nCounter_CNV_Data_Analysis_Guidelines
Contents
1. Hg18 chr22 42503372 42509471 _INVCONTROL 637 828 847 951 936 48 invariant INVCONTROL 5 Hg18 chr5 71261236 71261335 INVCONTROL 650 887 837 356 391 49 Invariant INVCONTROL 7 Hz18 chr7 41541153 41541252 _INVCONTROL 577 838 783 313 878 50 RestrictionSite RESTRICTIDNSITE A CTL AluI A _RESTRICTIONSITE 12 15 25 43 41 47 nanoStrin 8 T E c HN O LO G I E S NanoString Technologies PRODUCT MANUAL 2 Calculate a normalization factor for each assay by first calculating the mean of the average INV count values mean AVE INV across all lanes you wish to analyze Figure 1 6 FIGURE 1 6 Calculating the mean of the average INV count values 19 20 Code Class Name Accession 21 Positive POS A 128 ERCC 00117 1 21918 25182 22 Positive POS B 32 ERCC 00112 1 6608 7521 23 Positive POS C 8 ERCC 00002 1 1405 1627 24 Positive POS D 2 ERCC 00092 1 366 429 25 Positive POS E 0 5 ERCC_00035 1 139 161 26 Positive POS F 0 125 ERCC 00034 1 65 72 27 Negative NEG A 0 ERCC 00096 1 0 1 28 Negative NEG B 0 ERCC 00041 1 3 7 29 Negative NEG C 0 ERCC 00019 1 8 6 30 Negative NEG D 0 ERCC 00076 1 3 2 31 Negative NEG E 0 ERCC 00098 1 5 11 32 Negative NEG F 0 ERCC 00126 1 1 2 33 Negative NEG G 0 ERCC 00144 1 4 7 34 Negative NEG H 0 ERCC 00154 1 0 2 35 36 mean INV counts 695 485 37 AVERAGE D3b 136 38 Norm factor d 20426 4640 1351 284 242 87 wo 16 11 10 13 696 0 97 22. NEG H 0 ERCC 00154 1 0 4 In the scatter plot shown in Figure 1 3 the axes are represented in logarithmic scale and the linear regression R is shown in the inset The R value for the POS control probes should be gt 0 95 Restriction Fragmentation Controls The Custom CNV Assay Kit comes with a set of four DNA controls that when added to your genomic DNA sample prior to fragmentation will monitor the efficiency of enzymatic digestion and heat denaturation The DNA targets for probes labeled RESTRICTIONSITE A and RESTRICTIONSITE B contain an Alul restriction site such that after complete digestion the target site will be cleaved by the enzyme and low probe count will be observed The DNA targets for probes labeled RESTRICTIONSITE C and RESTRICTIONSITE D do not contain Alul sites and will generate probe counts even in the absence of fragmentation These targets will serve as controls for proper addition of the control DNA to the sample and proper heat denaturation If the DNA sample is not denatured prior to hybridization you will observe low counts generally 200 for RESTRICTIONSITE C and RESTRICTIONSITE D probes When the genomic DNA sample is completely digested with Alul enzyme and denatured you should observe at least a 10 fold difference in counts between RESTRICTIONSITE Probes A and B and RESTRICTIONSITE probes Probes C and D FIGURE 1 4 Restriction Fragmentation Controls 50 RestrictionSite RESTRICTIONSITE A CTL
3. Alul A RESTRICTIONSITE 12 15 25 43 41 47 51 RestrictionSite _RESTRICTIONSITE 8 CTL Alul B RESTRICTIONSITE 10 15 34 71 83 47 52 RestrictionSite RESTRICTIONSITE C CTL Alul C _RESTRICTIONSITE 1255 1399 1144 1331 1295 1120 53 RestrictionSite RESTRICTIONSITE D CTL Alul D RESTRICTIONSITE 975 1115 1336 1548 1541 1305 Molecules That Count 7 PRODUCT MANUAL nCounter Data Analysis Guidelines for CNV Normalization to Invariant Probes Each nCounter Custom CNV CodeSet contains probes designed to invariant regions of 10 autosomes It is assumed that these 10 regions will represent 2 chromosomal copies in a vast majority of samples analyzed Therefore normalizing data to the counts obtained from these 10 invariant probes should correct for any differences in sample to sample genomic DNA input arising from pipetting error or inaccuracies in DNA quantitation Normalization is performed automatically by the nCounter CNV Collector Tool but can be done manually as follows 1 To normalize data to the Invariant probes calculate the average count value for the 10 INV probes AVE INV in the first lane sample as shown in Figure 1 5 FIGURE 1 5 Calculating the average count value gra A B C E pd amp H l 20 Code Closs Nome Accession 21 Positive POS A 128 ERCC 00117 1 21918 25182 20426 25194 25293 22480 22 Positive POS B 32 ERCC 00112 1 6608 7521 4640 6107 6070 5367 23 Pasitive POS C 8 ERCC 00002 1 1
