Fluidigm® D3™ Assay Design User Guide (PN 100
Contents
1. 5 d d x rint sat Rd 17 ADDING TARGETS FOR DEEPASORNE AS3B N MGE Donde Ege m PRU ved pm e bp ARM TURNS 21 ADDING TARGETS FOR SNPIY PE ASSA VO phia eniai 25 CHAPTER DS BEST PRAECTIGES rena ERU MEDIEN EPUM 29 E NL MR oc 29 po or rcc 30 IVIPROVING COVERAGE FOR YOUR ASSAY IR RV ME 34 SECTION III CHAPTER 7 CHAPTER 8 REVIEWING AND ORDERING dS T 33 SUEMITUNGA DE GM di Wiz e Tc 55 55 ee eee ee ene E eee eee re eee 33 fm 33 PREPARI 5 5 taro MEN IUNII UNUM UN 36 y umb mq SP eee ere 36 PES mm r 40 SECTION IV FAQ AND TROUBLESHOOTING CHAPTER SF eR I CAP mr i M M 43 QUESTIONS AND COMMENTS ABOUT REGISTRATION AND LOGGING ass2. 43 QUESTIONS AND COMMENTS ABOUT DESIGNING ASSA Vo 4 4 45 5 125 00 aaea 44 QUESTIONS ARBCDNBIENTSAABODTORDERIDI 46 4 5 3 48 APPENDICES APPENDIX A GETTIN G HELP oscinina ea eaaa eee aain al APPENDIX E a 52 Fe cal ad ol Gas Bay Ge 86 9 c ee eee eee 53 SECTION GETTING STARTED FLUIDIGM D3 ASSAY DESIGN Define Design Deliver Fluidigm D3 Assay Design or D3 is a web based tool to streamline and speed up assay design for NGS library preparation qPCR and genotyping studies Its intuitive user interface enables researchers to design with only a few clicks high coverage assay panels for gene expression genotyping or targeted sequencing experiments Backed always by expert specialized technical support from the Fluidigm Assay Design Group D3 ensures that researchers can Minimize time and resources expended on primer design and assay formulation Rapidly design a single assay or many high throughput batches of assays Customize assays leveraging D3 s ability to control and fine tune design parameters Track hundreds of assay design projects with
3. dbSNP 137 all dbSNP 137 with gt 196 frequency N and flagged as clinically associated dis dbSNP 137 with gt 1 frequency None dbSNP 137 with gt 196 frequency and flagged as dbSNP 137 with gt 196 frequency and flagged as clinically associated e wild type dbSNP 137 with gt 1 frequency e wild type None None 5 Enter any Notes that will help you locate this project easily in future or distinguish it from other versions of the same project 6 When you finish click Create Project Fluidigm Assay Design User Guide 13 Project or Version what s the difference Projects define top level areas of investigation Depending on the experiment project definition may be by genome disease function or biochemical pathway Projects can intuitively be grouped in alignment with specific laboratory projects or areas of investigation thereby maintaining a separation between studies Versions help track the history of a project Since the assay design process is typically iterative versions enable a researcher to preserve a snapshot of the design process allowing tracking not only of design changes but also of order status and details When a new project is created in D3 it is automatically designated as Version 1 of that project Its version description should be written so as to indicate how it is unique or different from other versions in the same project Use
4. Status Created Retrieve from Archive There are currently no targets NOTE Target entry is not case sensitive rg Lim m 5 2 2 2 Fluidigm Assay Design User Guide 16 When targets entered into the appropriate field D3 responds real time with a selectable list of suggestions like this By Gene or RefSeq By Genome Coordinate By Target Sequence By Fluidigm Assay ID From Existing Project By File Upload By Delimited Text CESTP1 CES1P2 CESZ CES3 CES4A CES5A CESSAP1 ADDING TARGETS FOR ACCESS ARRAY ASSAYS e Users can submit RefSeq IDs gene symbols or genome coordinates e f genome coordinates are entered each alias must be defined and unique Aliases used to name primers For e g the primers for alias ex3 will be named 3 1 2 and 3 e Targets with genome coordinates must not exceed 5 000 base pairs bps in length Genome coordinate regions greater than 5 000 bps can be entered as separate targets ACCESS ARRAY ASSAYS ADDING TARGETS BY GENE NAME or RefSeq ID PM des When you finish click Add Fluidigm Assay Design User Guide Enter the gene name or the RefSeq ID Only NCBI NM Accession IDs are accepted Enter the number of bases upstream of the transcription start site TSS of the gene of interest Click the checkboxes to determine whether the design should cover
5. ORDER P O Processed Shipped You are also welcome to contact SalesAdmin fluidigm com for an update on your order Q How soon after send in my P O can expect to receive my primers Once Fluidigm receives and processes your P O manufacturing of your assay products begins Your assays will be delivered to you within 4 weeks for products provided non wet tested and within 6 weeks for wet tested products Q Can my order be changed Unfortunately manufacturing begins once you have reviewed a quote and submitted a P O for a specific project or version After this point your order is locked and cannot be changed You can make edits before you submit a P O for a specific version Q How reorder a previously purchased assay You can reorder a previously purchased assay by selecting its specific version in your order history tab Copy the version to a new project version and then request a quote for the copy Fluidigm Assay Design User Guide 47 Q How is assay coverage defined Does it mean covered by amplicon or between two primers TROUBLESHOOTING AN ASSAY DESIGN The D3 coverage report calculates coverage based on the inner amplicon or true amplification sequence between primers It excludes regions covered by synthetic oligonucleotides Occasionally two three or more primers may be required to cover the specific region In such cases coverage is accomplished by tiling inner amplicons
6. 1 to another project Edit this version Fluidigm Assay Design User Guide Edit a project Share this version Download txt file with version details 15 e ADDING TARGETS D3 lets users add assay targets in many ways Depending on the assay type a user can e Add targets Reference sequence RefSeq IDs e g 000181 Genome coordinate ranges e g chr21 33 043 963 33 044 163 Target sequences e g GAGGCCAC TCTCCACCCC A Fluidigm Assay ID numbers e g 00007XXX e Copy targets by gene symbols from another D3 project one that the user created earlier or that the Fluidigm Assay Design Group shared with the user e Download a template spreadsheet from the Add Targets screen complete the required fields and upload the spreadsheet back into the project e and paste multiple targets in comma or tab delimited formats The Add Targets screen looks like this DELTAgene o 1 ADD TARGETS Assays New project saved DELTAgene assay project Assay Type DELTAgene Please select a method for target entry Species Human Homo sapiens Created 27 Aug 2013 By Gene or RefSeq Gene or RefSeq Name required By Genome Add VERSION 1 Coordinate Target Sequence Status Created Last Modified 27 Aug 2013 By Fluidigm Assay ID Targets 0 From Existing Project Assays 0 By File Upload VERSIONS By Delimited Text Last Modified Version 1
