User Guide: Small RNA pipeline for Ion Torrent™ in Partek® Flow®
Contents
1. GEN 64 breast BC2 GEN 64 breast BC1 placenta y 9 GEN 64 breast BC3 GEN 64 breast BC2 ppreasmmss GEN 64 breast BC3 Add samples Edit sample attributes Assign sample attributes from a file y changes Add a system wide attribute column Manage attributes Project output directory home flow FlowData miRNA project miRNA project_output 276 25 GB free Change Discard changes Figure 11 Sample management table showing a column for a newly defined attributes The Edit sample attributes button allows editing of the table using the dropdown options inset An example of a completed sample management table is shown below in Figure 12 Each sample must be assigned a category for every attribute fa Help Settings Log out Project actions DID 378 placenta BC3 DID 378 placenta BC6 DID 378 placenta BC9 GEN 63 breast BC1 GEN 63 breast BC2 GEN 63 breast BC3 GEN 64 breast BC1 GEN 64 breast BC2 GEN 64 breast BC3 Add samples Edit sample attributes Assign sample attributes from a file Add a system wide attribute column Manage attributes Project output directory home flow FlowData miRNA project miRNA project_output 276 23 GB free Change Figure 12 Sample management table with sample attributes assigned User Guide Small RNA pipeline for lon Torrent in Partek Flow 12 Importing the pipel2. placenta Add comparison tissue vs tissue 1 breast vs placenta Advanced options Default Configure No comparisons selected No p value or fold change data will be produced Advanced options Default Configure Beck Bei Select Bowtie 2 index for Bowtie 2 Genome build Homo sapiens human hg19 Index y Whole genome miRNASeq pipeline miRBase20 GENCODE Genes release 19 x New index ay e a Select Annotation file for Quantify Partek E M Genome build Homo sapiens human hg19 Annotation model GENCODE Genes release 19 Back Finish Figure 16 Setting the options for running the Small RNA pipeline for lon Torrent User Guide Small RNA pipeline for lon Torrent in Partek Flow 15 The screen will return to the Analyses tab and the pipeline will start running Figure 17 The progress of the pipeline can be seen by the changing colors lighter faded shades are queued while darker colors are completed e jsmith Help Settings Log out Poot cions E E Data Analyses Queue Collaborators Expand collapse pipelines Align reads Coverage report Unaligned reads Aligned reads Quantify to Gene specific transcriptome Quantification analysis t n reads Ca ba nt Unaligned oiae aade L 2 version 2 1 9 Status Running Quantify to Unaligned reads transcriptome Quantification Ma
3. 770 95 22 15 29 13 255 SD 0 42 8 DID 378 placenta BC9 1 201 616 53 23 53 23 5 96E 4 788 01 SD 6 066 13 22 15 28 89 255 SD 0 42 87 GEN 63 breastBC1 3 266 398 26 5 26 5 6 88E 4 8892 SD7 60503 21 31 2457 255 SD0 43 85 GEN 63 breastBC2 2 349 690 30 25 30 25 5 7E 4 871 01 SD 5 466 65 21 07 24 57 255 SD 0 45 47 GEN 63 breastBC3 5 305 239 26 28 28 28 9 17E 4 1 170 SD867925 2157 24 71 255 SD 0 42 92 GEN 64 breastBC1 1 336 619 51 18 51 18 7 1E 4 694 49 SD3 570 6 21 71 25 59 255 SD 0 44 18 GEN 64 breastBC2 2 552 637 47 28 47 28 7 49E 4 1 181 96 SD 14 347 1 22 12 25 88 255 SD 0 42 28 GEN 64 breastBC3 1 096 738 50 01 50 01 6 33E 4 634 86 SD 5 498 33 22 07 25 82 255 SD 0 43 99 a Alignment breakdown Coverage 100 6 000 000 0 0 0 0 0 0 378 plac 378 plac 378 plac 63 breas 63 breas 63 breas 64 breas 64 breas 64 breas enta BC3 enta BC6 enta BC9 Genome coverage Quality score lt GEN 63 breast BC1 lt GEN 64 breast BC1 Average quality score per position lt GEN 64 breast BC2 lt gt DID 378 placenta BC3 gt DID 378 placenta BC6 gt DID 378 placenta BC9 lt gt GEN 63 breast BC2 lt GEN 63 breast BC3 lt GEN 64 breast BC3 Percentage of reads Average quality score per read Average Phred quality score DID 378 placenta
4. Aligned reads Bowtie 2 version 2 1 0 placenta gg placenta m Junction reads PO 5961 3974 a hsaet To So 17912169 Reference genome A T T CATOCOC y ii i i LE Sai TU YAR A rT on Cm CC CW 47912144 17912156 17912169 17912181 17912194 Figure 26 Chromosome view focusing on a selected microRNA The Isoform proportion track uses the annotation miRNASeg pipeline mirbase20 which was padded 10 bases on each side The regions will appear wider than the regions where the reads actually aligned shown in the first two tracks List of additional documents For more information please see the Partek Flow User Manual Copyright 2014 by Partek Incorporated All Rights Reserved Reproduction of this material without express written consent from Partek Incorporated is strictly prohibited User Guide Small RNA pipeline for lon Torrent in Partek Flow 23