4. CNV Assay Manual Contacting Support For questions about the nCounter CNV Assay nCounter CNV Collector Tool or data analysis please contact support nanostring com Phone 206 378 NANO or contact your NanoString Field Applications Scientist Molecules That Count 15 PRODUCT MANUAL nCounter Data Analysis Guidelines for CNV nanostring TECHN OL Oo GIE S NanoString Technologies Inc CONTACT US SALES CONTACTS 530 Fairview Ave N info nanostring com United States us sales nanostring com Suite 2000 Tel 888 358 6266 Europe europe sales nanostring com Seattle Washington 98109 Fax 206 378 6288 Japan japan sales nanostring com www nanostring com Other Regions info nanostring com 2011 NanoString Technologies Inc All rights reserved NanoString NanoString Technologies nCounter and Molecules That Count are registered trademarks of NanoString Technologies Inc NanoString in the United States and or other countries All other trademarks and or service marks not owned by NanoString that appear in this document are the property of their respective owners The manufacture use and or sale of NanoString product s may be subject to one or more patents or pending patent applications owned by NanoString or licensed to NanoString from Life Technologies Corporation and other third parties FOR RESEARCH USE ONLY Not for use in diagnostic procedures MAN C0014 02
5. If the reference sample copy number is 1 for a particular genomic region the formula for the corresponding probes can be altered by deleting the multiplication factor of 2 In the example below the reference sample for probel Chr1 is changed to Sample2 Column E and the copy number formula is adjusted for a copy number of 1 NOTE It may not be necessary to change the reference sample only to alter the known copy number of the original reference sample following the same method outlined here FIGURE 1 13 A A B Cc E F G H I T 22 Code Class Name Accession 23 Invariant _INVCONTROL 1 Hg18 chr1 97239055 97239154 _INVCONTROL 2 0 2 2 2 2 2 2 2 4 2 1 24 Invariant _INVCONTROL 10 Hg18 chr10 76411622 76411718 _INVCONTROL 2 0 2 4 1 8 L9 19 13 25 Invariant INVCONTROL 16 Hg18 chr16 11026537 11026636 _INVCONTROL 2 0 1 7 1 8 1 7 1 6 1 6 26 Invariant _INVCONTROL 17 Hg18 chr17 60617472 60617571 _INVCONTROL 2 0 2 1 2 2 2 0 A rA 27 Invariant INVCONTROL 19 Hg18 chr19 38818850 38818949 _INVCONTROL 2 0 2 0 1 8 2 9 2 0 2 1 28 Invariant _INVCONTROL 2 Hg18 chr2 137221998 137222092 _INVCONTROL 2 0 2 0 1 9 2 0 1 8 2 0 29 invariant _INVCONTROL 20 Hg18 chr20 10504898 10504992 _INVCONTROL 2 0 2 1 1 9 1 8 23 2 0 30 Invariant _INVCONTROL 22 Hg18 chr22 42509372 42509471 _INVCONTROL 2 0 2 0 2 2 2 1 2 0 rA 1 Invariant INVCONTROL 5 Hg18 chr5 71261236 71261335 _INVCONTROL 2 0 1 8 4i 2 1 2 1 2 0 2 Invariant INVCONTROL 7 Hg1
6. offer helpful suggestions alternative procedures methods and or shortcuts NOTE This note type emphasizes general information IMPORTANT GD This note type presents essential content indicating that the potential exists for assay failure diminished data quality and or a loss of data if the information presented is ignored WARNING This note type indicates that a potential hazard to your personal safety or the potential for equipment damage exists BOLD When appearing in text or in a procedure the bold text serves to highlight a specific button key stroke or menu option available Bold text may appear elsewhere to highlight important text or terms Green text is used to help the reader identify active hyperlinks ITALICS Used to emphasize an important word or expression within the text Formatting of a book title journal or other documentation Used to indicate the special or unusual meaning of a word or phrase Contact Information NanoString Technologies Inc 550 Fairview Ave N Suite 2000 Seattle Washington 98109 USA Tel 206 5 8 6266 888 558 NANO 6266 Fax 206 578 0288 E mail support nanostring com nanostring 4 T E c HN O LO G I E S PRODUCT MANUAL Introduction The nCounter Custom Copy Number Variation CNV Assay utilizes NanoString s unique direct and multiplexed detection of nucleic acids in solution to generate estimates of copy number variation for hundreds of loci in a single
7. reaction Each NanoString Reporter and Capture Probe pair is complementary to 100 nt of contiguous genomic DNA sequence at a user specified locus Genomic DNA is fragmented into small pieces 200 800 bp and denatured to produce single strands The Custom CNV CodeSet is then hybridized to the fragmented denatured DNA sample in a single multiplexed reaction up to 800 genomic loci per CodeSet Hybridized DNA CodeSet complexes are purified by the fully automated nCounter Prep Station and Reporters are counted by the nCounter Digital Analyzer The results of an nCounter Custom CNV Assay experiment are compiled and displayed using the CNV Collector Tool software included with the assay kit Detailed instructions for using this tool are in the CNV Collector Tool User Manual provided with the software The following Data Analysis Guidelines for CNV are intended as a supplement to the nCounter CNV Collector Tool User Manual They provide instructions and additional information for those who wish to do further QC and or data manipulations with the data output from the CNV Collector Tool such as e Modify the normalization method Apply additional assay quality control metrics Change reference samples within a single data set Use reference samples with known copy numbers that differ from 2 nCounter Custom CNV Data Output Raw Data The basic output of the Custom CNV Assay is a spreadsheet containing the CodeSet probe identifiers sample id