7. Existing Project 1803246 By File Upload 1803387 By Delimited Text Add 3 When you finish click Add ACCESS ARRAY ASSAYS ADDING TARGETS FROM EXISTING PROJECTS Eee 1 ADD TARGETS Please select a method for target entry Import targets from another project or version By Gene or RefSeq Please choose By Genome Coordinate Or From Existing Project Import targets from the Fluidigm catalog Please choose By File Upload By Delimited Text 1 Add targets from another project or version that you have created on D3 or from the Fluidigm catalog where applicable 2 When you finish click Add Fluidigm Assay Design User Guide 19 ACCESS ARRAY ASSAYS ADDING TARGETS UPLOADING A FILE ee lt lt 1 ADD TARGETS Please select a method for target entry By Gene or RefSeq 1 Download Template By Genome Coordinate 2 Choose File No chosen From Existing Project 3 Import By File Upload By Delimited Text ee ee FO ba T NET T Click Download Template An Excel spreadsheet downloads to your desktop Open the spreadsheet It contains three tabs like this Read the Instructions under the first tab and then enter your targets under the appropriate tab When you finish save as a tab delimited file 5 Go back to D3 browse to the file you just saved and click Impor
8. FOR DELTAgene ASSAYS e Users must ensure that Assay IDs genome coordinates and sequence submissions refer to the intended target species D3 cannot check if all targets entered belong to the same species e Assays are designed to target all known isoforms of the gene If an assay does not target all known isoforms a design comment will be displayed next to the assay along with a list of detectable isoforms NOTE Please be sure to provide genome coordinates corresponding to the currently supported genome build The supported genome build is identified in the Genome Build field located in your project properties FOR SNPtype ASSAYS e When targets are entered by sequence users must indicate the targeted SNP using square brackets separated by a forward slash For example A G e Aminimum of 60 bps is recommended both upstream and downstream of the targeted SNP with a maximum of 250bps to increase the probability of receiving a design e Any relevant additional non targeted SNPs should be indicated as IUPAC codes IUPAC codes are tabulated in Appendix C Fluidigm D3 Assay Design User Guide 29 RefSNP RS numbers must be specified A sequence is retrieved using the most recent build on human NCBI dbSNP and adjacent SNP with a reported allele frequency gt 196 excluding SNPs with no frequency data e f both sequence and RS number are provided the assay is designed to the sequence no additional sequence is retrieved from
9. Generally but not Fluidigm Assay Design User Guide always a dominant allele 52 IUPAC CODES Appendix 8 The table below lists the IUPAC standard representation of DNA bases by single characters that specify either a single base e g G for guanine A for adenine or a set of bases e g R for either G or A SYMBOL MEANING G G A A T T C C R GorA Y TorC M A or C K GorT S GorC W AorT H A or Cor T B GorTorC V or Cor A D GorAorT N GorAorTorC Fluidigm D3 Assay Design User Guide 53 Fluidigm World Headquarters 7000 Shoreline Court Suite 100 South San Francisco CA 94080 USA Fluidigm Europe BV Luna Arena Herikerbergweg 238 1101 CM Amsterdam Zuidoost The Netherlands Tel 31 0 20 578 88 53 Fax 31 0 20 203 11 11 Fluidigm France Sarl Les Conqu rants Bat Kilimandj aro 1 avenue de l Atlantique 91940 Les Ulis Tel 33 0 1 60 92 42 40 Fax 33 0 1 60 92 11 31 Fluidigm Singapore Pte Ltd Block 1026 07 3532 Tai Seng Avenue Singapore 534413 Singapore Tel 465 6858 7316 Fax 31 0 20 203 1111 Fluidigm J apan KK Luminous 2nd Floor 15 19 Nihonbashi Kodenmacho Chuo ku Tokyo 103 0001 J apan Tel 81 3 3662 2150 Fax 481 3 3662 2154 Fluidigm Shanghai Instrument Technology Co Ltd Room 2907 B Building Far East International Plaza No 317 XianXia Road 200051 Shanghai China Tel 486 21 3255 8368 Fax 486 21 3255 8369 Technical Support
10. Plate button Filter by Window Size Smal Medium Large Target Name 10 I GAPUN GAFPUH 4 Plate 1 Plate 2 5 New Plate GAPDH GAPDH 5 T GAPDH A GAPDH_6 T GAPDH A GAPDH_7 GAPDH GAPDH 8 GAPDH A GAPDH 9 2113 13 10 2113 A IL3 11 13 A 103 12 2113 13 13 13 A 13 14 16 17 A A A A 15 A A A 2113 18 A 1172 4 3 Create your custom layout by following these steps Fluidigm Assay Design User Guide 38 Mier try Window Hie 3 ese Large Tig Ay sas Step 1 Drag select or Ctrl select multiple targets Step 2 Drag select or Ctrl select well locations by column Step 3 Click to assign targets to selected well locations 4 When all assays are assigned to wells click Request Quote NOTE As soon as you request a quote for a version the version gets locked and can no longer be modified If you wish to modify targets assays or the layout clone the locked version and then modify the clone PLATING ON MULTIPLE PLATES You can add assays to as many plates as you require To include additional plates 1 Click New Plate at top right of your plate layout screen Filter
11. Receive a design review through D3 from the Fluidigm Assay Design Group Submit quote request Iterations if required FIRST STEPS WITH D3 This chapter shows you how to register as a D3 user and navigate the website REGISTERING AS A D3 USER 1 Open browser window and navigate to https d3 fluidigm com 2 Inthe Sign in panel click Register Sign in Email Password hm Remember Me Lost Password Signin Don t have an account 3 Enter your details in the Register panel The Phone number field is optional but all other fields are mandatory Register Email address Password Retype Password First Name Last Name Institution Phone number optional ZIP Postal Code Please choose a country 7 have read and agree to the Fluidigm user agreement oc EE Fluidigm Assay Design User Guide 4 Select a password Your password must contain at least e Eight characters e One uppercase character e lowercase character e Onenumber or special character like amp or 96 There should be no spaces between characters For example LOVE D3 is nota valid password because it contains a space and has lowercase character ILoveD3 is a valid password as it meets all criteria NOTE If you are located outside the United States please enter your posta
12. TARGETS Please select a method for target entry Import targets from another project or version Please choose 7 By Genome Coordinate By Gene or RefSeq Or From Existing Project Import targets from the Fluidigm catalog By File Upload Please choose By Delimited Text amm a he he qi epo 1 Addtargets from another project or version that you have created on D3 or from the Fluidigm catalog where applicable 2 When you finish click Add Fluidigm Assay Design User Guide 27 SNPtype ASSAYS ADDING TARGETS BY UPLOADING A FILE 1 ADD TARGETS Please select a method for target entry By RS 1 Download Template By Target Sequence 2 Mo file chosen By Fluidigm Assay ID From Existing Project impart By File Upload By Delimited Text Click Download Template An Excel spreadsheet downloads to your desktop 2 Openthe spreadsheet It contains four tabs like this Instructions Targets by 54 Targets by Sequence Targets by Fluidigm Assay ID 2 g 3 Readthe instructions under the first tab and then enter your targets under the appropriate tab When you finish save as a tab delimited file 5 Go back to D3 browse to the file you just saved and click Import SNPtype ASSAYS ADDING TARGETS BY DELIMITED TEXT ENTRY Two text entry delimiters tabs or commas are allowed in SNPtype assay design The entries themselve
