
Figure 7-7: Upon clicking Finish, the NMDP ambiguity code wizard asks to verify the input
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SironaQuant
Click on the SironaQuant button to open the wizard to compute balancing metrics used
in the MIA FORA NGS HLA Typing Kit (Figure 7-8). The SironaQuant program is
designed to seamlessly balance a sample plate so amplicons from all loci are
represented in sufficient quantity in the pooled library during sequencing.
Figure 7-8: Welcome page for the SironaQuant wizard
Each sample is amplified individually for all genes. The user first quantifies the
concentration of the sample with the Picogreen quantification assay, and then
generates a file containing the fluorescence data for each well. The dialog window is
shown in Figure 7-9.
The file names are expected to have three or four parts – project name, column name,
and user comments - separated by underscores (“_”). For example, the following two
file names are acceptable:
Project01_COLUMN1_Date.txt
Project_01_COLUMN1_Date.txt
Once a file for each of the three-sample plates has been uploaded into the SironaQuant
program, the user chooses a picomole value for normalization. The program then
extracts the fluorescence data from the files and balances the genes, if possible with the
chosen picomole amount.
Figure 7-9: Filled information page of the SironaQuant wizard
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The input files need to be text tab delimited if you are using Windows operating system,
or Windows Formatted Text if you are using Mac OSX operating system. The sixth
column contains the fluorescence values ordered as shown by the well location as
shown in Figure 7-10. If the user uses the Victor X3 system, the file is automatically
generated in the proper format. For other fluorescence readers the user must load the
data into the template file provided with the MIA FORA NGS system.
Figure 7-10: Sample input files for the SironaQuant wizard
The fluorescence data for the standard curve is first extracted from the first sample data
file. The slope, y-intercept and r-squared value are calculated using an average of the
fluorescence data for all replicates of each standard. The concentration of each sample
is calculated by applying the standard curve values to the fluorescence value. Then, the
molarity of the sample is calculated and the volume needed to pool at the desired
picomole value is calculated.
This process repeats for all genes. After all of the transfer volumes have been
calculated, the volume is calculated to verify that the total volume for all genes for each
sample does not exceed 55 µL, the maximum volume dictated by the protocol. After the
individual pooling volumes are determined, the volume of buffer needed to bring the
total volume to 55 µL is calculated.
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Figure 7-11: Conclusion page for the SironaQuant wizard
Once the transfer volumes have been calculated for all genes, the program outputs a
summary file (Figure 7-12) that contains, the normalization transfer volume for
normalization, the dilution volume (if any), the picomole(pmol) value that the gene was
normalized to, the total amount of pooled amplicons in nanogram (ng), the length of the
gene in base pairs, and identifiers for samples that have been excluded from the
average for each locus. The summary file also displays the standard curve slope, y-
intercept, and r-squared value, and the volume of buffer required to bring the total
pooled volume to 55 µL. Thus the balancing program provide a way for all the
amplicons to be represented in the sequencing to facilitate HLA typing of all loci, even
the ones that have allelic imbalance. It also serves to warn the user of failures early in
the sample-preparation process so that adjustments can be made prior to sequencing.
Warnings Display:
1. If the total volume exceeds the limit (55ul), an alert is displayed to the user and
the program does not proceed. The user then chooses a lower picomole value.
The minimum recommended picomole value is 0.0009 and the maximum is
0.0035.
2. If there are more than 8 samples with very low fluorescence values, an alert is
displayed to the user Figure 7-13. This alert is only as a warning to the user that
there are many failures in the plate and can be bypassed if the user choses to
proceed by clicking on the “Ignore warnings” option. If there are fewer than 8
samples with a low concentration failure, but more than 1, they are listed as
outliers in the output file. If the user chooses, they can redo the PCR for the
failed loci with remaining reagents in the kit. There is sufficient reagent to redo at
least 4 PCR reactions per locus.
3. If fluorescence values are not 25% larger than the negative control of the
corresponding gene, a warning message suggests user to redo PCR before
proceeding with library construction.
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These warning messages should not be ignored in order to get a good
representation of all the loci. Ignoring warnings may result in “insufficient data”
for these loci.
Figure 7-12: Sample output file for the Sirona Quant wizard
Figure 7-13: Sample Warning Message from the Sirona Quant wizard; click OK to get back to the
input page, and input a different pmole value or check “Ignore Warnings“ to continue
Figure 7-14: Filled information page of the Sirona Quant wizard with checked “Ignore Warnings”
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Appendix A: Software Color Codes Description
Several color codes are used throughout the software. Below is a description of different
color codes.