8. the average of copy number estimate values using the AVERAGE function as described in Normalization to Invariant Probes on page 5 Generating Integer Copy Number Calls To convert copy number estimates to integer copy number predictions the copy number estimate values must be rounded The simplest method is to round each estimated value to the nearest integer using an IF function in Excel In the following example we demonstrate an integer copy number 0 4 although each investigator should determine the appropriate rounding criteria based on their own specific data analysis requirements NOTE Copy number estimates that are half integer values e g 1 5 2 5 3 5 will require further interpretation by the investigator to determine the integer copy number value In the following example such values are returned as the decimal copy number estimate value and are not rounded to the nearest integer nanoStrin 12 T E c HN O LO G I E S NanoString Technologies PRODUCT MANUAL On a new sheet enter an IF formula in the cell corresponding to the first probe and first sample following the example shown below The values entered in the formula can be adjusted to alter the rounding criteria For example values between 0 8 and 1 2 rather than 0 6 and 1 4 can be rounded to 1 Once the formula has been entered click on the lower right corner of the cell and drag the formula down to apply to all Invariant and Endogenous probes then ac
9. 205 E C H AL REN c LC O LM ES nCounter CNV nCounter Data Analysis Guidelines for Copy Number Variation CNV NanoString Technologies Inc 550 Fairview Ave N Suite 2000 Seattle Washington 98109 www nanostring com Tel 206 5 8 6266 888 558 6266 E mail info nanostring com Molecules That Count Translational Research Gene Expression miRNA Expression Copy Number Variation MAN C0014 02 PRODUCT MANUAL nCounter Data Analysis Guidelines for CNV FOR RESEARCH USE ONLY Not for use in diagnostic procedures Intellectual Property Rights This nCounter Analysis System manual and its contents are the property of NanoString Technologies Inc NanoString and is intended solely for the use of NanoString customers for the purpose of operating the nCounter Analysis System The nCounter Analysis System including both its software and hardware components and this User Guide and any other documentation provided to you by NanoString in connection therewith are subject to patents copyright trade secret rights and other intellectual property rights owned by or licensed to NanoString No part of the software or hardware may be reproduced transmitted transcribed stored in a retrieval system or translated into other languages without the prior written consent of NanoString Limited License Subject to the terms and conditions of the nCounter Analysis System contained in the product quotation N
10. 405 1627 1351 1023 1637 1464 24 Positive POS D 2 ERCC_00092 1 366 425 284 406 402 331 25 Positive POS E 0 5 ERCC 00035 1 139 161 242 201 270 241 26 Positive POS F 0 125 ERCC 00034 1 65 72 87 80 79 80 27 Negative NEG A 0 ERCC 00096 1 0 1 6 B 14 7 28 Negative NEG B 0 ERCC 00041 1 3 7 9 il 13 15 29 Negative NEG C 0 ERCC 00019 1 8 6 9 10 5 10 30 Negative NEG D 0 ERCC 00076 1 3 2 16 20 15 12 3i Negative NEG E 0 ERCC 00098 1 5 11 11 7 9 11 32 Negative NEG F 0 ERCC 00126 1 1 2 15 13 15 12 33 Negative NEG G 0 ERCC 00144 1 4 7 10 B 13 15 34 Negative NEG_H 0 ERCC 00154 1 0 2 13 14 19 13 35 6 AVERAGE D40049 485 696 574 758 754 37 average GMrRocrmmmmert number Jj 38 Norm actr 097 1 40 0 97 1 00 0 80 0 90 39 40 Invariant INVCONTROL i Hgl8 chri 97239055 97239154 INVCONTROL 632 906 856 880 985 41 Invariant _INVCONTROL 10 Hg18 chr10 76411522 76411718 INVCONTROL 226 414 388 435 AAD 42 invariant INVCONTROL 18 Hglg chr18 11025537 11026636 INVCONTROL 356 495 500 575 571 43 invariant _INVCONTROL 17 Hg18 chr17 60617472 60617571 INVCONTROL 530 732 761 213 832 44 Invariant INVCONTROL 19 Hg18 chr19 38818850 38818949 INVCONTROL ABA 715 853 702 684 45 Invariant INVCONTROL 2 Hg18 chr2 127221938 137222092 INVCONTROL ala 636 585 739 663 46 Invariant _INVCONTROL 20 Hg18 chr20 10504898 10504992 _INVCONTROL 320 511 527 565 538 47 Invariant INVCONTROL 22