13. TARGETS BY TARGET SEQUENCE 1 ADD TARGETS Please select a method for target entry By RS sequence Name required By Target Sequence sequence required By Fluidigm Assay ID From Existing Project By File Upload By Delimited Text Enter the sequence name Enter up to 1000 bases of sequence Optionally place square brackets around the targeted bi allelic SNP for primer design Assays will be designed to target the bracketed area For example Bi allelic SNP GTGTGAGT C A TGACAGC Deletion GGTAATGAC G GTACGSG Insertion GTGACAG G TGAAGAGA 4 When you finish click Add NOTE Non bi allelic target SNP sequences are not accepted Fluidigm Assay Design User Guide 26 SNPtype ASSAYS ADDING TARGETS BY FLUIDIGM ASSAY ID An easy way to reorder assays from a previous order is to enter the associated Fluidigm Assay ID Assay IDs are available in the informatics packet that is supplied with your assays 1 ADD TARGETS 4 Please select a method for target entry By RS Name E Enter Assay IDs here with a carriage return between entries By Fluidigm Assay ID From Existing Project By File Upload By Delimited Text 4 4 1 Referring to your previously ordered assays copy and paste the RS and the Fluidigm Assay ID or just the Fluidigm Assay ID 2 When you finish click Add SNPtype ASSAYS ADDING TARGETS FROM EXISTING PROJECTS 1 ADD
14. across the region Q How determine the total amplicon length of my assays and what is Unique Coverage The total amplicon length is displayed under column Amplicon Length to the right of the Assay ID in the assays table This value represents the total amplicon length of an assay from locus specific forward primer to locus specific reverse primer The Unique Coverage Value is a representation of the genomic coverage provided by an assay not including overlapping assays This value is important for tiling designs where particular regions maybe covered by two or more assays Coverage Target Region ma mM Target Region GC 96 Repest Track SNP Assays Assay Assay ID BRAF_1 SAAA0044670 BRAF 2 0050790 BRAF 3 SAAA0046246 si Amplicon Length 185 196 189 Fluidigm Assay Design User Guide Unique Coverage 78 3 8 6 24 7 140 434 570 GC 49 36 31 Design Rank High Medium High Design Comments 48 Q How tell what genome build to use to submit my coordinate designs The information for the genome build of designs is displayed in the projects section below the Assay Type and Species field Access Array DEMO Access Array DEMO Q How is primer specificity tested Primer specificity is tested in silico against the latest genome build available at the time of release for the selected species Q The web
15. are ideal for analysis across focused genomic regions to better understand human genetic variation Primers are designed for quick and specific amplification of targets incorporation of sample specific barcodes and addition of sequencer specific adaptors all at the same time All Access Array assays are Flexible up to 480 plex sample e Universal barcodes and adaptors for Illumina lon Torrent and Roche sequencers e Accurate highly uniform sensitive and specific amplicon libraries DELTAgene qPCR GENE EXPRESSION ASSAYS DELTAgene Assays enable users to take full advantage of the BioMark HD System Experiment setup and validation times are minimized Biologically related gene lists are available to assist with identifying gene targets of interest All DELTAgene Assays are e High quality sensitivity and linearity similar to probe based assays compliant assay primer sequences are always provided e Economical low startup and running costs SNPtype GENOTYPING ASSAYS SNPtype Assays are custom high throughput and low cost SNP genotyping solutions for rapid screening Based on allele specific PCR SNP detection SNPtype Assays combine the advantages of minimum setup time and flexible assay choice with the reliability of Dynamic Array Integrated Fluidic Circuits IFCs They employ tagged allele specific PCR primers and a common reverse primer A universal probe set is used in every reaction producin
16. by Window Size Smali Medium Large 4 TargetName AssaylD 1 4 2 2 13 A IL3 10 GAPDH f GAPDH 17 13 5 T 13 A IL3 11 13 A IL3 12 GAPDH T IL3 13 18 36 T 13 A IL3 13 L 13 A 13 14 GAPDH T IL3 113 15 TEM 1 1 13 A IL3 16 TFI 1 C7 T IL3 A IL3 1 GAPDH 1 T IL3 132 K 13 A IL3 4 1 iss LITT TT TT i a m Jj 13 138 13 A IL3 9 T DIZIA Evand A i c p Fluidigm Assay Design User Guide 2 Toggle between plates to assign assays to wells CAUTION To remove assays from a plate select the assays and then click If you click e assays from all plates are removed 3 When you complete plate layout click Save PLACING AN ORDER 1 To begin the order placement process click Request Quote Targets Assays 09 1110 48 Plate Layout Default Custom Edit 5 Request Quote You currently do not have any offers NOTE You will be able to include notes and promotional codes and verify your contact information before you request a quote for your project 2 Onthe screen that appears several purchase options are available Fluidigm Assay Design User Guide 40 Request Quote Purchase Options Promotion Code Blanket Com
17. check shows assays may hybridize to multiple loci 6 Non Standard Assay Designed with amplicon lengths outside of specified lengths 7 Non Standard Assay Designed outside of specifications FOR DELTAgene ASSAYS 1 Non Standard Assay Designed to customer provided sequence or genome coordinate 2 Non Standard Assay Designed to non NM accession ID 3 Non Standard Assay Not designed to gene level 4 Non Standard Assay Designed outside of specifications Fluidigm D3 Assay Design User Guide 30 FOR SNPtype ASSAYS 1 Non Standard Assay GC content is outside of the product specification 2 Non Standard Assay Target is an insertion deletion or not a SNP 3 Non Standard Assay Designed outside of specifications IMPROVING COVERAGE FOR YOUR ASSAY DESIGN Access Array coverage is calculated as the total coverage of included assays inner amplicons divided by the total target region within the given version The coverage wheel provides a visual indicator of coverage which changes dynamically as targets are added or removed from a project and as assays are included or removed from the assays list A representative coverage wheel is depicted here ENT Selected Assays 215 215 Designed Targets 30 30 Design Rankings High 185 Medium 33 STANDARD vs NON STANDARD ASSAYS D3 designates assays as either standard or non standard depending on the design
18. dbSNP e Only one SNP can be identified per target number e Insertions or deletions gt 10 bps should not be included Non bi allelic SNPs should not be included e Targets with adjacent SNPs within 20 bps on both sides of the target SNP will not be designed Fluidigm recommends removal of an adjacent SNP if it is not pertinent within the study population e Sequences are filtered based GC content Sequences that bear greater than 65 GC will automatically be assigned a medium design rank Design Ranks what do they mean The design review process assigns a rank to your design The rank is not an indicator of the quality of your assay design However it does indicate what you can expect terms of support High ranked assays are designed to product specifications and do not include a design comment Medium ranked assays fall outside design criteria They are offered with limited support and may not meet product specifications They include a design comment providing a rationale for the ranking Design comments are specific to each assay type and are listed below DESIGN COMMENTS FOR ACCESS ARRAY ASSAYS 1 Non Standard Assay GC content is outside of the product specification 2 Non Standard Assay One primer is designed within a repeat region 3 Non Standard Assay Both primers are designed within a repeat region 4 Non Standard Assay Designed without SNP and repeat annotation 5 Non Standard Assay In silico