5. BC3 lt DID 378 placenta BC6 o lt GEN 63 breast BC2 lt GEN 64 breast BC2 GEN 63 breast BC1 GEN 64 breast BC1 DI1D 378 placenta BC9 GEN 63 breast BC3 GEN 64 breast BC3 a gt Figure 20 Post alignment QAQC report project level User Guide Small RNA pipeline for lon Torrent in Partek Flow 18 Home gt miRNA project gt Post alignment QA QC report gt DID 378 placenta BC3 Sample name _ DID 378 placenta BC3 Total reads 1 469 805 Average read length 22 17 Read length std dev 1 37 Average read quality 28 88 Quality score format Phred 33 0 Alignments per read Read breakdown Aligned 49 81 738 000 590 400 442 800 295 200 Reads 147 600 0 Alignments Base composition Average quality score per position H Ft A AA N NA NA NAN M MAM AMA Percentage of A C G T N H N D D eo 8 oo 62 Oo G amp 3 5 i 7 35 K Quality score d N Ly D e A N a 3 5 9 3 5 7 o S 3 19 3 5 5 Distribution of quality scores Mapping Quality per Alignment 800 000 640 000 480 000 320 000 Percentage of reads Number of alignments 160 000 255 Average Phred quality score Mapping quality Distribution of read lengths 300 000 240 000 180 000 120 000 Number of reads 60 000 p Tiis his dio hio dio Me dio hr he h f N a pap N M o A o oa rn QO nn A QO FT W ial val aa N N N N N N N
6. N N N m ag m m m i Length base pairs Figure 21 Post alignment QAQC report sample level Quantification report The Quantification report shows the estimation of abundance of reads that aligned to the annotation models In this pipeline there are two quantification reports for the two alignments 1 to mature RNA sequences and 2 to transcriptome annotation The report shows a mapping summary table as well as graphical summary column or pie charts visible when mousing over View column At bottom page of the task report are options for the user to Download gene level read counts or Download transcript level read counts as text files For the microRNA output the two text files would have the same values as each miRNA is considered a gene and transcript The transcript level read counts give additional information about the location of the annotation features used in the quantification of the microRNAS User Guide Small RNA pipeline for lon Torrent in Partek Flow 19 Figure 22 shows a sample quantification report for the first alignment of the pipeline alignment to mature microRNA sequences Although the pipeline shows two tasks named Quantification to transcriptome the first quantification only refers to quantification of microRNAs Fully within a feature DID 378 placenta BC3 732 159 Partly within a feature Not In a feature Incompatible paired end View Total junctions Compati
7. are not intended to be merged User Guide Small RNA pipeline for lon Torrent in Partek Flow 2 Files required for the pipeline Aside from the data to be analyzed and the system files for the Partek Flow software the Small RNA pipeline for lon Torrent requires three additional files 1 Small RNA pipeline for lon Torrent pipeline This is the pre built pipeline file downloadable from the Partek website at the following URL http www partek com pipelines Partek Flow now uses the extension pipeline for the pipeline files 2 An annnotation file containing mature microRNA sequences where the coordinates are padded 10 bases upstream and downstream of the mature microRNA For Homo sapiens hg19 Partek Flow distributes the annotation miRNASeq pipeline miRBase20 which is based on the mature microRNAs of miRBase 20 with the coordinates padded 10 bases upstream and downstream For more information visit http www mirbase org 3 An annotation file for the whole transcriptome For Homo sapiens hg19 Partek Flow hosts several whole transcriptome annotation files However the Small RNA pipeline for lon Torrent was validated using Gencode Genes release 19 It is based on the GENCODE 19 annotation with the addition of the tRNAs For more information visit http www gencodegenes org stats html For other species or genome builds users may upload their own annotation files Partek Flow accepts p
8. configuration Add a new annotation model miRBase mature microRNAs version 21 BaseSpace settings Bonis indas gt miRBase precursor microRNAs version 19 P en ane miRBase precursor microRNAs version 20 L Bowtie colorspace index yr miRBase precursor microRNAs version 21 Filter management Bowtie 2 index y RefSeq Transcripts 2012 08 03 Library file management TMAP index RefSeq Transcripts 2012 10 30 K a RefSeq Transcripts 2013 01 04 Option ost management B RefSeq Transcripts 2013 05 10 Task version management SHRIMP 2 indur y RefSeq Transcripts 2013 09 03 ee SHRIMP 2 colorspace index i RefSeq Transcripts 2014 01 03 m GSNAP index v RefSeq Transcripts 2014 04 29 User management PEN gt RefSeq Transcripts 2014 07 30 ndex Group management Licensing i i ye Can be created Cannot be created R Scheduled Created Directory permissions Whole genome miRBase mature pad Select annotation file pannot gtf gff3 bed Select files from Partek Flow server My computer Annotation file iy Select file Choose File mmu_mirbase20_padded aff3 Bowtle index mRNA Browse the server computer for this Annotation data type ty Amplicon e colorspace index Annotation file and import it into Partek Flow s bi 0 a ther Whole genome miRBase mature pad Annotation file Bowtie index Bowtie colorspace index Bowtie 2 index TMAP index Build this Bowtie 2 index using the Genome BWA index sequence f