A.1. Variants Table
Amber colored boxes in the Variants table may appear in the Contig or Block columns.
In Contig1 and Contig2 columns the amber colored boxes indicate that the ratio of the
two alleles is outside the expected standard deviation (Figure A1-B). In the Block
column the amber colored box indicates the first polymorphism in a phasing block
(Figure A1-A).
A B
Figure A1: Variants table colors. A, amber box in block column showing the beginning of a phasing block; B, amber boxes showing three polymorphisms that have a ratio outside the
standard deviation.
A.2. Genotype Table
The PC column in the genotype table displays an indicator of the number of alleles
detected by phasing consensus (PC.) A green circle indicates that the number of PC
alleles matches the number of computed alleles. An amber circle indicates that the
number of PC alleles does not match the number of computed alleles. Regardless of
color, the reviewer should verify all genes with only a single allele listed.
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Figure A2: Display of Genotype Table colors. Numbers in PC column are the number of alleles detected
by phasing consensus, green means the PC number matches the computed allele number while amber
means the two numbers do not match. The pink highlighted boxes show alleles that should be manually
reviewed.
A.3. LD Suggestion Table
The yellow highlights indicate alleles that are listed in the genotype assignment table.
Figure A3: Highlight use within the LD Suggestion Table
A.4. Allele Candidate table
The check symbol in the candidate table could be either green or amber. The amber
check symbol suggests that the locus requires manual review and the reason is given in
the smart guide.
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Figure A4-1: Allele Candidate Table with green check marks.
Figure A4-2: Allele Candidate Table with amber check marks.
A.5. Smart Guide
Smart Guide provides guidelines for reviewing the HLA typing results. In the review
window, Smart Guide is shown in the lower left panel as a tab together with “Variants”
and “LD Info” tabs. Recommended actions are described to give instructions of review
and reasons may be given to explain why a user should check the specified information
in the review window.
Figure A5: Smart guide with listed reasons and actions
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Appendix B: Glossary
Allele: An allele is a variant form of a gene.
Assembly: The genome assembly is simply the genome sequence produced after
chromosomes have been fragmented, those fragments have been sequenced, and the
resulting sequences have been put back together.
Central Read: Central Reads are empirically defined as mapped reads for which the
ratio between the length of the left arm and that of the right arm related to a particular
point is between 0.5 and 2. When reads are mapped onto a correct reference
sequence, they form a continuous tiling pattern over the entire sequenced region.
Contig: A contig (from contiguous) is a set of overlapping DNA segments that together
represent a consensus region of DNA.
Coverage: the percentage of bases called at predetermined depth for a genomic region
of interest.
eRead: The number of unique reads mapped to the partial cDNA sequence containing
only Exons 2 and 3 for class I loci and only Exon 2 for Class II loci.
FASTQ File: A text based format for sequences data with corresponding quality
indicators for each base.
gRead: The number of unique reads mapped to the genomic reference
sequences.HLA: The human leukocyte antigen (HLA) system is the locus of genes
that encode for proteins on the surface of cells that are responsible for regulation of the
immune system in humans. HLAs corresponding to MHC class I (A, B, and C) present
peptides from inside the cell. HLAs corresponding to MHC class II (DP, DQ, and DR)
present antigens from outside of the cell to T-lymphocytes.
Locus: A locus (plural loci), in genetics, is the specific location or position of a gene,
DNA sequence, on a chromosome.
Mismatched Exon (MME): The number of mismatched nucleotides in the exon region
after comparing the best-matched contig. For DRB1, DRB3, DRB4 and DRB5, exon 1
is excluded in the calculation because the region may not have sufficient amplification
product.
Mismatched Intron (MMI): The number of mismatched nucleotides in the intron region
after comparing the best-matched contig.
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Q score (Q30): Q scores measures the probability of wrong base call which is
logarithmically related to the base calling error probabilities. A Q score of 30 (Q30) is
equivalent to the probability of an incorrect base call 1 in 1000 times.
Paired-end read mapping: A set of independent reads that are derived from the same
library fragment which can be used to identify structural rearrangements.
Phasing: Historically, whole-genome sequencing generated a single consensus
sequence without distinguishing between variants on homologous chromosomes.
Phased sequencing, or genome phasing, addresses this limitation by identifying alleles
on maternal and paternal chromosomes. This information is often important for
understanding gene expression patterns for genetic disease research.
