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Mapping short reads

If you are using genome as the reference for RNAseq reads, you will need to use a splice-aware aligner like Tophat2. If you are using cDNA as the reference, you can use a general purpose aligner like Bowtie2.

You need to do only one of the procedures based on what your group have been assigned to.

Step 1: Mapping with Tophat2

  1. Click on App.

  1. In the finder window type “Tophat”

  1. Select “Tophat2-PE”.

    _images/tophat_1.png

  1. As indicated in the figure, Name your analysis as you want.

  1. Select the output folder where your analysis is going to be.

  1. Click on “Input data”

    _images/tophat_2.png

  1. Click on the Green “+” sign.

8. Navigate to the folder where your samples are located. Select only the first read files. Click “OK”. You can select all three of your first read files.

_images/tophat_3.png

  1. Scroll down and click on the “+” below “Fastq file(s) (Read 2 of paired end reads):”

  1. Select “Reference Genome” and select the tomato genome sequence as input.

    _images/tophat_4.png

  1. Make sure quality is Sanger and leave rest of the default values as they are. Launch the analysis.

    _images/tophat_5.png


Step 2: Mapping with Bowtie2

  1. Click on App.

  1. In the finder window type “Bowtie”.

  1. Select Bowtie app indicated in the figure.

    _images/bowtie_1.png

  1. As indicated in the figure, Name your analysis as you want.

  1. Select the output folder where your analysis is going to be.

    _images/bowtie_2.png

  1. Click on “Input”

8. Navigate to the folder where your samples are located. Select first and second read files. You can only input one sample at a time.


  1. You need to name your output file carefully. For e.g., if it is heat1 sample, name the output as heat1.sam.

    _images/bowtie_4.png

  1. Select “Reference Index” and select the tomato cDNA sequence as input.

    img/bowtie_3.png

  1. Select options. Set “Minimum fragment length” as 100 and “Maximum fragment length” as 600. Launch the analysis.

    _images/bowtie_5.png