9. User Guide Small RNA pipeline for lon Torrent in Partek Flow This is a guide to the analysis of microRNA Next Generation Sequencing data using the Small RNA pipeline for lon Torrent in the Partek Flow software It covers the following e Overview of the pipeline e Files required for the pipeline e Library file management e Creating a project in Partek Flow e Assigning attributes to samples e Importing the pipeline into Partek Flow e Running the pipeline e Viewing the results of the pipeline e List of additional documents Overview of the pipeline g jsmith Help Settings Log out a oo Rename Delete i Analyses Queue Collaborators Expand collapse pipelines Primary alignment to mature microRNA sequences Secondary alignment to transcriptome Make a pipeline Import a pipeline Figure 1 The Small RNA pipeline for lon Torrent The Small RNA pipeline for lon Torrent Figure 1 is a Bowtie2 based pipeline for the quantitation of mature microRNAs The pipeline was developed and tested on research data and annotation for Homo sapiens hg19 However it can be used for any species provided the reference genome and the the required annotation files are available User Guide Small RNA pipeline for lon Torrent in Partek Flow 1 Primary alignment to mature microRNAs miRBase The reads are aligned to known locations and sequences of mature microRNA sequences using Bowtie 2 The referenc
10. a let 7a 3p 588 0 38 0 47 1 41 lt a Gene symbol hsa let 7a 5p 121 423 1 43E 3 0 01 2 44 Total reads hsa let 7b 3p 3 334 2 01E 4 1 49E 3 6 67 P value hsa let 7b 5p 162 477 1 37E 5 1 88E 4 22 53 FDR step up hsa let 7c 3p 51 0 01 0 02 4 21 hsa let 7c 5p 30 420 6 19E 6 1 01E4 20 65 hsa let 7d 3p 1 479 1 83E 5 2 24E 4 7 04 hsa let 7d 5p 20 954 1 76E 5 2 19E 4 3 34 hsa let 7e 3p 144 0 86 0 9 1 07 hsa let 7e 5p 34 766 2 13E 4 1 54E 3 5 78 Saved filters Be Rows per page 10 10f85 gt Ratio Fold change Low Expressed OO Mini oi an amp WN Save filter Clear filter o 3 al el Bosse ee lee te te te tre tre te No saved filters available Figure 24 The Gene specific analysis Project report The Total Reads column show the total number of reads raw counts that aligned to the region User Guide Small RNA pipeline for lon Torrent in Partek Flow 21 The View column provides visualization for the microRNAs in the table Details of the GSA for each microRNA can be viewed by clicking the report icon El To generate a dot plot showing expression values of each microRNA click the dot plot icon 2 Each Sample is represented by a colored dot and the y axis shows the normalized read counts Figure 25 The samples can be grouped and colored by the different categorical attributes Box plots can be added to show the interquartile range with the line in the middle representin
11. a tab information about the samples can be added to the sample management table and can be either numeric i e continuous attributes or categorical attributes To use the Small RNA pipeline for lon Torrent there must be at least one categorical attribute and a category assigned to each sample This is a requirement to perform Statistical comparisons between groups and to detect differentially expressed microRNAs Categorical attributes must be assigned before running the Small RNA pipeline for lon Torrent To create a categorical attribute click Manage attributes from the Data tab Figure 9 The Manage attributes page will display Figure 10 Click Add new attribute and enter a name for the attribute and leave the Attribute type as Categorical In the next dialog box enter the categories of the attribute in the New category field User Guide Small RNA pipeline for lon Torrent in Partek Flow 10 Pew tiote gt 3 0 Home gt miRNA project gt Manage attributes g jsmith Help Settings Log out Project specific categorical attributes Add new attribute x Name Options No attributes found System wide categorical attributes Attribute type Categorical Numeric Name Options No attributes found a i Visibility Project specific System wide Numeric attributes _Add amp Name Options No attributes found Manage attribute Attribute tissue X Import ter
12. annot gff gtf and bed files See the Adding custom annotations section of this user guide User Guide Small RNA pipeline for lon Torrent in Partek Flow 3 Library file management The library files need to be copied to the Partek Flow server and indexed before running the pipeline for the first time Partek Flow s Library file management tool provides an easy way to create process and organize reference genome files aligner indexes and annotation files To access this tool click Settings in the upper right corner of Partek Flow and then click Library file management on the left panel Figure 2 Personal settings Genome build Homo sapiens human hg19 My profile Reference genome My preferences Genome sequence f Cytoband f Variant database gt System settings System information System queue l System resources Annotation models and aligner indexes Flow components Fitter management ne ee Library file management Option set management Bowtie index Access management Bowtie colorspace index Group management Bowtie 2 index TMAP index BWA index SHRIMP 2 index SHRIMP 2 colorspace index GSNAP index STAR index Can be created Cannotbe created X Scheduled Created Figure 2 Library file management tool To access this page click Settings top right arrow and then select Library file management left red arrow Selecting the genome reference Select the speci