Read Alignment: Position DNA sequence reads along the genome in relation to a
reference sequence.
Reference Sequence: A fully sequenced and assembled genome that acts as a
scaffold against which new sequence reads are aligned and compared.
xRead: The number of unique reads mapped to partial genomic reference sequences
except Exons 2 and 3 for Class I and Exon 2 for Class II.
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Appendix C: Best Practices
1. To mitigate the risk of any mismatch between a sample sheet and the MiSeq
data, the name of the corresponding fastq files must begin with the unique
complete project name created in the MIA FORA software. Users must create
complete project name before initiating the MiSeq sequence run.
2. Once Analysis indicator is green, first navigate to Statistics window to review
quality of run (a) invalid read percentage should be less than 10% (b) average
total read for entire project should be >200,000 (c) the quality score should be
above Q30. Due to the complex nature of HLA typing, qualified personnel should
review data interpretation and typing assignments.
3. After reviewing Statistics, navigate to the Summary window to review genotype
calls for HLA genes for each sample in the project. Alleles require manual review
if highlighted pink in the summary table. A Smart Guide tooltip window will show
when users hover the mouse cursor over the highlighted allele call.
4. When reviewing genotype results, any homozygous locus must be reviewed.
Recommended review process includes but not limited to (a) review candidate
pair table for top ranked candidate pair for presence of second allele, (b) review
status of PC indicator for inconsistency (c) if possible second allele is present,
select Allele 1 and Allele 2 and click both coverage plot and central read
coverage plot to confirm uniform coverage above baseline (d) if coverage plot of
second allele display uniform coverage, review mapped sequence using
alignment browser and phased resolved de novo contig for second allele using
the consensus browser.
5. When reviewing genotype results, review any samples that have amber diamond
indicating non-CWD alleles. Recommended review process includes but not
limited to (a) click both coverage plot and central read coverage plot to confirm
uniform coverage above baseline (b) if coverage plot of allele display uniform
coverage, then review mapped sequence using alignment browser and phased
resolved de novo contig for second allele using the consensus browser.
6. When reviewing genotype results, review any samples that have amber hexagon
indicating inconsistency in LD data. Recommended review process includes but
not limited to (a) click both coverage plot and central read coverage plot to
confirm uniform coverage above baseline (b) if coverage plot of allele display
uniform coverage, then review mapped sequence using alignment browser and
phased resolved de novo contig for allele using the consensus browser.
7. When reviewing genotype results, review any samples that have amber circle
containing a ? symbol indicating discordance between the automatic allele call
and phase resolved consensus. Recommended review process includes but not
limited to (a) review MME column for selected allele (b) identify the mismatched
position(s) between the select allele and reference sequence in the exon region
using consensus alignment browser (c) then review mapped sequence using
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alignment browser and phased resolved de novo contig allele using the
consensus browser.
8. When reviewing genotype results, review any samples that have amber circle in
the PC column indicating inconsistency in phase resolved consensus and
mapped sequence. Recommended review process includes but not limited to (a)
review candidate pair table for top ranked candidate pair (b) click both coverage
plot and central read coverage plot to confirm uniform coverage above baseline
(c) if coverage plot of allele display uniform coverage, then review mapped
sequence using alignment browser and phased resolved de novo contig for allele
using the consensus browser.
9. When navigating the allele candidate table, users should pay attention to the
minimum coverage column (Cov) and minimum central read coverage column
(Cen) of cDNA (cRead).
10. After manual review of each sample, select Approve and Confirm in order to
activate the Report feature. The sample must be Confirmed for the Approval
Status to display on Project window.
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Appendix D: Clickable Functions
C.1. Main MIA FORA NGS Project Window:
# Action Intention
1. Left Click on any heading; i.e. Project Name, Created On, Analyzed On, Lot, Received on, or Operator
Organize project list by the specific criteria clicked on
2. Left Click on the up or down arrows in the extreme top right corner of the frame
Maximize or minimize MIA FORA NGS window
3. Mouse over the top center of the VNC frame
Reveals dropdown menu for specifying destination file for transferring files, maximizing the window, exiting, etc.