19. for quote has been submitted As soon as you request a quote by pressing the Order button the version is locked and cannot be edited If you decide not to purchase the assays as presented in the version and wish to change your order please do the following Navigate to the locked version you wish to edit Copy the version within the same project and create a new version Navigate to the version you just created m mm Modify the design as you wish and once you are satisfied with the design submit a fresh request for a quote Q I want to share my design with my colleagues Make sure you and your colleagues have sharing enabled To enable sharing 1 Click the Account Settings link at top right of your screen Account Settings Logout 2 Scroll down and check the checkbox to enable sharing Sharing Version sharing allows users to grant read only access of a version to other registered users by entering thei e 9 egistered account on D3 Read more here edite share projects from other users 3 Navigate to the project version you want to share 4 Click the options icon for that version and then click the Share icon circled below Fluidigm Assay Design User Guide 45 Read and agree to the conditions of the Fluidigm sharing agreement Enter email addresses for users you want to share with Click Confirm Your project version is now shared with the users you designated A sample shared version is
20. multiple versions per project making D3 a convenient secure referable and reusable repository for any laboratory s designs Fluidigm custom designed assays support a variety of genome designs for model or non model organisms Supported genomes are listed here Assay types include Fluidigm Assay Design User Guide Access Array Target Specific Primer Assays These assays are designed to the region of interest with the necessary sequence tags for fast and easy incorporation of barcodes and NGS adaptors Targeted resequencing primers offer low amplicon dropout and high map to target rate DELTAgene Assays These are MIQE compliant qPCR gene expression assays designed to high specificity and sensitivity SNPtype Assays High throughput yet low cost these allele specific assays enable rapid single nucleotide polymorphism SNP screening by PCR They facilitate quick cost effective development and implementation of top quality SNP genotyping panels e THE D3 WORKFLOW Tailored for simplicity speed and accuracy across a diverse set of primers D3 is geared to span expertise levels from broad targeting at the gene level to specific target coordinates D3 users follow five fundamental steps Fluidigm Assay Design User Guide Create an assay project of the desired assay type Select targets for automatic validation in D3 Submit a design request for the validated assay project O
21. the 5 UTR the 3 UTR or both 17 S 1 ADD TARGETS Please select a method for target entry ByGeneorRefSeq GAPDH By Genome Coordinate Upstream TSS By File Upload Add By Delimited Text NOTE D3 validates NM Accession IDs and official gene names Only valid targets can be added non NM Accession IDs are not supported Please refer to the NCBI database to determine if an entry is valid ACCESS ARRAY ASSAYS ADDING TARGETS BY GENOME COORDINATES NOTE Please be sure to provide genome coordinates corresponding to the currently supported genome build The supported genome build is identified in the Genome Build field located in your project properties 1 ADD TARGETS Please select a method for target entry By Gene or RefSeq Name required By Genome Chromosome required Coordinate req From Existing Project Start required By File Upload End required By Delimited Text Fluidigm Assay Design User Guide 18 1 Enter the gene name or target alias and the chromosome For example Chr7 chr7 or 7 2 Enter the start and end chromosome coordinates making sure that the end coordinate is larger than the start coordinate A coordinate can be entered in the format 123456789 or 123 456 789 but not in the format 123 456 789 1 ADD TARGETS Please select a method for target entry By Gene or RefSeq BRCA2 By Genome Coordinate chr4 From
22. Fluidigm PN 100 6812 REV A2 USER GUIDE D3 Assay Design Copyright 2013 Fluidigm Corporation All rights reserved Fluidigm the Fluidigm logo Access Array BioMark C D3 Assay Design DELTAgene Dynamic Array and SNPtype are trademarks or registered trademarks of Fluidigm Corporation in the U S and or other countries For Research Use Only Not for use in diagnostic procedures PN 100 6812 Rev A2 Contacting Fluidigm On the internet www fluidigm com support techsupport fluidigm com By phone In the United States 1 866 FLUIDLINE 1 866 358 4354 Outside the United States 1 650 266 6100 By mail Fluidigm Corporation 7000 Shoreline Court Suite 100 South San Francisco CA 94080 USA SECTION I CHAPTER 1 CHAPTER 2 CHAPTER 3 SECTION CONTENTS GETTING STARTED oj Mm rer ur ND P TC 6 jj 25 8 8 7 sed se ocho Ny secrets es ett E A 8 A A WP 5 4 4 MN eT Pe 8 DESIGNING ON D3 uidi e Piet 12 PSF MN LC ERIS TN w 12 Ars dI em PER LET EET 9 Av 13 CHAPTER 3 ADDING TARGET UII DAI inter alise 16
23. In the unlikely event that you still have issues with password registration please email us copying or forwarding any error messages you are receiving State what browser you are using and be sure to provide your contact information Q do not recall what email address used to register on D3 If you registered for an account but have forgotten the email address you registered with we can help Please email your name and the name of your institution including the date you registered and we ll do our best to locate your account Fluidigm Assay Design User Guide 43 Q have moved labs employer and want to transfer my account We are unable to transfer ownership or consolidate customer accounts at this time If you would like to disable or delete your old account you can do that by logging a request here Please provide us the details of the old account including your full name institution and email address We ll do our best to delete the account and will notify you of the deletion If you need immediate access to D3 you may choose to create a new account with your new profile information and email address Q I have multiple accounts that need to consolidate To maintain data integrity and confidentiality D3 does not permit account consolidation We recommend that you maintain a single account and control experimental separation through the use of projects and versions To delete unwanted accounts review all your acc