13. ble junctions DID 378 placenta BC6 815 794 DID 378 placenta BC9 639 570 GEN 63 breast BC1 865 734 GEN 63 breast BC2 710 796 GEN 63 breastBC3 1 500 241 GEN 64 breastBC1 684 123 GEN 64 breastBC2 1 206 954 GEN 64 breastBC3 548 517 eceecaeacaeaces oro oo o o o o o o d_i ee a oo ool ol o o o o o Summary t 7 703 888 100 80 60 40 20 Percent 0 DID DID DID ta BC3 ta BC6 Ila Fully within a feature 378 placen 378 placen 378 placen ta BC9 Coverage breakdown GEN 63 breast BC1 Partly within a feature GEN 63 breast BC2 Sample GEN 63 breast BC3 Not in a feature GEN 64 breast BC1 GEN 64 breast BC2 GEN 64 breast BC3 Summary Incompatible paired end Normalized reads PCA mapping 46 41 18 3137 PC 2 17 65 37 7452 0 PC 1 28 76 enlarge chart view in 3D Color by Size by Fixed size x Per Y Pe2 tissue Color w Type breast placenta Figure 22 Quantification report from the alignment to mature microRNAs The links to download the read counts are shown in the red box A principal components analysis PCA plot is calculated based on normalized reads showing the first two principal components A three dimensional PCA plot can be invoked by clicking view in 3D Figure 23 Normalizatio
14. e for the Homo sapiens microRNAs was obtained from miRBase www miRBase org For improved alignment using Bowtie2 the chromosomal coordinates from miRBase version 20 was padded 10 bases upstream and 10 bases downstream to create a new annotation file Alignment coverage report post alignment QAQC for this pipeline is based on this annotation The aligned reads are quantified to give total counts of each mature microRNA hsa mir which can be downloaded as text files in the quantitation report Differentially expressed microRNAs are determined by comparing between Sample groups Secondary alignment to GENCODE transcripts Reads which do not map to the mature microRNA reference are then aligned to the GENCODE v19 whole transcriptome reference www gencodegenes org stats html in an attempt to rescue more alignments to better characterize a small RNA library This can have value in terms of evaluating QC QA of your sample library Moreover since the library has been enriched for smaller transcripts the quantitation step after alignment can reveal presence of other RNA species such as snoRNAs snRNA and processed pseudogenes among others As with the microRNA quantitation step the gene level or transcript level read counts may be downloaded using the links at the bottom of the quantitation report The purpose of the secondary alignment is to see where the rest of the reads align to the transcriptome The results of the two alignments
15. es and build for your project under the Genome build section Figure 2 red box Confirm that the icon next to the Genome sequence is a green check mark If it is showing a blue flag the genome reference needs to be downloaded Click the blue flag and then select the option to download You may also take this opportunity to download the Cytoband file as well This is required in order to use the chromosome viewer in Partek Flow However it may be also be downloaded after running the pipeline User Guide Small RNA pipeline for lon Torrent in Partek Flow 4 Adding a Partek distributed microRNA annotation To add the annotation model for mature microRNAs Figure 3 1 Click the tab in the Annotation models and aligner indexes section 2 Inthe dialog box select the Annotation model miRNASeq pipeline miRBase20 and then click Add A new tab will be created hover the mouse cursor over the tab to reveal the complete name 3 Download the annotation by clicking the blue flag next to Annotation file Wait for the icon to become a green check 4 Build the annotation index by clicking the blue flag next to the Bowtie2 index Wait for the hourglass 2 to become a green check Fora 6 core 8 GB RAM machine the last step takes about 30 minutes Genome build Homo sapiens human hg19 Reference genome Genome sequence f Cytoband f Variant database Add annota
16. ex for mature microRNAs User Guide Small RNA pipeline for lon Torrent in Partek Flow 5 Adding a Partek distributed transcriptome annotation To add the annotation model for the transcriptome Figure 4 1 Click the tab in the Annotation models and aligner indexes section 2 Inthe dialog box select the Annotation model Gencode Genes release 19 and then click Add A new tab will be created hover the mouse cursor over the tab to reveal the complete name 3 Download the annotation by clicking the blue flag next to Annotation file Wait for the icon to become a green check 4 Build the annotation index by clicking the blue flag next to the Bowtie2 index Wait for the hourglass 2 to become a green check Fora 6 core 8 GB RAM machine the last step takes about 2 5 hours Genome bulld Homo sapiens human hgl9 4 Reference genome Genome sequence Cytoband f Variant database Annotation models and aligner indexes Add annotation model Species Homo sapiens human Genome build hgi9 Annotation mode COSMIC 2013 01 18 COSMIC v69 2014 07 16 Add AceView Transcripts Ensembl Transcripts release 69 Ensembl Transcripts release 70 Ensembl Transcripts release 71 Ensembl Transcripts release 72 Ensembl Transcripts release 73 Ensembl Transcripts release 74 Ensembl Transcripts release 75 GENCODE Genes release 19 miRBase microRNAs version 17 miRBase microRNAs