11. 6194 6107 1623 261 11 10 20 13 14 674 1 00 25293 6070 1637 402 270 79 14 13 Dh wo U uw 19 758 22480 5367 1464 331 241 DBHRER SG 754 To generate a normalization factor divide the mean average value mean AVE INV from Step 2 by the average count value AVE INV from Step 1 for each lane FIGURE 1 7 Generating a Normalization Factor EL a B c 19 20 Code Class Name Accession 21 Positive POS A 128 ERCC 00117 1 22 Positive POS B 32 ERCC 00112 1 23 Positive POS C amp ERCC 00002 1 24 Positive POS D 2 ERCC 00092 1 25 Positive POS E 0 5 ERCC 00035 1 26 Positive POS F 0 125 ERCC 00034 1 27 Negative NEG A 0 ERCC 00096 1 28 Negative NEG B 0 ERCC 00041 1 29 Negative MEG C 0 ERCC 00019 1 30 Negative NEG D 0 ERCC 00076 1 31 Negative NEG E 0 ERCC 00098 1 32 Negative NEG F 0 ERCC 00126 1 33 Negative NEG G 0 ERCC 00144 1 34 Negative NEG H 0 ERCC 00154 1 35 36 mean INV counts 37 average 38 Norm factor Molecules That Count 21918 1405 139 65 0 3 8 3 5 1 A 0 20182 7521 1627 429 161 72 en 11 j 1 40 20420 1351 242 of wo 16 11 10 13 0 97 PRODUCT MANUAL nCounter Data Analysis Guidelines for CNV 3 Calculate INV normalized counts for each probe On a new sheet generate normalized counts for each probe in the CodeSet by multiplying the RAW counts for each probe by the normali
12. 8 chr7 41541153 41541252 _INVCONTROL 2 0 1 9 1 9 2 0 1 9 2 0 33 Endogenous probe1 Hg18 chri coordinates CNV region 1 zv norm example D33 INV norm example SE33 3 9 M Endogenous probe2 Hg18 chr8 coordinates CNV region 2 20 3 1 0 7 1 0 1 0 1 4 35 Endogenous probe3 Hg18 chr10 coordinates CNV region 3 2 0 3 0 1 4 1 9 2 1 2 7 36 Endogenous probe4 Hg18 chr12 coordinates CNV region 4 2 0 3 2 1 0 1 3 La 1 8 37 Endogenous probe5 Hg18 chrX 111441962 111442061 CNV region 5 2 0 1 2 1 4 0 9 2 1 rA 38 Endogenous probe6 Hg18 chrYv 2715200 2715287 CNV region 6 0 0 1 0 1 0 11 0 0 0 0 nanoStrin T E c HN O LO G I E S NanoString Technologies PRODUCT MANUAL If the copy number of the new reference is 3 the formula can be adjusted by changing the multiplication factor to 3 to generate the correct copy number In the following example the reference sample is Sample 1 but the formula is adjusted by adding a multiplication factor of 5 to reflect 5 copies of probe regionl in the reference sample FIGURE 1 14 ud A pad B ee S DD E EOS PE E 21 22 Code Class Name Accession 23 Invariant _INVCONTROL 1 Hg18 chr1 97239055 97239154 _INVCONTROL 2 0 2 2 2 2 2 2 24 Invariant INVCONTROL 10 Hg18 chr10 76411622 76411718 _INVCONTROL 2 0 24 1 8 1 9 25 Invariant _INVCONTROL 16 Hg18 chr16 11026537 11026636 _INVCONTROL 2 0 1 7 1 8 1 7 26 Invariant INVCONTROL 17 Hg18 chr17 60617472 60617571 _INVCONTROL 2 0 2 1 2 2 2 0 2 In
13. 898 10504992 INVCONTROL 2 0 21 1 9 1 8 1 9 2 0 30 Invariant INVCONTROL 22 Hg18 chr22 42509372 42509471 _INVCONTROL 2 0 2 0 2 2 2 1 2 0 2 1 31 Invariant _INVCONTROL 5 Hg18 chr5 71261236 71261335 _INVCONTROL 2 0 1 8 2 1 2 1 2 1 2 0 32 Invariant _INVCONTROL 7 Hg18 chr7 41541153 41541252 _INVCONTROL 2 0 1 9 1 9 2 0 L9 2 0 33 Endogenous probel Hg18 chr1 coordinates CNV region 1 0 6 3 2 2 2 2 6 3 0 3 9 34 Endogenous probe2 Hg18 chr8 coordinates CNV region 2 2 0 3 1 0 7 1 0 1 0 1 4 35 Endogenous probe3 Hg18 chr10 coordinates CNV region 3 2 0 3 0 1 4 1 9 2 1 2 7 36 Endogenous probe4 Hg18 chr12 coordinates CNV region 4 2 0 3 2 1 0 1 3 1 3 1 8 37 Endogenous probe5 Hg18 chrX 111441962 111442061 CNV region 5 2 0 1 2 14 0 9 2 1 2 1 38 Endogenous probe6 Hg18 chrY 2715200 2715287 CNV ret INV norm example E38 IN norm example E38 0 0 0 0 Averaging Copy Number Estimates by Genomic Region If your nCounter Custom CNV CodeSet contains multiple probes for a single genomic locus it may be desirable to generate an average copy number estimate value based on all probes for that particular locus This average value can then be used to generate an integer copy number assignment If you are using a single probe per region proceed to Generating Integer Copy Number Calls below for instructions on how to round the estimated copy number To average probes for each locus create a new spreadsheet For each genomic region locus calculate
14. AN I nnm eri oe c nns de pcs INV norm example CN estimate example Integer Copy Number example For copy number analysis it is not necessary to normalize the data for POS NEG or Restriction Site controls For simplicity these probes can be left out of the INV normalization spreadsheet The INV normalization procedure should be carried out on the Invariant control and Endogenous code classes e CAUTION Copy number data generated by the Custom CNV Assay can be negatively affected when DNA input amounts are too low at which point sampling error can introduce unacceptably high levels of variation in the data NanoString recommends a minimum of 100 counts for the average of the 10 Invariant control probes in the INV normalized data set to ensure reliable copy number estimation This is particularly important for the reference sample s since accurate copy number calculations depend upon high quality reference sample data A poor quality reference sample will adversely affect copy number calls for all samples A poor quality test sample will result in unreliable copy number call for that sample alone In general for purified genomic DNA free of RNA contamination 100 INV normalized counts will correspond to 2 100 ng genomic DNA Calculating Copy Number Estimates Reference Sample Selection The basic data analysis strategy for determining copy numbers with the nCounter Custom CNV Assay is to calculate a copy number estimate for each pr
