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Basic Analysis

Use the Experiments window to perform basic data analysis and view the results (Data & Analysis>Experiments).

Experiment Window

The Experiments table shows all the records created by or shared with the user’s laboratory. The datasets can be filtered by many variables, such as folder, owner and experiment type. The table shows the unique dataset identification number (“#”) and owner of the record (“Author”), description parameters (“Genome”, “Type”, “Cells/Condition”, “Alias”) and some mapping statistics in the form of a pie chart (“Mapping statistics”). Pictograms show the status of the record: downloading stage ( , , ) and processing stage ( , , ). Please note that the status is updated every 10 minutes and that the system will not start downloading the record immediately. Depending on the number of new records entered at the same time, the records will have to wait in the queue for download or analysis.

Records can be viewed, duplicated or deleted using the three pictograms in the last column ( , , ). Unneeded records can be deleted by clicking the delete icon .

Adding Experiment Record

Step 1 - Open Experiments

Go to Data & Analysis>Experiments.

Step 2 - Open Experiment Window

Press the “New” button in the toolbar of "Laboratory data" window.

Tip: If the record to be entered is similar to one that already exists, it can be created by clicking the “Duplicate record” icon located in the last column of the table.

Step 3 - Fill in the Experiment form

Experiment form has four tabs: “General info”, “Protocol”, “Notes” and “Advanced”.
Experiment Form

General info tab

  • "Experiment Description" fieldset
    The following parameters in "General info" tab are needed to describe your sample: Cells, Conditions, Genome Type, Experiment Type, Fragmentation and Experiment Date. Donor field is not in use currently, but will be used for paired statistical analysis in the future.
    • Parameters for RNA-Seq or ChIP-Seq
      1. Genome Type

        Select the genome to which the data will be mapped: Use the “+ spike” option only if you are using ERCC controls for RNA-Seq.
      2. Experiment Type
        • Select DNA-Seq for ChIP-Seq, DNase-Seq, MNase-Seq and all other experiments in which the data need to be mapped to the genome; select RNA-Seq for experiments in which mapping to a transcriptome is preferred.
        • Select paired-end (“pair”) or single read. If paired-end read is selected, BioWardrobe will expect to match two FASTQ files from core or one SRA from external source. If the stranded RNA-Seq protocol was used, please select “dUTP”, as this will affect RPKM calculations.
      3. Fragmentation
        Select which fragmentation type was used for library preparation.
    • ChIP-Seq additional parameters
      When you choose "DNA-Seq" or "DNA-Seq pair" as an "Experiment type" additional parameters will be shown.
      • Crosslink
      • Antibody
        Select your ChIP antibody or other protocol (e.g. DNaseI). This selection will affect the pipeline parameters and island calling (e.g. narrow peaks vs. broad peaks in MACS). The pipelines and parameters for each antibody can be set up in the antibody catalog by laboratory-level administrators. You can also add antibody catalog # for your records.
      • Background/Controls/Input

        If you want MACS2 to use an input library as a control during peak calling, you can specify the control record using the drop down menu. This menu contains all samples that has been designated as controls. You can designate a sample to be used as a control using a checkmark on the right. When adding control to a record that has already been processed, remember to click the “Force to repeat analysis” checkmark on the advanced tab and click Save.
  • "Experiment Arrangement" fieldset:
    1. Experiment’s short name:
      Select a short name for the experiment. This name will display on the genome browser and will be used to designate the sample during analysis. Please select a name that is UNIQUE and SHORT but DESCRIPTIVE. DO NOT use “_” or “-“ in lieu of spaces. The name can be edited after processing is complete.
    2. Folders/Genome Browser folders:
      Folders are used to organize data on the browser and to share data between laboratories. Folders can be set up and shared by laboratory-level administrators (see Wardrobe Settings section). If the folder is not selected, the data will be downloaded but not analyzed. You can only select folders that belong to or have been shared with your laboratory. The data can be moved to another folder at any time, but relocating data will affect data visibility for other laboratories.
    3. Checking the “Share data online?” box will deposit data on the external mirror of the genome browser if it has been set up. (Normally data are deposited on the browser available only on the internal network.)
  • "Data Source" fieldset:
    Choose “Download type”. Data can currently be obtained from a “Core facility” (for instance CCHMC sequencing core), the internet (a direct URL to a FASTQ or SRA file) (“Direct link”), or a local file uploaded to the server's shared drive (“Server upload directory”). Non-CCHMC users can only download data via an URL (Direct link).
    1. Core facility:
      Enter a filename or a code that will allow Wardrobe to find and download the file. Please note that the code needs to be unique (e.g. code such as AB1 will not work if there is also another sample named AB11).
    2. Direct link:
      Please enter a URL for the file. The URL must end with the file name and extension. Wardrobe can process .fastq and .sra files that are uncompressed or compressed with .gz, .bz2, or .zip. Currently, .tgz files cannot be processed. If there are several files (e.g. sequencing data from several lanes), the URLs can be separated by semicolons (;). For paired-end data, the URLs for the first and second FASTQ should be separated by semicolons. *Currently, paired-end data coming from several lanes require manual processing. How to find a link to .sra file in GEO
      Dropbox Instructions
  1. Server upload directory:
    The file should be copied to wardrobe upload share, for instance, \\EMS\WARDROBE-UPLOAD). After copying file, enter file name in the URL field.
    • Windows users go to Tools>Map network drive in Windows explorer.
    • Mac users go to finder, press command+k (In menu choose "Go -> Connect to Server") and type smb:\\EMS\WARDROBE-UPLOAD