17. f the screen Figure 15 _ Flow Ss e jemith Help Settings Log out W 30 Home gt miRNA project Data Analyses Collaborators x Unaligned reads gt QA QC gt Pre analysis tools gt Aligners v Pipelines Small RNA pipeline for lon Torrent Small RNA pipeline Bowtie2 based pipeline for quantitation of mature miRNAs Download data 2 GB Click a data node to reveal the toolbox Import a pipeline l l Figure 15 Analyses tab showing a selected data node the toolbox in red box and the Small RNA pipeline for lon Torrent listed in the Pipelines task section Click of the Pipelines section of the toolbox to expand the list and see the available pipelines The imported Small RNA pipeline for lon Torrent will appear on the list Moving your mouse cursor over the pipeline will display a description of the pipeline Pipelines can be previewed or downloaded by clicking the appropriate icon To run the pipeline click the Small RNA pipeline for lon Torrent from the toolbox You will be guided through six steps to set the options for pipeline Figure 16 For the first aligner select miRNASeq pipeline miRBase 20 Bowtie 2 index For the coverage report select miRNASeg pipeline miRBase 20 annotation For the first quantification select miRNASeg pipeline miRBase 20 annotation For the fourth step set the options for the differential expression task e Choose Attributes
18. from files A file browser will locate files compatible with Partek Flow within the current folder The files will be displayed and User Guide Small RNA pipeline for lon Torrent in Partek Flow 8 selected with a checkmark on the right panel Figure 8 When you have selected the correct folder and files click Create samples g jsmith Help Settings Log out Home gt miRNA project gt Create samples from files Select files from Partek Flow server My computer Select files Current directory home flow FlowData miRNA project v f Server Computer v home v flow v FlowData W Brain vs UHR sub gt fs library_files gt fms RP gt fa partek_flow Name Size DID 378 placenta BC3 fastq 98 85 MB DID 378 placenta BC6 fastq 108 6 MB DID 378 placenta BC9 fastq 79 69 MB GEN 63 breast BC 1 fastq 216 49 MB GEN 63 breast BC2 fastq 154 67 MB GEN 63 breast BC3 fastg 353 36 MB GEN 64 breast BC 1 fastq 88 05 MB GEN 64 breast BC2 fastq 170 68 MB wv Cy Cy Cy Cy Cy Cy wv Cy Cy GEN 64 breast BC3 fastq 72 36 MB Don t see your folder Refresh folder list Create samples Figure 8 File browser showing selected files on the right to be added into a Project Files may also be uploaded from the local computer by selecting My computer and locating the files to be uploaded However the success of the transfer depends on the speed and latency of the internet connection between the local computer and the Pa
19. g the median while the whiskers symbolize the 10 to 90 range Dataset Normalized reads Total Count Group by tissue Colorby tissue 4 Show shading Show box plots Save image hsa miR 424 3p 137 06 LJ _ Legend _ Q breast 4 placenta 107 43 gt a fo O T _ 2 D QO 8 2 ao D N 7 s A om 48 17 18 54 3 placenta breast Figure 25 Dot plot showing the expression levels of a microRNA Chromosome Viewer The chromosome viewer can be invoked by selecting the chromosome view 4 In an example in Figure 26 top two reads tracks Aligned reads in blue and red shows a pileup of the total reads from all samples in each category breast and placenta The bottom track Isoform proportion shows the relative proportion of each category In this case the selected miRNA is expressed much higher in breast than in placenta If the experiment is unbalanced i e different number of samples in each group the proportion seen in the top two tracks may not look consistent with the bottom isoform proportion track User Guide Small RNA pipeline for lon Torrent in Partek Flow 22 Pere 3 0 Home gt miRNA project gt Chromosome view hg19 Selecttracks Search Position 21 17912144 17912194 Reset view QO Aligned reads Bowtie 2 version 2 1 0 breast jg breast 5961 3974 1987 E i 0