4. Right Click on VNC icon at the bottom right corner of VNC frame
Shortcut to the VNC file transfer menu
C.2. Statistics Window:
# Action Intention
1. Right click on any Graph or Chart Option to Print appears, and the graph or chart can be saved or printed
2. Left click on any tab at the top of the statistics graphs/charts
Open specified tab
C.3. Review Window:
# Action Intention
1. Left Click on any heading in the Candidate Allele Table
Organize table by specified column
2. Right Click on any Table Option to save the table appears
3. Left Double-Click on Call column Toggle allele call on/off
4. Left Double-Click on Cmt column Comment window appears
5. Shift Left Click on Coverage Graphs Displays the allele sequence at the clicked region
6. Shift Left Double-Click on Coverage Graphs
Loads the Genomic or cDNA Alignment Browser for the region clicked on
7. Left Click, hold and drag cursor on Coverage Graphs
Zoom in on a specific region of Coverage Graphs (Double left click to zoom out)
8. Ctrl Left Click on Coverage Graphs Centers and displays selected region in contig alignment browser
9. Right Click on Coverage Graphs Option to save the graphs appears
10. Left Click on a polymorphism position (first column)
Open specified polymorphism location in the contig alignment browser
11. Hover over any grab bar of a window and left click, hold and drag
Increase or decrease the size of a window
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C.4. Summary Window:
# Action Intention
1. Right click anywhere on summary table Options to export and save the summary table are displayed
2. Hover over pink highlighted allele in summary table
A Smart Guide tooltip window will show when users hover the mouse cursor over the highlighted allele call.
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Appendix E: Additional Resources
MIA FORA NGS algorithm and coverage statistics: Chunlin Wang, Sujatha
Krishnakumar, Julie Wilhelmy, Farbod Babrzadeh, Lilit Stepanyan, Laura F. Su,
Douglas Levinson, Marcelo A. Fernandez-Viña, Ronald W. Davis, Mark M. Davis, and
Michael Mindrinos. High-throughput, high-fidelity HLA genotyping with deep
sequencing. PNAS 2012 109(22):8678-8681.
Relative proportions of DRB1-DRB3/4/5-DQB1 haplotypes in major ethnic groups of the
U.S. (selected, not random): Burdett L, Smith K, Tu B , Guiterrez M , Buck K. , Maiers
M., Ng J., Hartzman R. and Fernandez-Vina M. DRB-DQB1 diversity in the analysis of
4727 donors typed by SBT. (abstract) Hum Immunol. 2003 Oct;64(10 Suppl):S6.
HLA CWD allele Database: http://igdawg.org/cwd.html
International HLA and Immunogenetics Workshops: http://ihiws.org/
International Immunogenetics Information System: http://www.imgt.org
Online alignment tool: http://www.ebi.ac.uk/ipd/imgt/hla/align.html
Haplotype frequency table: https://bioinformatics.bethematchclinical.org/HLA-
Resources/Haplotype-Frequencies/
Online allele nomenclature lookup tool: http://www.marrow-donor.org/cgi-
bin/DNA/dnatyp.pl
VNC Viewer User Guide:
realvnc.com/products/vnc/documentation/5.2/guides/user/VNC_User_Guide.pdf
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Appendix F: Third Party Libraries’ Licenses
Trademark and Copyright Information
MIA FORA™ is a registered trademark of Sirona Genomics Inc. MIA FORA is copyright © Sirona
Genomics Inc, 2016. All rights reserved.
Sirona Genomics Inc.
1916A Old Middlefield Way, Mountain View, CA 94043
[email protected] +1 650-396-2409
http://www.sironagenomics.com
Other programs mentioned in this software are trademarked and copyrighted by their respective owners.
Portions of this software use Klib; Klib is a standalone and lightweight C library distributed under
MIT/X11 license.
Copyright (c) 2008, 2009, 2011 by Attractive Chaos <[email protected]>
Permission is hereby granted, free of charge, to any person obtaining a copy of this software and
associated documentation files (the "Software"), to deal in the Software without restriction, including
without limitation the rights to use, copy, modify, merge, publish, distribute, sublicense, and/or sell copies
of the Software, and to permit persons to whom the Software is furnished to do so, subject to the
following conditions:
The above copyright notice and this permission notice shall be included in all copies or substantial
portions of the Software.
THE SOFTWARE IS PROVIDED "AS IS", WITHOUT WARRANTY OF ANY KIND, EXPRESS OR
IMPLIED, INCLUDING BUT NOT LIMITED TO THE WARRANTIES OF MERCHANTABILITY, FITNESS
FOR A PARTICULAR PURPOSE AND NONINFRINGEMENT. IN NO EVENT SHALL THE AUTHORS
OR COPYRIGHT HOLDERS BE LIABLE FOR ANY CLAIM, DAMAGES OR OTHER LIABILITY,
WHETHER IN AN ACTION OF CONTRACT, TORT OR OTHERWISE, ARISING FROM, OUT OF OR IN
CONNECTION WITH THE SOFTWARE OR THE USE OR OTHER DEALINGS IN THE SOFTWARE.