How to find a link to .sra file in GEO

The full instructions can be found here

Protocol and Notes tabs

You can enter protocol information and notes (including images and hyperlinks) in these tabs.

Advanced tab

  • Experiment additional info, ChIP-seq specific parameters:
    Confirm or select the expected fragment size (“Expected Fragment Size”). In some cases, MACS does not determine the fragment size correctly. In these cases, you can force it to use the fragment size that you select by checking the “Force to use this fragment size?” box.
    Selecting the “Remove duplicates?” box will remove coincident reads (pairs of reads that start and end at the same position on the same strand) after alignment and before analysis.
  • Experiment additional info common part:
    Trim from the left/right:
    Selecting the “Trim from the left” or “Trim from the right” options allows getting rid of sequenced indices or poor-quality bases from either end of the read.
    If you change any of the advanced options after finishing the initial analysis, select the “Force to repeat experiment analysis?” box (only visible after the initial analysis) and click the “Save” button to implement these changes in the analysis.
    Make and save the changes only ONCE and let the reanalysis finish before further modifying any of the options, otherwise the pipeline will break.

Step 4 - Click “Save”

Quality Control Tab

Quality Control Window

Quality Control Tab can be divided into three groups of information Alignment Statistics, Base frequency plot and QC plot.

Alignment Statistics

RNA-Seq

For RNA-Seq type data next statistics are shown:

  • the total number of reads (“Tags total”)
  • the number of reads mapped in a unique fashion to transcriptome (“Tags mapped”) or genome outside of the transcriptome (“Outside annotation”)
  • multi-mapped (“Multi-mapped reads”, reads which are discarded there is more than one place on the genome where read can be aligned)
  • unmapped (percentage shown in red in the pie chart).
  • Ribosomal contamination shows the number of reads that can be mapped to ribosomal DNA repeat. This reflects the completeness of ribosomal RNA removal during library preparation.

A high percentage of reads outside of the transcriptome (percentage shown in orange in the pie chart) may be due to DNA contamination and can inflate RPKM values by several points; this contamination is often a result of insufficient, on-column DNAse digestion.

DNA-Seq

For DNA-Seq type data the system presents statistics:

  • the total number of reads (“Tags total”)
  • the number of reads mapped to the genome (“Tags mapped”)
  • multi-mapped (“Multi-mapped reads”, which are discarded)
  • mapped duplicates (“Mapped duplicates”, which may be discarded depending on the selected settings in the Advanced tab of the Experiment form tab)
  • unmapped (percentage shown in red in the pie chart).

In the case of single read sequencing fragment size is estimated by MACS (“Estimated fragment size”) from the distribution of reads mapping to the top and bottom strands.

Base frequency plot

Frequency plot

“Base frequency plot” shows how often each base is present at each position of the read. Spikiness of the base frequency plot can help detect adapter contamination during library construction. The ratio of CG/AT can also indicate the presence of DNA contamination – in the human genome, the CG/AT is ~60/40, whereas in the human transcriptome, it is closer to ~50/50.

QC plot


p(. The “QC” box plot shows the distribution of Phred quality scores for each base as reported in the FASTQ file.

Download BAM file

“File link” can be used to download the read mapping data (.bam) for use in other software. The same name can be used to find the sample’s folder on the Wardrobe server.