20. iRNASeg pipeline miRBase20 Sample name 1x 10x 20x Average coverage Average quality GEN 64 breast BC3 7 24 107 06 25 83 GEN 64 breast BC1 8 35 671 131 89 25 6 GEN 63 breast BC2 8 28 653 435 12 24 58 DID 378 placenta BC9 8 42 433 07 29 13 GEN 63 breast BC1 888 69 4 09 164 44 24 57 DID 378 placenta BC3 8 86 4 53 152 44 29 12 DID 378 placenta BC6 o 470 22 29 36 GEN 64 breast BC2 235 33 25 88 GEN 63 breast BC3 289 78 24 72 Task details e jsmith Help Settings Log out Region name oChromosome Start gt Stop Strand Length Reads e GC hsa miR 200b 5p 1 1102494 1102536 42 hsa miR 200b 3p 1 1102530 1102572 hsa miR 200a 3p 1 1103286 1103328 hsa miR 551a 1 3477264 3477305 E 1 9211742 9211784 hsa miR 34a 5p 1 9211784 9211826 1 1 1 1 100x SAverage coverage Average quality o 0 62 32 45 o 41 38 28 87 lo losz 33 88 0 1 54 0 9 83 54 76 106 31 o 12 o 0 44 0 0 hsa miR 34a 3p hsa miR 1273d 10287775 10287820 hsamiR 1290 19223562 19223601 hsa miR 1256 21314860 21314902 hsa miR 1976 26881055 26881095 o o o co o co o o o o fe y Se Se ae ee Se ee ee ee s s 3 2 Rows per page HOF 10f77 elm Figure 19 Coverage report table at the sample level User Guide Small RNA pi
21. ine into Partek Flow The pipeline can be downloaded from Partek website at the following URL http www partek com pipelines 1 Download the Small RNA pipeline for lon Torrent pipeline Save the file to a folder that is easily accessible on your local computer typically the Downloads folder Figure 13 2 Open a project and select the Analyses tab 3 Click Import a pipeline on the lower left side of the screen Figure 14 In the dialog box click Choose File button 4 Navigate to the local directory where the pipeline was downloaded Select the Small RNA pipeline for lon Torrent pipeline file and click Import Downloads m aoe a FAVORITES Name a DEVICES Small RNA pipeline for lon Torrent pipeline 1 KB SHARED TAGS Figure 13 Downloads folder showing the Small RNA pipeline for lon Torrent pipeline file actual appearance will depend on your operating system e jsmith Help Settings Log out Popeia EEE osc Collaborators Analyses Pipeline file Choose File No file chosen Task section Pipelines m Rename pipeline Import Cancel Import a aar Figure 14 Analyses tab showing a data node and the Import a pipeline link red box User Guide Small RNA pipeline for lon Torrent in Partek Flow 13 Running the pipeline In the Analyses tab select a data node containing Unaligned reads The toolbox a data specific menu will appear on the right side o
22. ke a pipeline Import a pipeline Figure 17 Small RNA pipeline for lon Torrent in progress Data nodes are circles and task nodes are shown as rectangles The pipeline is at the stage where the task node has a lighter shade of blue indicated by a red arrow The progress of the pipeline can also be monitored using the Queue tab where individual tasks and their status are listed When the pipeline is finished all the colors will have the same darker shade User Guide Small RNA pipeline for lon Torrent in Partek Flow 16 Viewing the results of the pipeline To view the results of the pipeline double click a node to show the Task report Coverage report task report The pipeline generates a coverage report to show the depth of sequencing of the known mature microRNA regions both at the project level Figure 18 and at the individual sample level Figure 19 In the project level the percentages of bases in the microRNA with coverage of 1x 10x 20x or 100x are presented as a table Average coverage is defined as the sum of read depths of each nucleotide base in the genomic feature regions divided by the length of genomic feature regions Average quality is defined as the sum of average quality of those bases that covered the genomic feature regions divided by the length of covered genomic feature regions Pete Flow gt g jsmith Help Settings Log out 3 0 Home gt miRNA project gt Coverage report Annotation model m
23. ms from SNOMED CT Add new atributa am now category b Ly Name Options Back to sample management table breast f Figure 10 Manage attributes page and the dialog boxes for adding an attribute middle inset and defining categories bottom right inset In the example shown in Figure 10 the categorical attribute tissue has two categories breast and placenta Repeat the process for additional attributes of the samples in your study When done click Back to sample management table The sample management table will now show a column of the new attribute Figure 11 The column will initially appear blank as the samples have not yet been categorized To edit the table click Edit sample attributes Each sample can be assigned a category using the drop down menu Figure 11 inset When done click Apply changes and the sample management table will be updated After editing the sample table make sure there are no fields with blank or N A values before proceeding User Guide Small RNA pipeline for lon Torrent in Partek Flow 11 Data Analyses Queve Collaborators Sample name Data Analyses Queue Collaborators on na 2 DID 378 placenta BC6 aa 3 DID 378 placenta BCO DID 378 placenta BC6 placenta 4 GENS3 breastBCt sra pecnta Bos a 8 GEN3 breast BC2 a eg 6 GEN 63 breast BC3 SEEE 7 GEN 64 breast BC1 GEN 63 breast BC3 i N A