Portions of this software use SSW; SSW is a fast implementation of the Smith-Waterman
algorithm distributed under MIT license.
Copyright (c) 2012-2015 Boston College
Permission is hereby granted, free of charge, to any person obtaining a copy of this software and
associated documentation files (the "Software"), to deal in the Software without restriction, including
without limitation the rights to use, copy, modify, merge, publish, distribute, sublicense, and/or sell copies
of the Software, and to permit persons to whom the Software is furnished to do so, subject to the
following conditions:
The above copyright notice and this permission notice shall be included in all copies or substantial
portions of the Software.
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THE SOFTWARE IS PROVIDED "AS IS", WITHOUT WARRANTY OF ANY KIND, EXPRESS OR
IMPLIED, INCLUDING BUT NOT LIMITED TO THE WARRANTIES OF MERCHANTABILITY, FITNESS
FOR A PARTICULAR PURPOSE AND NONINFRINGEMENT. IN NO EVENT SHALL THE AUTHORS
OR COPYRIGHT HOLDERS BE LIABLE FOR ANY CLAIM, DAMAGES OR OTHER LIABILITY,
WHETHER IN AN ACTION OF CONTRACT, TORT OR OTHERWISE, ARISING FROM, OUT OF OR IN
CONNECTION WITH THE SOFTWARE OR THE USE OR OTHER DEALINGS IN THE SOFTWARE.
Portions of this software use the work of the FreeType Project, copyright © 2015 The FreeType
Project.
The FreeType Project LICENSE
2006-Jan-27
Copyright 1996-2002, 2006 by
David Turner, Robert Wilhelm, and Werner Lemberg
Introduction
The FreeType Project is distributed in several archive packages; some of them may contain, in addition to
the FreeType font engine, various tools and contributions which rely on, or relate to, the FreeType
Project.
This license applies to all files found in such packages, and which do not fall under their own explicit
license. The license affects thus the FreeType font engine, the test programs, documentation and
makefiles, at the very least.
This license was inspired by the BSD, Artistic, and IJG (Independent JPEG Group) licenses, which all
encourage inclusion and use of free software in commercial and freeware products alike. As a
consequence, its main points are that:
o We don't promise that this software works. However, we will be interested in any kind of bug reports. (`as is' distribution)
o You can use this software for whatever you want, in parts or full form, without having to pay us. (`royalty-free' usage)
o You may not pretend that you wrote this software. If you use it, or only parts of it, in a program, you must acknowledge somewhere in your documentation that you have used the FreeType code. (`credits')
We specifically permit and encourage the inclusion of this software, with or without modifications, in
commercial products. We disclaim all warranties covering The FreeType Project and assume no liability
related to The FreeType Project.
Finally, many people asked us for a preferred form for a credit/disclaimer to use in compliance with this
license. We thus encourage you to use the following text:
"""
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Portions of this software are copyright 2015 The FreeType Project (www.freetype.org). All
rights reserved.
"""
Please replace <year> with the value from the FreeType version you actually use.
Legal Terms
0. Definitions Throughout this license, the terms `package', `FreeType Project', and `FreeType archive' refer to the
set of files originally distributed by the authors (David Turner, Robert Wilhelm, and Werner Lemberg)
as the `FreeType Project', be they named as alpha, beta or final release.
`You' refers to the licensee, or person using the project, where `using' is a generic term including
compiling the project's source code as well as linking it to form a `program' or `executable'.
This program is referred to as `a program using the FreeType engine'. This license applies to all files
distributed in the original FreeType Project, including all source code, binaries and documentation,
unless otherwise stated in the file in its original, unmodified form as distributed in the original archive.
If you are unsure whether or not a particular file is covered by this license, you must contact us to
verify this.
The FreeType Project is copyright (C) 1996-2000 by David Turner, Robert Wilhelm, and Werner
Lemberg. All rights reserved except as specified below.