15. CNV region 3 2 3 1 4217305 2 2 3 36 Endogenous probe4 Hg18 chr12 coordinates CNV region 4 2 3 1 1 1 2 37 Endogenous probe5 Hg18 chrX 111441962 111442061 CNV region 5 2 1 1 4052658 1 2 2 38 Endogenous probe6 Hg18 chrY 2715200 2715287 CNV region 6 0 1 1 1 0 0 Here we have presented a simple three step method for generating integer copy number calls from the nCounter CNV Assay raw data normalization to INV probe counts copy number estimation relative to a reference sample and conversion to integer copy number calls In the next section Reference Sample Adjustments we will consider situations when the copy number of the reference sample differs from 2 and when it is desirable to use multiple reference samples Molecules That Count 13 PRODUCT MANUAL nCounter Data Analysis Guidelines for CNV 14 Reference Sample Adjustments In some cases the reference sample selected may contain genomic regions with a copy number of O deletion 1 single copy or greater than 2 copies Since it is not possible to calculate copy number estimates using a reference sample with O copies at a particular locus it is necessary to change the reference to a sample that has counts registered for that specific region and that has a known copy number Adjusting the Copy Number Estimate Calculation After the appropriate reference sample has been identified it may be necessary to adjust the formula for calculating the copy number estimate
16. anoString grants you a limited non exclusive non transferable non sublicensable research use only license to use the proprietary nCounter Analysis System only in accordance with the manual and other written instructions provided by NanoString Except as expressly set forth in the terms and conditions no right or license whether express implied or statutory is granted by NanoString under any intellectual property right owned by or licensed to NanoString by virtue of the supply of the proprietary nCounter Analysis System Without limiting the foregoing no right or license whether express implied or statutory is granted by NanoString to use the nCounter Analysis System with any third party product not supplied or licensed to you by NanoString or recommended for use by NanoString in a manual or other written instruction provided by NanoString Trademarks NanoString Technologies NanoString nCounter and Molecules That Count are registered trademarks or trademarks of NanoString Technologies Inc in the United States and or other countries All other trademarks and or service marks not owned by NanoString that appear in this manual are the property of their respective owners Copyright 2011 NanoString Technologies Inc All rights reserved nanostring T E c HN O LO G I E S NanoString Technologies PRODUCT MANUAL CONTENTS ditis MACC RTTE 4 Conventions IS ae rte segs nat atone cea
17. ation reaction These probes monitor the nonspecific or background counts for every assay Each CodeSet contains a set of 10 probes INV designed to autosomal genomic regions predicted not to contain common CNVs Custom CNV CodeSets contain four control probes to monitor the efficiency of the DNA fragmentation and denaturation steps of the CNV Assay Protocol Probes A and B are designed to a DNA sequence containing an Alul restriction site and will return low count when the Alul fragmentation is working correctly Probes C and D are designed to sequences that lack an Alul restriction site and serve as controls for the presence of target DNA in the sample preparation step When used according to the assay manual these controls help identify problems in restriction enzyme fragmentation and denaturation steps of the assay The Custom CNV Assay probes specified by the user designed to specific regions of the genome Basic CNV Data Analysis Workflow FIGURE 1 2 CNV Data Analysis Workflow Step 1 Assay QC 4 Step 2 Data Normalization 4 Step 3 Copy Number Estimation Relative to Reference Sample v Step 4 Averaging Probes per Region if applicable nanostring T E c HN O LO G I E S PRODUCT MANUAL NanoString Technologies Assay Quality Control Check When used according to the assay manual each Custom CNV Assay contains controls which monitor hybridization efficiency sample DNA fragmentation denaturation and samp
