Importing your Exeter NGS data into Galaxy:

Transcription

1 Importing your Exeter NGS data into Galaxy: The aim of this tutorial is to show you how to import your raw Illumina FASTQ files and/or assemblies and remapping files into Galaxy. As of 1 st July 2011 Illumina data is provided in Sanger FASTQ format rather than the old Illumina 1.3+ format. As such any raw data imported into Galaxy should not require conversion before quality control. Rather than downloading large (>10Gb) files to your local machine and trying to upload them to Galaxy, we will instruct the Galaxy server to obtain the files directly from the FTP site. This saves a lot of time and effort, but is in places slightly fiddly. Before you start: Please make sure you have an account on the Exeter Galaxy server ( and that you have the notifiying you of your results. Once a run is complete you may wish to repeat an analysis provided by the Bioinformatics facility or do your own custom analysis. For details of how to perform a given analysis, please see the other tutorials in this series. In your notification once your run completes, you should receive details of the FTP server hosting your data. FTP stands for File Transfer Protocol a method of moving data between computers. Cut and paste the link (beginning ftp://) to the address bar in your browser. This will contain a list of all the projects you have. Click on the project you are interested in.

2 You should see a page similar to that shown below. Each directory contains results for a single sample within a given project. The precise layout will depend on the type of sequencing run for your sample (i.e. Denovo genomic. RNA-seq etc) and the browser you are using. Note Safari will not work. Click on the sample you wish to import results for.

3 Obtaining FASTQ Illumina reads: You should see a list of file similar to that below. Files with names ending in.fastq are the complete set of reads without any quality control or filtering applied. Those ending in.filtered have the quality control filters applied and any adaptor sequence trimmed off. Files ending.png are plots representing the quality scores and nucleotide distribution pre-filtering. Important: Files with names like 11_NoIndex_L006_R1_001.fastq mean that this is sample name 11 without any index sequence present (i.e. no multiplexing), in Lane 6 and Read 1. The file 11_NoIndex_L006_R2_001.fastq would be the same sample from read 2. If you are conducting a paired end analysis, make sure you download both the read 1 and read 2 files. If you want to import the raw FASTQ files into Galaxy then right click on the the file and select 'Copy link location'.

4 Instructing Galaxy to fetch your data: Create a Galaxy account if you do not already have one. Once you have logged in, select the right hand History menu and select 'Create New'. Rename your history to something appropriate. Click on 'Get Data' in the left-hand main menu.

5 In the URL/Text window you should paste the URL you copied from the FTP page. It should look something like this. Also note that you will need to do this for each file you want to import. It is probably only worth doing this for files which are larger than a few hundred megabytes. You can upload smaller files from your local machine if you prefer. Note: On some systems you may find that the above URL does not work. In this case you will need to change some of the %2E into dots in the URL e.g: ftp://my %40ex%2eac%2euk:ctzgdas@bioruby.ex.ac.uk/project_759/sample_11/raw_illumina_reads/11_noindex_l006_r1_001.fastq ftp://my %40ex.ac.uk:ctzgdas@bioruby.ex.ac.uk/project_759/sample_11/raw_illumina_reads/11_noindex_l006_r1_001.fastq Note that the %40, should be left in place.

6 You can now click on Execute and submit the data. Galaxy will now fetch the data direct from the FTP site and import it into your history. Depending on the size of the file this can take anywhere up to an hour per file so please be patient. Once complete, the item should turn green. It is recommended that you click on the pencil icon and change the name to something a bit more memorable. Once you have imported all the relevant files, you can begin your analysis! Konrad Paszkiewicz, Exeter Sequencing Service, 23/09/2011