24. ability of the assay Although every D3 assay type has its supported species list users can enter non supported species if the selected assay type allows sequence submission Non standard assays designed with relaxed algorithm parameters like relaxed requirements for GC filter amplicon length SNP filter or repeat filter or designed to customer provided sequences are always assigned a medium rank Assays designed with relaxed design parameters typically have a lower success rate than standard Fluidigm assays and yet offer a viable alternative for studying difficult targets Such assays are provided with limited support and may not meet product specifications Access Array Assays singleplex or multiplex Your choice of singleplex or multiplex depends on the needs of your experiment Access Array assays are performed on the Fluidigm 48 48 Access Array IFC Individual or singleplex Access Array reactions contain one forward and one reverse primer in each microfluidic reaction chamber on the chip and are provided with wet test data Multiplex reactions contain up to twelve sets of forward and reverse primers in each reaction chamber Assays for multiplex are singleplex assays arrayed in a format for easy pooling into your primer pools No wet test data is provided with this option Fluidigm D3 Assay Design User Guide 31 SECTION Ill REVIEWING AND ORDERING DESIGN REVIEW D3 is designed to automatically perform an in silico ve
25. chase order P O is received 3 Select the plate layout Your choices are either Default or Custom Fluidigm Assay Design User Guide 36 1 2 Targets Assays es 1 10 20 Plate Layout Default amp Custom Edit Z Request Quote You currently do not have any offers DEFAULT LAYOUT The Default Standard layout places your assays onto plates column by column sorted alphabetically by target name To accept the default layout simply click the Default radio button and then click Request Quote CUSTOM LAYOUT Custom layout allows you to assign your assays to specific wells To conform to the configuration of Fluidigm IFCs you must plate by columns not by rows NOTE All assays must be assigned to well positions before a quote can be requested NOTE When you order Multiplex Access Array assays Fluidigm will determine the optimal array of your assays for your multiplex PCR To create a custom layout 1 Select the Custom radio button and click Edit Fluidigm Assay Design User Guide 37 Targets Assays Me 65 Min 45 Plate Layout Default amp Custom Edit Rm At A Z Request Quote You currently do not have any offers 2 The system generates a blank 96 well plate template as seen below The left pane lets you filter your assays by either Target Name or Assay ID The right pane lets you adjust the Window Size or add more plates via the New
26. e l Or From Existing Project Import targets from the Fluidigm catalog By File Upload Please choose By Delimited Text amm aun 1 Addtargets from another project or version that you have created on D3 or from the Fluidigm catalog where applicable 2 When you finish click Add DELTAgene ASSAYS ADDING TARGETS BY UPLOADING A FILE 1 M H H 1 ADD TARGETS Please select a method for target entry By Gene or RefSeq 1 Download Template By Genome Coordinate 2 No file chosen By Target Sequence m 3 Import By Fluidigm Assay ID imas From Existing Project By File Upload Click Download Template An Excel spreadsheet downloads to your desktop 2 Open spreadsheet It contains five tabs like this Instructions Target by Gene or RefSeq Targets by Fluidigm Assay ID Targets by Sequence Targets by Coordinates 3 Readthe instructions under the first tab and then enter your targets under the appropriate tab When you finish save as a tab delimited file Go back to D3 browse to the file you just saved and click Import Fluidigm Assay Design User Guide 24 DELTAgene ASSAYS ADDING TARGETS BY DELIMITED TEXT ENTRY Two text entry delimiters tabs or commas are permitted The entries themselves can be either by reference sequence gene or by genome coordinates Entry formats vary depending on
27. e protein The region of DNA amplified between two synthetic primers does not include the primers Intron Non coding portion of a gene Marker DNA sequence with a known physical location on a chromosome Multiplexing Simultaneous analysis of more than one target in the same reaction Specific quantification of multiple targets that are amplified within a reaction can be performed using a differentially labeled primer or probes Amplicon or probe melting curve analysis allows multiplexing in allelic discrimination if a dsDNA binding dye is used as the detection chemistry NCBI The National Center for Biotechnology Information online at www ncbi nlm nih gov Phenotype An observable characteristic displayed by an organism Primer A short single stranded DNA sequence used in PCR In the PCR method a pair of primers is used to hybridize with the sample DNA and define the region of the DNA that will be amplified RefSeq Collection providing a comprehensive integrated non redundant well annotated set of sequences including genomic DNA transcripts and proteins SNP Single Nucleotide Polymorphism A single base difference found when the same DNA Total Amplicon sequence from two different individuals is compared The PCR product resulting from the amplification of DNA by two synthetic primers includes primer TSS Transcription Start Site UTR Untranslated Region Wild type The non mutant form of a gene encoding the normal genetic function
28. efinition Content T1 Overall Coverage Track A list of lines dynamically calculated by the coverage script as the user selects and deselects assays T2 Target Definition Defined target regions T3 Zoomed Target Definition Extracted from T2 determined by the zoomed coordinates T4 GC Content GC portion extracted from the full definition by the zoomed coordinates T5 Repeat Track Extracted from the full definition by the zoomed coordinates T6 SNPs Display of relative location of SNPs with associated dBSNP IDs T7 Inner Amplicon Inner amplicon minus primers of selected assays within the zoomed coordinates T8 Gap Track with Gap Coordinates Displays the coordinates and size of a gap a region not covered by designs ACCESS ARRAY COVERAGE COLOR CODING DEFINITIONS Red hash indicates Blue lines indicate Yellow lines indicate GAPS in coverage HIGH assays MEDIUM assays zi Coverage m m E m S _ F m ou mm m m o a Target Region HE E ee 1 41 277290 41 277 348 41 277 500 Target Region GC 96 Repest Track SS SNF CR E Assays ee Yellow lines indicate j MEDIUM assays G Blue lines indicate DECAT 8 HIGH assays Gap 1 17 41 277 571 41 277 576 Fluidigm D3 Assay Design User Guide 35 D3 offers the convenience of arraying assays using either default parameters your customized layout in standard 96 well plates P
29. ence required By Target Sequence By Fluidigm Assay ID Optional Place brackets around regions of interest for primer design Assays will be designed to target bracketed area From Existing Project By File Upload By Delimited Text Enter the sequence name Enter up to 1000 bases of sequence Optionally place square brackets around the regions of interest for primer design Assays will be designed to flank the bracketed area with a primer on either side of the bracket 4 When you finish click Add DELTAgene ASSAYS ADDING TARGETS BY FLUIDIGM ASSAY ID An easy way to reorder assays from a previous order is to enter the associated Fluidigm Assay ID Assay IDs are available in the informatics packet that is supplied with your assays 1 ADD TARGETS Please select a method for target entry By Gene or RefSeq Name By Genome Enter Assay IDs here with a carriage return between entries Coordinate 3 g By Target Sequence By Fluidigm Assay ID From Existing Project By File Upload By Delimited Text 1 Referring to your previously ordered assay copy and paste the gene names and the Fluidigm Assay IDs 2 When you finish click Add Fluidigm Assay Design User Guide 23 DELTAgene ASSAYS ADDING TARGETS FROM EXISTING PROJECTS SSE 1 ADD TARGETS Please select a method for target entry By Gene or RefSeq Import targets from another project or version Please choose By Genome Coordinat