18. entifiers and the digital counts recorded for each probe in each sample This file referred to as a Reporter Code Count RCC file can be uploaded to the nCounter CNV Collector Tool for automated normalization and analysis FIGURE I I Report Code Count RCC file Code Class Name Accession Positive POS A 128 ERCC 00117 1 21918 25182 20426 26194 25293 22480 Positive POS B 32 ERCC 00112 1 6608 7521 4640 6107 6070 5367 Positive POS C 8 ERCC 00002 1 1405 1627 1351 1623 1637 1464 Positive POS D 2 ERCC 00092 1 366 429 284 406 402 331 Positive POS E 0 5 ERCC 00035 1 139 161 242 261 270 241 Positive POS F 0 125 ERCC 00034 1 65 72 87 80 79 Negative NEG A 0 ERCC 00096 1 0 1 6 8 14 7 Negative NEG B 0 ERCC 00041 1 3 7 5 11 13 Negative NEG C 0 ERCC 00019 1 8 6 9 10 a 10 Negative NEG DIO ERCC 00076 1 3 2 16 20 15 12 Molecules That Count Translational Research Gene Expression miRNA Expression Copy Number Variation D PRODUCT MANUAL nCounter Data Analysis Guidelines for CNV Code Classes 1 POSITIVE 2 NEGATIVE 3 INVARIANT 4 RESTRICTION SITE 5 ENDOGENOUS nCounter Custom CNV CodeSets contains 6 positive dsDNA control probes each targeting a unique DNA sequence present in every assay The concentrations of DNA target range from 0 125 fM to 128 fM in the hybridization reaction nCounter Custom CNV CodeSets contain 8 negative control probes for which there is no DNA target present in the hybridiz
19. le DNA input amount Before continuing with CNV analysis first look at the Raw Data output file from the CNV Collector to gauge the performance of the assay Positive Controls The positive control POS DNA targets are added in a linear titration to each codeset to generate a standard curve The final concentration in the hybridization of each target in fM is indicated in parentheses next to the POS probe identifier To check the linearity of the POS control standard curve insert a new column in the Raw Data spreadsheet to the left of the first assay data column Add the POS target concentrations to the first 6 rows of this new column Then create a scatter plot of the target concentration vs raw data for each target Figure 1 3 FIGURE 1 3 Positive Controls A B C D E F Rn I i J K 20 Code Class Name Accession ju _ 21 Positive POS A 128 ERCC_00117 1 128 21913 199966 22 Positive POS B 32 ERCC 00112 1 ys pioa 23 Positive POS C 8 ERCC 00002 1 9006 7 24 Positive POS D 2 ERCC 00092 1 BT 25 Positive POS E 0 5 ERCC 00035 1 oe 26 Positive POS F 0 125 ERCC 00034 1 65 27 Negative NEG A 0 ERCC 00096 1 0 iiia i 28 Negative NEG B 0 ERCC 00041 1 3 PERDU 1 29 Negative NEG C 0 ERCC 00019 1 8 30 Negative NEG D 0 ERCC 00076 1 3 31 Negative NEG E 0 ERCC 00098 1 5 32 Negative NEG F 0 ERCC 00126 1 1 33 Negative NEG G 0 ERCC 00144 1 alil o4 i i ii 34 Negative
20. ndogenous probe4 Hg18 chr12 coordinates CNV region 4 2 0 3 2 1 0 1 3 1 3 1 8 Endogenous probe5 Hg18 chrX 111441962 111442061 CNV region 5 ENV nbrm example D37 INV norm example D37 2 2 1 38 Endogenous probe6 Hg18 chrY 2715200 2715287 CNV region 6 0 0 1 0 1 0 1 1 0 0 0 0 Molecules That Count ll PRODUCT MANUAL nCounter Data Analysis Guidelines for CNV In order to generate meaningful copy number estimates for the Y chromosome probe it is best to use a reference sample that contains a Y chromosome male The formula for Y chromosome probes can then be adjusted to use this male reference to calculate copy numbers for only the Y chromosome probe s In the example below the reference for the Y chromosome probe has been switched to Sample 2 Column E FIGURE L11 22 Code Class Name Accession 23 Invariant INVCONTROL 1 Hg18 chr1 97239055 97239154 _INVCONTROL 2 0 2 2 2 2 22 2 4 2 1 24 Invariant INVCONTROL 10 Hg18 chr10 76411622 76411718 _INVCONTROL 2 0 2 4 1 8 1 9 1 9 1 9 25 Invariant INVCONTROL 16 Hg18 chr16 11026537 11026636 _INVCONTROL 2 0 1 7 1 8 1 7 1 6 1 6 26 Invariant _INVCONTROL 17 Hg18 chr17 60617472 60617571 _INVCONTROL 2 0 v 2 2 2 0 AL 2 1 27 Invariant INVCONTROL 19 Hg18 chr19 38818850 38818949 _INVCONTROL 2 0 2 0 1 8 1 9 2 0 2 1 28 Invariant INVCONTROL 2 Hg18 chr2 137221998 137222092 _INVCONTROL 2 0 2 0 1 9 2 0 1 8 2 0 29 Invariant INVCONTROL 20 Hg18 chr20 10504