Genome browser Tab

Genome Browser Window

The data are displayed on the genome browser as coverage per million reads. For single-read DNA-Seq, coverage is calculated after extending the reads to the estimated fragment size. For pair-end reads, actual fragment sizes are used. Islands identified by MACS are shown under the signal track. For RNA-Seq, actual coverage by reads is used. For stranded RNA-Seq, reads mapping to the top and bottom strand are shown separately. Please note that for the dUTP method, the reads will map to the opposite strand.

RPKM list Tab

The RPKM tab displays for RNA-Seq type records and shows RPKM values for genes or isoforms. Total read number can also be shown (select the drop-down menu of one of the columns>Columns>TOT_R). Values are initially determined by our algorithm for each RefSeq by isoform; however, they can also be summed up for common TSS (for promoter activity studies, e.g. average tag density profiles) and for genes (for functional analysis, e.g. GO) using the drop-down menu.

The table can be sorted, searched and filtered by clicking on the appropriate column headings. The “Jump” button opens the genome browser tab on the selected gene. The “Save” button allows the table to be downloaded in .csv format.

Islands list Tab

Islands Tab

  The Islands list tab appearss for DNA-Seq type records and shows the list of islands as detected by MACS2, statistics and the nearest gene. Islands are classified (“region” column) as intersecting with promoter, upstream (less than 20 kb), exon, intron or intergenic regions (in this order). The promoter radius can be adjusted; click the “apply” button after changes in the promoter size are made. This will also modify the peak distribution plot in the Island Distribution tab (see below).
  If an island has multiple “summits” each summit coordinate (“start” and “end”) is shown as a separate row together with the position of the whole island (“txStart” and “txEnd”). Checking the “Show uniq islands?” box will show each island (start and end position; “txStart” and “txEnd”) only once and will not show summits. The “Jump” button opens the genome browser tab on the selected island. The “Save” button allows the table to be downloaded in .csv format.
  The headings of the table can be used to filter peaks: e.g. selecting peaks with q value better than a certain threshold or 10000 best peaks, etc. This selection can be saved using the retain filter button and will be used in downstream analysis e.g. MAnorm.
The “get fasta” button can be used to obtain sequence located within X bases around each summit in the selected peaks. This can be used to obtain sequence logos with MEME-ChIP or MelinaII. Please note that Mac/Unix and Windows use different “End of Line” symbols. For this reason the sequences will not look properly fasta formatted in Windows. However, MEME will still recognize the format.
http://meme.nbcr.net/meme/doc/meme-chip.html
http://melina2.hgc.jp/public/index.html

R Tab

R Tab ChIP

  R tab was included to allow for easy integration of specialized tools into Wardrobe. It can be used to run R code within Wardrobe. Currently default code creates pileup vs. peak rank graphs and peak length histogram for ChIP-Seq. Additionally, this window is used to output plots produced by MACS.

R Tab RNA

  For RNA-Seq, the default R code creates gene body coverage and RPKM histogram.

R Tab Sources

  Custom R code can be edited by administrators in the Source tab. Please see help file for the Wardrobe R library for reference.

Average Tag Density Tab

The Average Tag Density tab displays for DNA-Seq type records and shows the average tag density profile around all annotated TSS. Such graphs can be used to estimate the success of ChIP-Seq type experiments for some histone modifications (e.g. H3K4me).

Islands Distribution Tab

The Islands Distribution tab displays for DNA-Seq type records and shows distribution of islands between upstream, promoter, exon, intron and intergenic regions (in this order left to right). Area definitions in relation to the TSS are shown above the diagram for the upstream and promoter regions. The number and percentage of island counts per region are shown on each region in the diagram.

Fig1.png (58.7 KB) Andrey Kartashov, 01/24/2015 02:31 AM

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Fig10.png - Frequency plot (163 KB) Andrey Kartashov, 01/25/2015 11:27 PM

Fig2.png - Experiment Window (328 KB) Andrey Kartashov, 01/25/2015 11:32 PM

Fig8.png - Quality Control Window (250 KB) Andrey Kartashov, 01/25/2015 11:34 PM

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Fig13.png - Genome Browser Window (504 KB) Andrey Kartashov, 01/27/2015 01:18 AM

Fig15.png (131 KB) Andrey Kartashov, 01/27/2015 01:44 AM

Fig4_1.png (17.6 KB) Andrey Kartashov, 05/09/2015 03:23 PM

Fig4.png - Experiment Form (52.2 KB) Andrey Kartashov, 05/09/2015 03:23 PM

Fig14.png - Islands Tab (329 KB) Andrey Kartashov, 05/09/2015 03:46 PM