30. erage 100 100 100 100 100 2 Inthe Coverage column click coverage percentage to view coverage results for that target A multi panel track displays Tracks and panels are explained in the graphic that follows Fluidigm D3 Assay Design User Guide 33 opino 1951 usisaq Aessy VE ACCESS ARRAY COVERAGE RESULTS TRACK DEFINITIONS Slider bar for sizing and aligning to zoomed area T1 Overall coverage track Coverage gt gt e E c m 0000000000000 08M s 11 1811 lt 12 Target definition overall coverage for all selected assays Gap Gag T3 Zoomed target definition 41 277290 41 277 340 41 277 500 ar Target Region 14 GC portion extracted from full definition Mec am T5 Repeat track SNP x 4 T6 SNP locations with dbSNP IDs Panel displays zoomed area By default it covers about three assay lengths Assays SS 7 Inner amplicon minus primers of selected assays within zoomed coordinates Panel lists gaps in zoomed area with Gap 1 17 41 277 571 41 277 576 50 4 Gap track with gap coordinates genome coordinates that can be copy pasted into txt file Ellipsis punctuation denoting omission of sequence not relevant to the current design Display tracks in the coverage results are Track D
31. established blanket P O with Fluidigm your order can begin processing after submission without the need for a quote Typically assays that are not wet tested are delivered within four weeks wet tested assays are delivered within six weeks An informatics packet is emailed at the time of product shipment It includes primer sequences plate layout and instructions on where to download supporting documents like User Guides and QRCs Informatics packets are also available online within your D3 orders Timely updates are provided through the D3 website while assays are being manufactured An update is generated immediately upon the P O being processed and subsequently when assays are shipped Fluidigm Assay Design User Guide 41 SECTION IV FAQ AND TROUBLESHOOTING FAQ USING D3 QUESTIONS AND COMMENTS ABOUT REGISTRATION AND LOGGING IN Q The registration screen does not accept my password Your password must contain at least e Eight characters e One uppercase character e One lowercase character e One number or special character like amp or 96 There should be no spaces between characters If your password already meets all these criteria make sure that e Allfields on the registration screen are complete e User Agreement circled in the image below is checked Register First Name Last Name Institution Phone number optional ZIP Postal Code Please choose a count
32. g uniform fluorescence while significantly reducing startup and running costs All SNPtype assays are Economical low startup and running costs Robust locus specific primer sequences are provided e Flexible designed to your custom target list Fluidigm Assay Design User Guide 12 STARTING NEW D3 PROJECT 1 Onthe Projects page click New Project The Add New Project panel appears Add New Project Project Properties Version 1 Properties Name Notes optional ition Descriptio Design SNP Masking Default Assay Type Advanced Access Array Target Specific Primers DELTAgene Assays SNPtype Assays Species Homo sapiens Mus musculus Close Create Project 2 Enter a project Name and Description Any new project is automatically designated as Version 1 of that project 3 Select the Assay Type and the Species 4 For Access Array and SNPtype assays select SNP Masking preferences using the table below as a reference SUPPORTED GENOMES AND SNP MASKING OPTIONS Assay Type Access Array Target Specific Primers DELTAgene Gene Expression SNPtype SNP Genotyping Supported Genomes Human Human Mouse Human Mouse Other Default SNP Masking Option Advanced SNP Masking Options dbSNP 137 with gt 1 frequency dbSNP 137 with gt 196 frequency dbSNP 137 with gt 196 frequency and flagged as and flagged as clinically associated clinically associated
33. l or PIN code in the Zip Code field 5 Readthe user agreement check the user agreement box and click Register An activation email is sent to your email address 6 Click the link in your activation email and log in You will land at the Projects page an example of which is shown below From here you can begin creating your study specific assay design projects Fluidigm Design Deliver Logged In My Assay Projects New Project You currently do not have any projects Fluidigm Assay Design User Guide The icons along the left panel of the Projects page indicate Fluidigm Assay Design User Guide Goto D3 home page View your orders Navigate to your last project Read the user guide View online help topics Email Fluidigm Technical Support 10 SECTION II DESIGNING ON D3 O YOUR D3 PROJECT This chapter describes all your assay design options in D3 and shows you how to design your assays ASSAY DESIGN OPTIONS All assays are ready to use via Fluidigm standard protocols and are accompanied by a detailed informatics packet Singleplex Access Array Assays and DELTAgene Assays are additionally offered with or without wet lab testing ACCESS ARRAY TARGET SPECIFIC PRIMER ASSAYS Access Array Target Specific Primers used with the Fluidigm Access Array System allow fast simple and inexpensive preparation of large numbers of amplicons They
34. lated assays allow use of multi channel pipettes to reduce pipetting steps significantly reducing your effort and minimizing the possibility of pipetting errors REQUESTING A QUOTATION PREPARING TO ORDER Once design review is complete you are notified by email Assuming that no further changes are required the assays are ready for a quote request To request a quote 1 Follow the link in the notification email to access the project version that is ready for your review 2 Ifyou are satisfied with the assays click Order at top right of your screen Designed Targets Design Rankings iy Retrieve Archive Index Target Name 1 Apbb1 2 Atm 3 1 4 Brca2 2 REVIEW DESIGN High 68 Medium 0 Target Type Target Info Assay Assay ID Assay ID Assay ID GXA 00013 338 Q 00012 GXA 00012 820 Q 00009 624 Q Fluidigm Assay ID GER00013338 GER00012813 GER00012820 GER00009624 Design Rank High High High High Exon Junction 11 12 NM 009685 49 50 NM 007499 19 20 NM 009764 7 8 NM 001081001 Add 5 Design Isoform Coverage Comments NM 009685 2 NM 007499 2 NM 009764 3 NM 009765 2 NM 001081001 1 NOTE Clicking Order enables you to proceed towards ordering The first step in the order process is to request a quotation Assays are not actually manufactured until a customer pur