1. No Warranty THE FREETYPE PROJECT IS PROVIDED `AS IS' WITHOUT WARRANTY OF ANY KIND,
EITHER EXPRESS OR IMPLIED, INCLUDING, BUT NOT LIMITED TO, WARRANTIES OF
MERCHANTABILITY AND FITNESS FOR A PARTICULAR PURPOSE. IN NO EVENT WILL
ANY OF THE AUTHORS OR COPYRIGHT HOLDERS BE LIABLE FOR ANY DAMAGES
CAUSED BY THE USE OR THE INABILITY TO USE, OF THE FREETYPE PROJECT.
2. Redistribution This license grants a worldwide, royalty-free, perpetual and irrevocable right and license to use,
execute, perform, compile, display, copy, create derivative works of, distribute and sublicense the
FreeType Project (in both source and object code forms) and derivative works thereof for any
purpose; and to authorize others to exercise some or all of the rights granted herein, subject to
the following conditions:
o Redistribution of source code must retain this license file (`FTL.TXT') unaltered; any additions, deletions or changes to the original files must be clearly indicated in accompanying documentation. The copyright notices of the unaltered, original files must be preserved in all copies of source files.
o Redistribution in binary form must provide a disclaimer that states that the software is based in part of the work of the FreeType Team, in the distribution documentation. We also encourage you to put an URL to the FreeType web page in your documentation, though this isn't mandatory.
These conditions apply to any software derived from or based on the FreeType Project, not just
the unmodified files. If you use our work, you must acknowledge us. However, no fee need be
paid to us.
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3. Advertising
Neither the FreeType authors and contributors nor you shall use the name of the other for
commercial, advertising, or promotional purposes without specific prior written permission.
We suggest, but do not require, that you use one or more of the following phrases to refer to this
software in your documentation or advertising materials: `FreeType Project', `FreeType Engine',
`FreeType library', or `FreeType Distribution'.
As you have not signed this license, you are not required to accept it. However, as the FreeType
Project is copyrighted material, only this license, or another one contracted with the authors,
grants you the right to use, distribute, and modify it. Therefore, by using, distributing, or modifying
the FreeType Project, you indicate that you understand and accept all the terms of this license.
4. Contacts There are two mailing lists related to FreeType:
Discusses general use and applications of FreeType, as well as future and wanted
additions to the library and distribution. If you are looking for support, start in this list if
you haven't found anything to help you in the documentation.
Discusses bugs, as well as engine internals, design issues, specific licenses, porting, etc.
Our home page can be found at
http://www.freetype.org
Portions of this software use the work of the Independent JPEG Group. This software is copyright
© 1991-1998, Thomas G. Lane.
Independent JPEG Group License
LEGAL ISSUES
In plain English:
1. We don't promise that this software works. (But if you find any bugs, please let us know!)
2. You can use this software for whatever you want. You don't have to pay us.
3. You may not pretend that you wrote this software. If you use it in a program, you must acknowledge
somewhere in your documentation that you've used the IJG code.
In legalese:
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The authors make NO WARRANTY or representation, either express or implied, with respect to this
software, its quality, accuracy, merchantability, or fitness for a particular purpose. This software is
provided "AS IS", and you, its user, assume the entire risk as to its quality and accuracy.
This software is copyright (C) 1991-1998, Thomas G. Lane. All Rights Reserved except as specified
below.
Permission is hereby granted to use, copy, modify, and distribute this software (or portions thereof) for
any purpose, without fee, subject to these conditions:
(1) If any part of the source code for this software is distributed, then this README file must be
included, with this copyright and no-warranty notice unaltered; and any additions, deletions, or
changes to the original files must be clearly indicated in accompanying documentation.
(2) If only executable code is distributed, then the accompanying documentation must state that "this
software is based in part on the work of the Independent JPEG Group".
(3) Permission for use of this software is granted only if the user accepts full responsibility for any
undesirable consequences; the authors accept NO LIABILITY for damages of any kind.
These conditions apply to any software derived from or based on the IJG code, not just to the unmodified
library. If you use our work, you ought to acknowledge us.
Permission is NOT granted for the use of any IJG author's name or company name in advertising or
publicity relating to this software or products derived from it. This software may be referred to only as "the
Independent JPEG Group's software".
We specifically permit and encourage the use of this software as the basis of commercial products,
provided that all warranty or liability claims are assumed by the product vendor.
ansi2knr.c is included in this distribution by permission of L. Peter Deutsch, sole proprietor of its copyright
holder, Aladdin Enterprises of Menlo Park, CA. ansi2knr.c is NOT covered by the above copyright and
conditions, but instead by the usual distribution terms of the Free Software Foundation; principally, that
you must include source code if you redistribute it. (See the file ansi2knr.c for full details.) However, since
ansi2knr.c is not needed as part of any program generated from the IJG code, this does not limit you
more than the foregoing paragraphs do.