24. n is done using RPKM reads per kilobase of exon per million of mapped reads User Guide Small RNA pipeline for lon Torrent in Partek Flow 20 Color by tissue Size by Fixedsize ConnectBy None Show labels X PC1 Y PC2 mZ PC3 Save amp Reset tissue Color Type breast placenta xb ech os 74 l lt 2 PC3 14 46 2 7e 30 99 Figure 23 Principal components analysis 3D plot Gene Specific Analysis Project report The Gene Specific Analysis GSA algorithm is a statistical analysis used to compare differential expression of microRNAs among groups of samples The GSA report consists of a Statistical results section and an interactive Gene list table containing differentially expressed mature microRNAs Figure 24 The pie chart depicts the proportion of microRNAs with respect to the best model fit The different model types used in GSA are Lognormal linear model Poisson and Negative binomial The table shows the GSA output as a list of microRNAs and provides the total number of reads as well as the statistical results The table can be filtered using the fields on the left or sorted using the columns Statistical results Best model type Lognormal linear model 56 33 Negative binomial 21 18 S Lognormal linear model S Negative binomi Gene list Results 845 breast vs placenta lt 4 Filter Row Gene symbol Total reads P value gt FDRstepup Ratio hs
25. or mm 10 and the Annotation SHRIMP 2 index Pi model for miRBase mature padded mouse I Figure 5 Adding a custom annotation User Guide Small RNA pipeline for lon Torrent in Partek Flow 7 Creating a project in Partek Flow Analyses in Partek Flow are done within projects To create a project click the New Project icon from the Home page and enter a project name Figure 6 g jsmith Help Settings Log out New project Recent projects v Recent activity Project Activity Project No projects available No recent activity View all projects View all activity Status New project x Name miRNA project Create project _ D You have no tasks in the queue Figure 6 Partek Flow Home page and the dialog box for naming a project inset Once a project is created Partek Flow will display the Data tab To add samples to the project click Add samples Three options will be displayed Figure 7 g jsmith Help Settings Log out Papei ioe mcrae Once Data Analyses Queue Collaborators Add samples You ll need some samples before you can run an analysis Automatically create samples from files To get started click the Add samples button Add samples Import samples from another project Manage attributes Create a new blank sample Figure 7 Partek Flow Data tab and the options for adding samples inset Select Automatically create samples
26. pdown menu Enter a name click Add A new tab will be created hover the mouse cursor over the tab to reveal the complete name 4 Click the blue flag next to Annotation file and browse to the file Partek Flow accepts pannot gff gtf and bed files Set the Annotation file type as microRNA Click Finish Wait for the icon to become a check 5 Build the annotation index by clicking the blue flag next to the Bowtie2 index Wait for the hourglass 2 to become a green check This process is done by an Admin user and may take hours depending on the system PETTEE Home gt Settings gt Library file management Personal settings Genome build Mus musculus mouse mm10 Add annotation model i Mye Reference genome Species Mus musculus mouse Genome build mm10 Annotation mode y Ensembl Transcripts release 69 Ensembl Transcripts release 70 Ensembl Transcripts release 71 Ensembl Transcripts release 72 My preferences System settings System information Genome sequence ff Cytoband ff System queue Ensembl Transcripts release 73 System preferences ee ie Annotation models and aligner indexes Ensembl Transcripts release 74 Server configuration Ensembl Transcripts release 75 System resources Whole genome miRBase mature microRNAs version 19 f ay miRBase mature microRNAs version 20 LDAP
27. peline for lon Torrent in Partek Flow 17 Post alignment QA QC task report The pipeline generates a post alignment QA QC report for each of the alignments The output summarizes the efficiency of the alignment and the quality of the aligned reads at the project level Figure 20 Clicking a sample name on the table will show additional details of the individual sample Figure 21 Home gt miRNA project gt Post alignment QA QC report 80 60 40 20 Alignment percent 0 DID DID th Unique DID t BC2 t BC3 Sample Non unique gl Unaligned t BC1 GEN t BC2 lt Total reads GEN t BC3 4 800 000 3 600 000 2 400 000 1 200 000 GEN 378 plac 378 plac 378 plac 63 breas 63 breas 63 breas 64 breas 64 breas 64 breas enta BC3 enta BC6 enta BC9 t BC1 Total reads DID Average read depth in covered regions DID DID GEN GEN t BC1 t BC2 Sample GEN GEN GEN GEN t BC3 t BC1 t BC2 th Average read depth gt Genome coverage t BC3 ig Sample name sTotal reads gAligned Unique Coverage Avg coverage depth Avg length Avg quality SAvg mapping quality GC DID 378 placenta BC3 1 469 805 49 81 49 81 6 03E 4 893 32 SD 6 977 89 22 17 28 88 255 SD 0 42 94 DID 378 placenta BC6 1 619 088 50 39 50 39 6 49E 4 923 72 SD 7