35. ments 4 Wet tested Access Array DELTAgene Contact Information Please review your contact information below Any necessary changes should be made through your account setting Note Shipping destination is based on your payment specification Be sure to specify a bill to and ship to addresses if different with your payment method Close Hequest Quote Customer Name e Promotion Code Blanket PO If you have a valid promotional code or a blanket P O number please enter it here To expedite processing please submit only valid promotional codes or established blanket P O numbers with Fluidigm e Comments This enables you to include any notes for your order A note may for example read Please include a quote for 10 units of part number AA M 48 48 e Wet tested Fluidigm offers an optional wet lab testing for Access Array and DELTAgene assays Please note that there is an additional charge for this option and an incremental two weeks turnaround time e Contact information Review your contact information CAUTION To avoid delays with processing and quotation generation only include valid Fluidigm Promotional Codes or valid Blanket P O numbers SUBMITTING A QUOTE REQUEST Once your request for a quote is received your local Fluidigm sales representative or distributor will provide a quote and handle all ordering logistics Manufacturing of your assays commences only after your P O is received In case you have an
36. ounts and decide which one you want to keep From the rest download record any information you require and then delete them Remember that once you delete an account all projects versions assays and quotes related to that account are automatically deleted QUESTIONS AND COMMENTS ABOUT DESIGNING ASSAYS Q I did not receive confirmatory emails from the website Although we do everything we can to ensure that you receive our emails there are many reasons why you may not receive them Here are a few things to check 1 Checkif D3 emails are going into your spam or junk folder Mark them as not spam and redirect them to your Inbox 2 Confirm that you have received emails from fluidigm com in the past 3 Validate with your IT department that D3 emails are not being blocked due to any blacklisting or spam filters at the company server level 4 Validate that your D3 account is active and accessible If you still have issues we re happy to help Please email us to help resolve this issue Q I cannot modify a version in my project Once you submit a design request the version associated with that request is locked until your design review is complete or until you cancel the design request After you submit a request for a quote the version gets permanently locked You are free to make a copy into a new version or project and then modify that copy Fluidigm D3 Assay Design User Guide 44 Q How edit a design after a request
37. our assay designs System and chemistry user guides QRCs MSDSs are available via www fluidigm com product documents html The following help options are available to all D3 users HELP WITH ASSAY DESIGN OR GENERAL TECHNICAL SUPPORT Fluidigm has several resources to support you in your assay designs System and chemistry user guides QRCs and MSDSs are available via www fluidigm com product documents html If you need help with assay design or have questions about product specifications please email techsupport fluidigm com or call Fluidigm Technical Support HELP WITH ORDERS To know the status of your order please email SalesAdmin fluidigm com or call 650 266 6170 HELP WITH THE WEBSITE Please email webmaster fluidigm com Fluidigm Assay Design User Guide 51 GLOSSARY Appendix For exhaustive glossary of genomics terms please visit www genome gov TERM OR ACRONYM DEFINITION Base pair bp Two chemical bases bonded to one another forming a rung of the DNA ladder CDS Coding sequence Ensembl Genome browser online at www ensembl org Exon Portion of a gene that codes for amino acids Gene expression Inner Amplicon Process by which the information encoded in a gene is used to direct the assembly of a protein molecule The cell reads the sequence of the gene in groups of three bases Each group of three bases codon corresponds to one of 20 different amino acids used to build th
38. rification after the user has added all targets If a design passes verification the user can submit it for design Custom assays are generated and the user notified by email once the design is ready for review A design review response can be expected within 48 72 hours SUBMITTING A DESIGN FOR REVIEW 1 Checkthe list of targets to make sure you have added all targets you want to include in this version of your design You can always add more later 2 Click Submit for Design Retrieve from Archive Index Target Name Target Target Info Fluidigm Assay ID 1 rs46196775 c1 Sequence CCA C T GAC Q Not submitted 2 552441230 2 Sequence ACA C TJATA Q Not submitted 3 533767644 c3 Sequence _ AAG C T TTG Q Not submitted 4 rs47642237 c4 Sequence TTC C G TTG Q Not submitted 3 Review the confirmatory email that you receive from the system Your design will typically be returned within 48 72 hours THE ACCESS ARRAY COVERAGE DISPLAY For Access Array assays D3 graphically displays the coverage results for each target This includes an alignment of the genomic region with all relevant information SNPs GC content and repeat regions for each assay design A complete target region and a zoom window enable users to examine coverage at the assay level To access the coverage display 1 Navigate to the Design Review screen of your Access Array design Cov
39. rs wanting to make small changes to a design staying with the same species and a majority of the same targets should create a new version of an existing project If the species is changed a new project should be created INCREMENTAL SETUP REQUIREMENTS FOR ACCESS ARRAY ASSAYS D3 supports the Illumina Roche 454 and lon Torrent sequencing platforms To create a new Access Array project you must follow these additional steps Add New Project Project Properties 6 Version 1 Properties 6 Name Notes optional TSP1 Description Design SNP Masking Exons 1 4 amp Default Assay Type e Advanced Access Array Target Specific Primers Platform DELTAgene Assays SNPtype Assays Roche 454 Species lon Torrent Homo sapiens Amplicon Length 1 Select the sequencing Platform 2 Select the Amplicon Length NOTE An asterisk next to an amplicon length option indicates that the particular length is within specifications for that sequencing platform Selecting longer amplicons will result in a design that is ranked medium during the design review process Details on design rank are provided here Fluidigm Assay Design User Guide 14 PROJECT AND VERSION CONTROL SETTINGS D3 projects and versions can be controlled via the left panel buttons in the project version view Project level controls are Delete a project Version level controls are Copy this version VERSION
40. rt list HERMES 1 ADD TARGETS Please select a method for target entry Gene or RefSeq Name required By Gene or RefSeq By Genome Coordinate Add By Target Sequence By Fluidigm Assay ID From Existing Project By File Upload By Delimited Text Fluidigm Assay Design User Guide 21 DELTAgene ASSAYS ADDING TARGETS BY GENOME COORDINATES NOTE Please be sure to provide genome coordinates corresponding to the currently supported genome build The supported genome build is identified in the Genome Build field located in your project properties 1 ADD TARGETS Please select a method for target entry By Gene or RefSeq BRCA2 By Genome chr4 1803246 1803387 Coordinate By Target Sequence Add By Fluidigm Assay ID From Existing Project By File Upload By Delimited Text 1 Enterthe gene name or target alias and the coordinates keeping in mind the upper limit of 5000 bps 2 Click Add NOTE Optionally place square brackets around the regions of interest for primer design Assays will be designed to flank the bracketed area with a primer on either side of the bracket For example chr4 1803246 1803387 chr4 1803388 1803435 Fluidigm Assay Design User Guide 22 DELTAgene ASSAYS ADDING TARGETS BY TARGET SEQUENCE 1 ADD TARGETS Please select a method for target entry By Gene or RefSeq Sequence Name required By Genome Coordinate Sequ