21. obe relative to a reference sample or samples Each probe in the Custom CNV CodeSet is a unique sequence and bar code and as a result small variations in probe efficiency can result in count variation between probes even when targeting genomic regions of equal copy number However this difference in counting efficiency will be constant for a given probe over all samples analyzed Therefore highly accurate copy number estimates can be generated by simply taking the ratio of counts from test samples to the counts of a fixed reference sample s and calculating copy numbers relative to that reference sample The example analysis that follows will assume a copy number of 2 for all genomic loci being assayed in the reference sample In a later section Reference Sample Adjustments on page 10 we will consider alternative analysis strategies for genomic loci that differ from 2 copies in the reference sample s nanoStrin 10 T E c HN O LO G I E S NanoString Technologies PRODUCT MANUAL NOTE If the copy number of your reference sample is not known the analysis method presented here will only estimate the copy number relative to the unknown reference an absolute integer copy number prediction will not be possible First start a new sheet for copy number calculations and select your reference sample In the example below the reference sample will be Sample 1 Column D Next divide each test samples probe value INV normalized counts by the co
22. ross to apply to all samples FIGURE 1 12 La A B c E F G H l 21 22 Code Class Name Accession 23 Invariant _INVCONTROL 1 Hg18 chr1 97239055 97239154 _INVCONTROL ir cN estimate example D23 0 4 0 IF AND CN estimate example D23 gt 0 6 24 Invariant INVCONTROL 10 Hg18 chr10 76411622 76411718 INVCONTROL CN estimate example D23 1 4 1 IF AND CN estimate example D23 gt 1 6 CN 25 Invariant INVCONTROL 16 Hg18 chr16 11026537 11026636 _INVCONTROL estimate example D23 2 4 2 IF AND CN estimate example D23 gt 2 6 CN 26 Invariant INVCONTROL 17 Hg18 chr17 60617472 60617571 _INVCONTROL estimate example D23 3 4 3 IF CN estimate example D23 gt 3 6 4 CN 27 Invariant INVCONTROL 19 Hgi8 chr19 38818850 38818949 _INVCONTROL estimate example D23 28 Invariant JINVCONTROL 2 Hg18 chr2 137221998 137222092 INVCONTROL IF logical test value if true value if false 2 2 29 Invariant JINVCONTROL 20 Hg18 chr20 10504898 10504992 INVCONTROL 2 2 2 2 2 2 30 Invariant _INVCONTROL 22 Hg18 chr22 42509372 42509471 INVCONTROL 2 2 2 2 2 2 31 Invariant _INVCONTROL 5 Hg18 chr5 71261236 71261335 _INVCONTROL 2 2 2 2 2 2 32 Invariant _INVCONTROL 7 Hg18 chr7 41541153 41541252 _INVCONTROL 2 2 2 2 2 2 33 Endogenous probe1 Hg18 chr1 coordinates CNV region 1 1 3 2 3 3 4 34 Endogenous probe2 Hg18 chr8 coordinates CNV region 2 2 3 1 1 1 1 4418876 35 Endogenous probe3 Hg18 chr10 coordinates
23. rresponding probes in the reference samples INV normalized counts For autosomal chromosomes 1 22 multiply this quotient by 2 to account for the presence of two chromosomal copies in the diploid reference sample Hint Use the shortcut command to hold the reference sample column constant in the formula FIGURE 1 9 22 Code Class Name Accession 23 Invariant _INVCONTROL 1 Hg18 chr1 97239055 97239154 _INVCONTROL Env norm example D23 INV norm example D23 2 2 1 24 Invariant _INVCONTROL 10 Hg18 chr10 76411622 76411718 _INVCONTROL 2 0 2 4 1 8 1 9 LS 13 25 Invariant _INVCONTROL 16 Hg18 chr16 11026537 11026636 _INVCONTROL 2 0 1 7 1 8 1 7 1 6 1 6 26 Invariant _INVCONTROL 17 Hg18 chr17 60617472 60617571 _INVCONTROL 2 0 re 2 2 2 0 2 1 2 1 27 Invariant _INVCONTROL 19 Hg18 chr19 38818850 38818949 _INVCONTROL 2 0 2 0 1 8 1 9 2 0 2 1 28 Invariant _INVCONTROL 2 Hg18 chr2 137221998 137222092 _INVCONTROL 2 0 2 0 1 9 2 0 1 8 2 0 a Invariant INVCONTROL 20 Hg18 chr20 10504898 10504992 _INVCONTROL 2 0 2 1 1 9 1 8 15 2 0 30 Invariant _INVCONTROL 22 Hg18 chr22 42509372 42509471 _INVCONTROL 2 0 2 0 22 2 1 2 0 2 1 31 Invariant _INVCONTROL 5 Hg18 chr5 71261236 71261335 _INVCONTROL 2 0 1 8 2i 2 1 2 1 2 0 32 Invariant _INVCONTROL 7 Hg18 chr7 41541153 41541252 _INVCONTROL 2 0 1 9 1 9 2 0 1 9 2 0 X and Y Chromosome Copy Number Calculation The formula for determining copy number of the X and Y chromosomes must be adj
24. se ent te mid inso east Ounce FM M EREE ESRA a 4 Contecr IntoEmiaEIOBI soise E EE Tes Sube DIU RUFEN LUI EE SU E CITED 4 CHAPTER 1 Introduction m ET 5 15 nCounter Custom CNV Data Output sssesee eene eret reete reete ER E EEEa 5 E AE i T e m 5 Goo LE EA E EE AE N E EE AE A E E AEE E E A P 6 Basic CNV Data Analysis Workflow sse erre eere tnn 6 PS Sey Ci Il ENT e Rm 6 Normalization to Invariant Probes sscncsccsniaisdssensesshsnecdasessnseacesecnies snsdbeaniedasadaashbinbiedotaetacwkeadassucanidenkssandsassatenss 8 Caleulating Copy Number ESCH SDSS zits icccsterccice restet Hoe nE E Ennii 10 X and Y Chromosome Copy Number Calculation 11 Averaging Copy Number Estimates by Genomic Region esscr 12 Generating Integer Copy Number Calls sseeeeeeeeenenneene nnne 12 Reference Sample Adjustments ssssssssemeeemenmeneerememenenr eren eren 14 Adjusting the Copy Number Estimate Calculation nnrir 14 Reference Documents and Support sssssssmnennmeeeeenne nene 15 Molecules That Count 3 PRODUCT MANUAL nCounter Data Analysis Guidelines for CNV PREFACE Conventions Used The following conventions are used throughout this manual and are described below for your reference Note Types Special font formatting is used in this manual Such formatting conventions are used in specific instances as described below TIP Information contained in a Tip may