The Unix configuration script "configure" was produced with GNU Autoconf. It is copyright by the Free
Software Foundation but is freely distributable. The same holds for its supporting scripts (config.guess,
config.sub, ltconfig, ltmain.sh). Another support script, install-sh, is copyright by M.I.T. but is also freely
distributable.
It appears that the arithmetic coding option of the JPEG spec is covered by patents owned by IBM, AT&T,
and Mitsubishi. Hence arithmetic coding cannot legally be used without obtaining one or more licenses.
For this reason, support for arithmetic coding has been removed from the free JPEG software. (Since
arithmetic coding provides only a marginal gain over the unpatented Huffman mode, it is unlikely that very
many implementations will support it.) So far as we are aware, there are no patent restrictions on the
remaining code.
The IJG distribution formerly included code to read and write GIF files. To avoid entanglement with the
Unisys LZW patent, GIF reading support has been removed altogether, and the GIF writer has been
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simplified to produce "uncompressed GIFs". This technique does not use the LZW algorithm; the resulting
GIF files are larger than usual, but are readable by all standard GIF decoders.
We are required to state that
"The Graphics Interchange Format(c) is the Copyright property of CompuServe Incorporated. GIF(sm) is
a Service Mark property of CompuServe Incorporated."
Portions of this software use libpng; libpng versions 1.0.7, July 1, 2000, through 1.6.18, July 23,
2015, are Copyright (c) 2000-2002, 2004, 2006-2015 Glenn Randers-Pehrson, and are distributed
according to the same disclaimer and license as libpng-1.0.6 with the following individuals added
to the list of Contributing Authors: Simon-Pierre Cadieux, Eric S. Raymond, Mans Rullgard,
Cosmin Truta, Gilles Vollant, James Yu and with the following additions to the disclaimer: There is
no warranty against interference with your enjoyment of the library or against infringement. There
is no warranty that our efforts or the library will fulfill any of your particular purposes or needs.
This library is provided with all faults, and the entire risk of satisfactory quality, performance,
accuracy, and effort is with the user.
This copy of the libpng notices is provided for your convenience. In case of any discrepancy between this
copy and the notices in the file png.h that is included in the libpng distribution, the latter shall prevail.
COPYRIGHT NOTICE, DISCLAIMER, and LICENSE:
If you modify libpng you may insert additional notices immediately following this sentence.
This code is released under the libpng license.
libpng versions 1.0.7, July 1, 2000, through 1.6.19, November 12, 2015, are Copyright (c) 2000-2002,
2004, 2006-2015 Glenn Randers-Pehrson, are derived from libpng-1.0.6, and are distributed according to
the same disclaimer and license as libpng-1.0.6 with the following individuals added to the list of
Contributing Authors:
Simon-Pierre Cadieux
Eric S. Raymond
Mans Rullgard
Cosmin Truta
Gilles Vollant
James Yu
and with the following additions to the disclaimer:
There is no warranty against interference with your enjoyment of the library or against infringement.
There is no warranty that our efforts or the library will fulfill any of your particular purposes or needs.
This library is provided with all faults, and the entire risk of satisfactory quality, performance, accuracy,
and effort is with the user.
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libpng versions 0.97, January 1998, through 1.0.6, March 20, 2000, are Copyright (c) 1998-2000 Glenn
Randers-Pehrson, are derived from libpng-0.96, and are distributed according to the same disclaimer and
license as libpng-0.96, with the following individuals added to the list of Contributing Authors:
Tom Lane
Glenn Randers-Pehrson
Willem van Schaik
libpng versions 0.89, June 1996, through 0.96, May 1997, are Copyright (c) 1996-1997 Andreas Dilger,
are derived from libpng-0.88, and are distributed according to the same disclaimer and license as libpng-
0.88, with the following individuals added to the list of Contributing Authors:
John Bowler
Kevin Bracey
Sam Bushell
Magnus Holmgren
Greg Roelofs
Tom Tanner
libpng versions 0.5, May 1995, through 0.88, January 1996, are Copyright (c) 1995-1996 Guy Eric
Schalnat, Group 42, Inc.