28. rtek Flow server Two other methods of adding samples to a project are shown in Figure 7 inset Samples from previously created projects within the same Partek Flow server can be associated with a new project by selecting Import samples from another project Samples can also be added manually by selecting Create a new blank sample After adding samples The Data tab would now contain a sample management table Figure 9 User Guide Small RNA pipeline for lon Torrent in Partek Flow 9 g jsmith Help Settings Log out Project actions Analyses Collaborators Sample name DID 378 placenta BC3 DID 378 placenta BC6 DID 378 placenta BC9 GEN 63 breast BC1 GEN 63 breast BC2 GEN 63 breast BC3 GEN 64 breast BC1 GEN 64 breast BC2 GEN 64 breast BC3 4 2 3 4 5 6 rf 8 9 Add samples Assign sample attributes from a file Add a system wide attribute column Manage attributes Project output directory home flow FlowData miRNA project miRNA project_output 278 22 GB free Change Figure 9 The sample management table in the Data tab Sample names are automatically generated based on the filenames and can be edited by selecting the gear i icon next to it The expand or collapse A triangles will display or hide the file names associated with each sample respectively Once a project is created it can be shared with other Partek Flow users using the Collaborators tab Assigning attributes to samples In the Dat
29. tion model Species Homo sapiens human Genome build hg19 Annotation mode V COSMIC 2013 01 18 COSMIC v69 2014 07 16 AceView Transcripts Ensembl Transcripts release 69 Ensembl Transcripts release 70 Ensembl Transcripts release 71 Ensembl Transcripts release 72 Ensembl Transcripts release 73 Ensembl Transcripts release 74 Annotatio Ensembl Transcripts release 75 GENCODE Genes release 19 miRBase microRNAs version 17 miRBase microRNAs version 18 miRNASeg pipeline miRBase20 N miRBase mature microRNAs version 19 Annotation models and aligner indexes Add Whole genome e Add a new annotation model Bowtie index Bowtie colorspace index Bowtie 2 index TMAP index BWA index SHRIMP 2 index SHRIMP 2 colorspace index GSNAP index STAR index AVAVAVV44F canbe created Cannotbecreated Scheduled Created Whole genome miRNASegq pipeline Annotation file iss Bowtie index gt B ia a ae s Download this Annotation file from the Internet owtie colorspace index Bowtie 2 index gt gt Whole genome miRNASeq pipeline Annotation file Bowtie index Bowtie colorspace index Bowtie 2 index TMAP index Build this Bowtie 2 index using the Genome BWA index ig sequence for hg19 and the Annotation model SHRIMP 2 index Pifor hg19_mirbase_20_v2 Figure 3 Adding the annotation and building the Bowtie2 ind
30. to include in your test and click Next If there is only one categorical attribute it will be automatically selected e Select the groups by categories to compare then click Add comparison e Set up the comparisons that you need then click Next Make sure to click the Add comparison button oS 5 For the second aligner select GENCODE Genes release 19 Bowtie 2 index 6 For the second quantification select Select GENCODE Genes release 19 annotation n general first three options have the same answer miRNASegq pipeline mirBase 20 And the last two options have the same answer GENCODE Genes release 19 User Guide Small RNA pipeline for lon Torrent in Partek Flow 14 Po row DD E io i seunas Logout 3 0 Home gt miRNA project gt miRNASeq Select Bowtie 2 index for Bowtie 2 Genome build Homo sapiens human hg19 index Whole genome GENCODE Genes release 19 g New index Select Annotation file for Coverage report Genome build Homo sapiens human hg19 Annotation model miRNASeq pipeline miRBase20 Seok aa Select Annotation file for Quantify Partek E M Genome build Homo sapiens human hg19 Annotation model miRNASeq pipeline miRBase20 Comparison selector tissue breast C placenta tissue Mark categories to breast placenta compare then click Add comparison
31. version 18 miRBase mature microRNAs version 19 Whole genome miIRNASeq pipeline 8 Add a new annotation model Annotation file Bowtie index Bowtie colorspace index Bowtie 2 index TMAP index BWA index SHRIMP 2 index SHRIMP 2 colorspace index GSNAP index STAR index AAAAAAN AAS gt Can be created Cannot be created R Scheduled Created Whole genome GENCODE Genes re miRNASeg pipeline Annotation file Bowtie index Download this Annotation file from the Internet Bowtie colorspace index x T Bowtie 2 index Whole genome GENCODE Genes re miRNASegq pipeline Annotation file Bowtie index Bowtie colorspace index Bowtie 2 index ARENON Build this Bowtie 2 index using the Genome BWA index sequence for hg19 and the Annotation model SHRIMP 2 index fOr hg19_gencode_19_v2 Figure 4 Adding the annotation model for the transcriptome User Guide Small RNA pipeline for lon Torrent in Partek Flow 6 Adding custom annotations For custom annotations the library files need to be managed by a user with Administrator privileges Adding custom annotation for mature microRNAs is shown in Figure 5 The steps can be adapted for adding a custom transcriptome annotation 1 Select species and Genome build The Genome sequence must be checked 2 Click the tab in the Annotation models and aligner indexes section 3 Select Other from the dro
Download Pdf Manuals
Related Search