41. s can be either by reference sequence gene or by genome coordinates As tabulated below entry formats vary depending on the parameters selected Tab Delimited Target Name lt tab gt Assay ID Target Name tab Sequence Comma Delimited Target Name lt comma gt Assay ID Target Name lt comma gt Sequence Fluidigm D3 Assay Design User Guide 28 O D3 BEST PRACTICES Adherence to best practices optimizes the D3 user experience This chapter provides design recommendations with pointers to increase assay coverage DESIGN RECOMMENDATIONS FOR ACCESS ARRAY ASSAYS list targets by genomic coordinates the format should be Chromosome Start Coordinates End Coordinates For example chr4 1803246 1803387 This can be used for genomic region resequencing SNP calling or selecting specific exons e When genomic coordinates are provided aliases must be added An alias should not exceed 15 characters Aliases are used to name primers For e g the primers for alias APC_ex3 will be named ex3 1 3 2 and ex3 3 e The optimal size range to obtain uniform coverage across amplicons is to design assays targeting regions 2150 bps However amplicon lengths should be within 2596 of the average in a given library NOTE Please be sure to provide genome coordinates corresponding to the currently supported genome build The supported genome build is identified in the Genome Build field located in your project properties
42. send email to TechSupport fluidigm com Phone in United States 1 866 FLUIDLINE 1 866 358 4354 Outside the United States 650 266 6100 On the Internet www fluidigm com support Visit our website at www fluidigm com
43. shown here ps E D Ium VERSION 5 Status Design Completed Last Modified 09 Sep 2013 Targets 6 Assays 139 Platform Illumina Amplicon Size 200 G Shared to shareduserl company com shareduserez company com sharedusers company com VERSIONS Last Modified Version 5 Status Design Completed To stop a version from being shared delete the shared email addresses and confirm the change QUESTIONS AND COMMENTS ABOUT ORDERING Why am unable to place an order An order can be placed only after design review of your assay is complete After you select your targets and send a design review request you will be notified within 48 72 hours that review is complete At that point you can work iteratively if you want to improve coverage by adding modifying targets and requesting another review Your design is ready to order once targets meet your satisfaction failed targets are removed minimum requirements are met and no more iteration is required Fluidigm Assay Design User Guide 46 Q How do determine the status of my order The design or order status of your project is automatically updated in D3 Update notices include Status Notification Created An empty project has been created without targets DESIGN In Progress Targets have been added and or targets have been submitted for design Completed Designs are completed and ready for your review Quote Request Submitted Quote Request Received
44. site does not accept my gene name Use the NCBI library to identify the official gene symbol If the gene name is not accepted try to enter aliases It is recommended that you always use NCBI RefSeq IDs Q If there are two RefSeq IDs for a single gene which one shall enter into the website Access Array designs can be designed to a specific gene isoform by entering the RefSeq ID which is always a unique identifier For gene level designs users can annotate using an asterisk DELTAgene designs are designed to the gene level whenever possible If designs do not detect all known isoforms a design comment will be included during design review Q Assay coverage for Access Array designs does not show gene direction Is the direction left to right or the orientation on the genome Access Array designs reference the plus strand of the human genome reference and do not imply gene transcription directionality Q am unable to change amplicon size for my Access Array design You can change the amplicon size only when there is no design associated with any target in that version Q would like to send some suggestions feedback Your feedback is always appreciated To provide feedback on assays or chemistry talk to your Field Applications Specialist or email techsupport fluidigm com Fluidigm Assay Design User Guide 49 5 GETTING HELP Appendix O Fluidigm has several resources to support you in y
45. t ACCESS ARRAY ASSAYS ADDING TARGETS BY DELIMITED TEXT ENTRY Two text entry delimiters tabs or commas are permitted The entries themselves can be either by reference sequence gene or by genome coordinates Entry formats vary depending on the parameters selected as tabulated here 22 DATA ENTRY FORMATS By RefSeq Gene By Genome Coordinates RefSeq or Gene Name lt tab gt Upstream TSS 0 Target Name lt tab gt Coordinate String 500 lt tab gt 5 UTR Y N lt tab gt 3 UTR Y N Tab Delimited RefSeq or Gene Name lt comma gt Upstream TSS 0 Target Name lt comma gt Coordinate String 500 lt comma gt 5 UTR Y N lt comma gt 3 UTR Y N Comma Delimited Fluidigm Assay Design User Guide 20 ADDING TARGETS FOR DELTAgene ASSAYS lf the species is in the supported list users add targets by RefSeq ID gene symbol genome coordinate target sequence or Fluidigm Assay ID Otherwise only Target Sequence or Fluidigm Assay ID are valid entry methods e If genome coordinates are entered each alias must be defined and unique e f Assay IDs not entered they are automatically populated when the design query is complete assuming there is a design for the target DELTAgene ASSAYS ADDING TARGETS BY GENE NAME OR RefSeq ID 1 Enter the gene name or RefSeq ID NCBI nomenclatures are acceptable 2 When you finish click Add NOTE If a gene cannot be located it may be known by a different alias in the suppo
46. the parameters selected as tabulated here By Assay ID By RefSeq Gene Tab Target Name lt tab gt Assay RefSeq or Gene Delimited ID Name lt tab gt Comma Target RefSeq or Gene Delimited Name comma Assay ID Name lt tab gt ADDING TARGETS FOR SNPtype ASSAYS For SNPtype Assays the following parameters apply By Sequence Target Name tab Sequence Target Name lt comma gt Sequen ce By Coordinates Target Name lt tab gt Coordinate String Target Name lt comma gt Coordinate String e fthe species is in the supported list users can add targets by 5 target sequence or Fluidigm Assay ID e fthe species is not in the supported list only Target Sequence or Fluidigm Assay ID are valid entry methods e lf either Target Sequence or Fluidigm Assay ID is entered the SNP name must be defined e If the Fludigm Assay ID is not entered it is automatically populated when the design query is complete assuming that there is a design for the target SNPtype ASSAYS ADDING TARGETS BY RS 1 Enter the RS or the polymorphism dbSNP nomenclatures are acceptable 2 When you finish click Add 1 ADD TARGETS Please select a method for target entry By RS rs139589944 rs7574220 By Target Sequence rs6433410 rs10242197 By Fluidigm Assay ID 516919668 56493973 From Existing Project By File Upload By Delimited Text Add Fluidigm Assay Design User Guide 25 SNPtype ASSAYS ADDING
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