25. usted depending on the gender of the reference sample If the reference sample is female the formulas should reflect two copies of the X chromosome and O copies of the Y chromosome as shown on Figure 1 10 FIGURE 1 10 21 22 Code Class Name Accession 23 Invariant _INVCONTROL 1 Hg18 chr1 97239055 97239154 _INVCONTROL 2 0 2 2 2 2 2 2 2 4 2i 24 Invariant _INVCONTROL 10 Hg18 chr10 76411622 76411718 _INVCONTROL 2 0 2 4 1 8 135 13 1 9 25 Invariant _INVCONTROL 16 Hg18 chr16 11026537 11026636 _INVCONTROL 2 0 1 7 1 8 1 7 1 6 1 6 26 Invariant _INVCONTROL 17 Hg18 chr17 60617472 60617571 _INVCONTROL 2 0 2 1 2 2 2 0 21 2 1 27 Invariant INVCONTROL 19 Hg18 chr19 38818850 38818949 _INVCONTROL 2 0 2 0 1 8 1 9 2 0 2 1 28 Invariant _INVCONTROL 2 Hg18 chr2 137221998 137222092 _INVCONTROL 2 0 2 0 15 2 0 1 8 2 0 29 Invariant INVCONTROL 20 Hg18 chr20 10504898 10504992 _INVCONTROL 2 0 2 1 1 9 1 8 1 9 2 0 30 Invariant INVCONTROL 22 Hg18 chr22 42509372 42509471 _INVCONTROL 2 0 2 0 2 2 P 2 0 2 1 31 Invariant INVCONTROL 5 Hg18 chr5 71261236 71261335 _INVCONTROL 2 0 1 8 2 1 2 1 2 1 2 0 32 Invariant _INVCONTROL 7 Hg18 chr7 41541153 41541252 _INVCONTROL 2 0 1 9 1 9 2 0 19 2 0 33 Endogenous probel Hg18 chr1 coordinates CNV region 1 0 6 3 2 2 2 2 6 3 0 3 9 34 Endogenous probe2 Hg18 chr8 coordinates CNV region 2 2 0 3 1 0 7 1 0 1 0 1 4 35 Endogenous probe3 Hg18 chr10 coordinates CNV region 3 2 0 3 0 14 15 2 1 2 7 36 E
26. variant INVCONTROL 19 Hg18 chr19 38818850 38818949 _INVCONTROL 2 0 2 0 1 8 1 9 28 Invariant _INVCONTROL 2 Hg18 chr2 137221998 137222092 _INVCONTROL 2 0 2 0 1 9 2 0 29 Invariant INVCONTROL 20 Hg18 chr20 10504898 10504992 _INVCONTROL 2 0 2 1 1 9 1 8 30 Invariant _INVCONTROL 22 Hg18 chr22 42509372 42509471 _INVCONTROL 2 0 2 0 2 2 2 1 31 Invariant INVCONTROL 5 Hg18 chr5 71261236 71261335 _INVCONTROL 2 0 1 8 J 1 2 1 32 Invariant _INVCONTROL 7 He18 chr7 41541153 41541252 _INVCONTROL 2 0 1 9 1 9 2 0 33 Endogenous probe1 Hg18 chr1 coordinates CNV region 1 ziNv horm example D33 INV norm example D33 3 34 Endogenous probe2 Hg18 chr coordinates CNV region 2 20 3 1 0 7 1 0 35 Endogenous probe3 Hg18 chri0 coordinates CNV region 3 2 0 3 0 14 1 9 36 Endogenous probe4 Hg18 chri2 coordinates CNV region 4 2 0 3 2 1 0 1 3 37 Endogenous probe5 Hg18 chrxX 111441962 111442061 CNV region 5 2 0 1 2 14 0 9 38 Endogenous probe6 Hg18 chr 2715200 2715287 CNV region 6 0 0 1 0 1 0 1 1 The adjusted formula can then be copied to the appropriate cells for each probe by clicking on the lower right corner of the highlighted cell and dragging over the cells you would like to change Reference Documents and Support For additional information on the nCounter CNV Assay and data analysis please refer to these documents available for download at www nanostring com e nCounter CNV Collector Tool User Manual nCounter Custom
27. zation factor for the lane as follows FIGURE 1 8 E A B C D E F G H TE 21 22 Code Class Name Accession Invariant INVCONTROL 1 Hg18 chr1 97239055 97239154 _INVCONTROL Raw data example D40 Raw data example D 38 Invariant _INVCONTROL 10 Hg18 chr10 76411622 76411718 _INVCONTROL 372 316 403 389 389 Invariant _INVCONTROL 16 Hg18 chr16 11026537 11026636 _INVCONTROL 422 497 481 502 514 Invariant _INVCONTROL 17 Hg18 chr17 60617472 60617571 _INVCONTROL 772 740 712 764 Invariant _INVCONTROL 19 Hg18 chr19 38818850 38818949 _INVCONTROL 639 648 695 655 Invariant _INVCONTROL 2 Hg18 chr2 137221998 137222092 _INVCONTROL 589 578 618 588 Invariant _INVCONTROL 20 Hg18 chr20 10504898 10504992 _INVCONTROL 475 447 497 529 Invariant _INVCONTROL 22 Hg18 chr22 42509372 42509471 _INVCONTROL 872 890 805 850 Invariant _INVCONTROL 5 Hg18 chr5 71261236 71261335 _INVCONTROL 889 964 862 840 Invariant _INVCONTROL 7 Hg18 chr7 41541153 41541252 _INVCONTROL 779 806 815 792 Endogenous probe1 Hg18 chr coordinates CNV region 1 204 330 225 269 Endogenous probe2 Hgi18 chr8 coordinates CNV region 2 456 711 155 225 Endogenous probe3 Hg18 chr10 coordinates CNV region 3 399 609 284 377 Endogenous probe4 Hg18 chr12 coordinates CNV region 4 480 779 232 317 37 Endogenous probe5 Hg18 chrX 111441962 111442061 CNV region 5 444 265 312 207 1 s Te Tes e eT S S 11424301 421 7 2074 c i 7175203 i 3 cel ecl O
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