Integrative Genomics Viewer介紹 國家基因體醫學中心 葉爾瞻 準備IGV guide網頁 教學檔網頁

課程目標 能自行安裝、設定與啟動IGV軟體 能使用IGV瀏覽variants(.vcf) 能使用IGV瀏覽alignments(.sam/.bam) 能使用IGV開啟外部資料庫做為參考 IGV工具似乎是比較偏研究人員使用的工具。我們資訊人員的角度用的比較沒那麼深入。今天的課程只能著眼在功能性的介紹。

Overview http://www.broadinstitute.org/software/igv/home high-performance visualization tool for interactive exploration of large, integrated genomic datasets. supports a wide variety of data types, including array- based and next-generation sequence data, and genomic annotations.

Citing IGV Helga Thorvaldsdottir, James T. Robinson, Jill P. Mesirov. Integrative Genomics Viewer (IGV): high-performance genomics data visualization and exploration. Briefings in Bioinformatics 2012. James T. Robinson, Helga Thorvaldsdóttir, Wendy Winckler, Mitchell Guttman, Eric S. Lander, Gad Getz, Jill P. Mesirov. Integrative Genomics Viewer. Nature Biotechnology 29, 24–26 (2011)

準備Java環境 IGV是Java軟體，可以透過Java Web Start啟動。系 統需要有Java 1.6以上的環境。 系統需要javaws.exe才能使用Java Web Start 開始->在“搜尋程式及檔案”方塊中輸入javaws.exe 叫出命令提示字元，輸入命令java –version確認 版本在1.6以上 如果需要安裝java環境，請從http://www.java.com 下載並安裝。 如果是64-bit作業系統，建議安裝64-bit版本的Java。 64-bit的Java不能安裝在32-bit的作業系統中。

下載、安裝與啟動 IGV下載網址：(需先註冊) http://www.broadinstitute.org/software/igv/download Web Start：依照本機的記憶體大小下載對應的檔 案 2Gb或更少：使用750Mb版本 (http://www.broadinstitute.org/igv/projects/current/igv.jnlp) 2Gb以上，32-bit 作業系統：使用1.2Gb的版本 (http://www.broadinstitute.org/igv/projects/current/igv_mm.jnlp) 更大的記憶體需要在64-bit的Java環境才能使用。 或者下載Binary Distribution，直接解壓縮即可使用。 今天的課程因為人數較多，我們用助教提供的檔案安裝。

下載、安裝與啟動 Binary Distribution設定 使用文字編輯器打開igv.bat(Mac user請打開igv.sh) 將-Xmx750m 的750改為適合您的大小，存檔。 執行igv.bat(Mac 使用者請執行igv.sh) 搭配早上確認過的記憶體大小，在這裡可以決定要開多大的記憶體。也可以先不要動，保留預設的設定。如果發現記憶體不夠再來考慮加大。

IGV文件 官網首頁有完整的文件 使用手冊： http://www.broadinstitute.org/software/igv/UserGuide 新版(2.0)功能說明 http://www.broadinstitute.org/software/igv/Version2.0G uide Forum：https://groups.google.com/forum/#!forum/igv- help 所支援的各種檔案各有說明文件 本投影片的功能說明，大多節錄自使用手冊

Reference Genome IGV有預設的許多物種的Reference Genomes可以選擇： http://www.broadinstitute.org/softwar e/igv/Genomes 我們這裡使用1000 genomes project 使用的genome(1kg, b37+decoy)。 如果自己要的reference genome沒有 在清單中，也可以透過import genome的功能建立

Viewing the Reference Genome Note that the sequence and the arrow are only displayed when zoomed in to a sufficiently small region. You can change the strand that is displayed by clicking on the arrow in the title to the left of the track. you can optionally display a 3-band track that shows a 3-frame translation of the amino acid sequence for the corresponding nucleotide sequence. 以下的投影片大多摘錄自User Guide以避免投影片和官方文件的風格與用語不協調。投影片的大部分內容只要copy paste到google就可以找出原文。

Viewing the Reference Genome Feature Track(Genes and Transcripts) Collapsed Squished Expanded Query by gene name

Loading Data and Attributes Load from File see IGV File Formats for supported file formats Load from URL Load from Server Load from a Distributed Annotation System (DAS) 很多大型計劃的資料都可以直接下載在IGV中瀏覽。如1000 genomes的vcf檔。最好連index一起下載。

Removing Tracks and Attributes To remove all tracks and attributes: Click File>New Session. This is essentially the same as restarting IGV.  To remove specific tracks: Right-click a track name and select Remove Tracks in the pop-up menu. Control-click track names for multiple selections

Viewing Variants (vcf) Open sample vcf file (from 1000 genomes project) ALL.chrMT.phase1.20101123.vcf build index with igv tools select File>Run igvtools select index as Command specify the location of the vcf file Run Set locus to MT Zoom in/ Zoom out Go back/Go forward Set Feature Visibility Windows

Viewing Variants (vcf) shows the allele fraction for a single locus. the genotypes for each locus in each sample. Dark blue = heterozygous Cyan = homozygous Grey = reference. To change the color coding of the plot, select Color By>Allele

About VCF format less {sample vcf file}. VCF Format 4.1 Spec Or try to open vcf file with excel VCF Format 4.1 Spec http://www.1000genomes.org/wiki/Analysis/Variant%20 Call%20Format/vcf-variant-call-format-version-41 VCF Poster http://vcftools.sourceforge.net/VCF-poster.pdf Fixed Fields Genotype Fields (Optional) Meta-information lines

Viewing Alignments Remove 1000 genomes track(or Click File>New Session) Load sample files from URL(Ctrl-click for multi-select) 1_149003461-149460645.bam 1_149003461-149460645.vcf Set locus to 1:149022000-149023000 View>Preferences for alignments view settings downsampling Can be Collapsed, Squished or Expanded

Viewing Alignments Control+click a read to find its mate Split Screen View Right-click over an alignment and select View mate region in split screen from the drop-down list Return to Normal View To return to the “normal view”, double-click the name panel at the top of one of the panes, or right-click in a name panel and select Switch to standard view.

Interpreting Color by Insert Size Red for an inferred insert size that is larger than expected (deletion) Blue for an inferred insert size that is smaller than expected (insertion) Other colors for for paired end reads that are coded by the chromosome on which their mates can be found(Inter-chromosomal Rearrangement) 開啟官方文件

Interpreting Color by Pair Orientation The orientation of paired reads can be used to detect structural events including: inversions, duplications and translocations

(*)Splice Junctions The splice junction view displays an alternative representation of .bed files encoding splice junctions, such as the "junctions.bed" file produced by the TopHat program.

About SAM(BAM) format SAM/BAM spec A human-readable summary http://samtools.sourceforge.net/SAM1.pdf A human-readable summary http://genome.sph.umich.edu/wiki/SAM Explan SAM flags http://picard.sourceforge.net/explain-flags.html

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