For the purposes of this copyright and license, "Contributing Authors" is defined as the following set of
individuals:
Andreas Dilger
Dave Martindale
Guy Eric Schalnat
Paul Schmidt
Tim Wegner
The PNG Reference Library is supplied "AS IS". The Contributing Authors and Group 42, Inc. disclaim all
warranties, expressed or implied, including, without limitation, the warranties of merchantability and of
fitness for any purpose. The Contributing Authors and Group 42, Inc. assume no liability for direct,
indirect, incidental, special, exemplary, or consequential damages, which may result from the use of the
PNG Reference Library, even if advised of the possibility of such damage.
Permission is hereby granted to use, copy, modify, and distribute this source code, or portions hereof, for
any purpose, without fee, subject to the following restrictions:
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1. The origin of this source code must not be misrepresented.
2. Altered versions must be plainly marked as such and must not be misrepresented as being the original source.
3. This Copyright notice may not be removed or altered from any source or altered source distribution.
The Contributing Authors and Group 42, Inc. specifically permit, without fee, and encourage the use of
this source code as a component to supporting the PNG file format in commercial products. If you use
this source code in a product, acknowledgment is not required but would be appreciated.
Portions of this software use PCRE; PCRE is a library of functions to support regular expressions
whose syntax and semantics are as close as possible to those of the Perl 5 language. THE BASIC
LIBRARY FUNCTIONS were written by Philip Hazel [email protected]. Copyright © 1997-2012
University of Cambridge. All rights reserved.
PCRE2 LICENCE
PCRE2 is a library of functions to support regular expressions whose syntax and semantics are as close
as possible to those of the Perl 5 language.
Release 10 of PCRE2 is distributed under the terms of the "BSD" license, as specified below. The
documentation for PCRE2, supplied in the "doc" directory, is distributed under the same terms as the
software itself. The data in the testdata directory is not copyrighted and is in the public domain.
The basic library functions are written in C and are freestanding. Also included in the distribution is a just-
in-time compiler that can be used to optimize pattern matching. This is an optional feature that can be
omitted when the library is built.
THE BASIC LIBRARY FUNCTIONS
Written by: Philip Hazel
Email local part: ph10
Email domain: cam.ac.uk
University of Cambridge Computing Service, Cambridge, England.
Copyright (c) 1997-2015 University of Cambridge All rights reserved.
PCRE2 JUST-IN-TIME COMPILATION SUPPORT
Written by: Zoltan Herczeg
Email local part: hzmester
Email domain: freemail.hu
Copyright(c) 2010-2015 Zoltan Herczeg All rights reserved.
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STACK-LESS JUST-IN-TIME COMPILER
Written by: Zoltan Herczeg
Email local part: hzmester
Email domain: freemail.hu
Copyright(c) 2009-2015 Zoltan Herczeg All rights reserved.
THE "BSD" LICENCE
Redistribution and use in source and binary forms, with or without modification, are permitted provided
that the following conditions are met:
Redistributions of source code must retain the above copyright notice, this list of conditions and the following disclaimer.
Redistributions in binary form must reproduce the above copyright notice, this list of conditions and the following disclaimer in the documentation and/or other materials provided with the distribution.
Neither the name of the University of Cambridge nor the names of any contributors may be used to endorse or promote products derived from this software without specific prior written permission.
THIS SOFTWARE IS PROVIDED BY THE COPYRIGHT HOLDERS AND CONTRIBUTORS "AS IS" AND
ANY EXPRESS OR IMPLIED WARRANTIES, INCLUDING, BUT NOT LIMITED TO, THE IMPLIED
WARRANTIES OF MERCHANTABILITY AND FITNESS FOR A PARTICULAR PURPOSE ARE
DISCLAIMED. IN NO EVENT SHALL THE COPYRIGHT OWNER OR CONTRIBUTORS BE LIABLE
FOR ANY DIRECT, INDIRECT, INCIDENTAL, SPECIAL, EXEMPLARY, OR CONSEQUENTIAL
DAMAGES (INCLUDING, BUT NOT LIMITED TO, PROCUREMENT OF SUBSTITUTE GOODS OR
SERVICES; LOSS OF USE, DATA, OR PROFITS; OR BUSINESS INTERRUPTION) HOWEVER
CAUSED AND ON ANY THEORY OF LIABILITY, WHETHER IN CONTRACT, STRICT LIABILITY, OR
TORT (INCLUDING NEGLIGENCE OR OTHERWISE) ARISING IN ANY WAY OUT OF THE USE OF
THIS SOFTWARE, EVEN IF ADVISED OF THE POSSIBILITY OF SUCH DAMAGE.