Page tree

Versions Compared

Key

  • This line was added.
  • This line was removed.
  • Formatting was changed.
Comment: dostosowanie tutoriala do nowego galaxy

...

PL-Grid instance of Galaxy provides all the tools necessary for quality control of your raw reads, using FASTQC software, and  for preparation to alignment to the reference throughout trimming and filtering, using Flexbar software. This tutorial will give you an introduction to how to use these tools and it will guide you through the process.

Image RemovedImage Removed

For the reference, the figures above show where to quickly find the used tools.

Input Dataset

In this tutorial we will use sample single-end (SE) reads from total RNA sequencing of two different chicken lines.

The dataset is available in the list of published histories (https://galaxy.plgrid.pl/history/list_published) - look for the Adapters historyYou may import it to your working space from there.

 

 Image Removed

 

we  will use the file ‘test_dataset.fastq’ which is available for download from the Bismark homepage (it contains 10,000 reads in FastQ format, Phred33 qualities, 50 bp long reads, from a human directional BS-Seq library).

Link to input dataset:

http://www.bioinformatics.babraham.ac.uk/projects/bismark/test_data.fastq

 

Also, you may try using your own input files. In such a case, please use the Upload File tool.

...

File format: fastqsanger [HINT: this is important, because galaxy will recognize all kinds of fastq files as generic fastq format. However, most tools require more specific fastqsanger format]

Image Removed

You could do it also later using "Edit Attributes" in your history Data/History window.

...

Info
iconfalse
titleTools
NGS: QC and manipulation -> FASTQC: FASTQ/SAM/BAM -> FastQC: Read QC Quality reports using FastQC
  • Short read data from your current history: select uploaded fastq file.

You could also add the "Contaminant list" if you know basic assumptions of library preparation step, or add the list provided us by FASTQC authors (https://github.com/csf-ngs/fastqc/blob/master/Contaminants/contaminant_list.txt).


Image RemovedImage Added

The FASTQC results should be available in your history window (click the eye icon near the name of the history step related to the executed FastQC run):

Image RemovedImage Added

 

Some of these results are presented below:

...

Flexbar software demultiplexes barcoded runs and removes adapter sequences. Moreover, trimming and filtering features are provided. Flexbar increases read mapping rates and improves genome and transcriptome assemblies. It supports next-generation sequencing data in fasta/q and csfasta/q format from Illumina, Roche 454, and the SOLiD platform.

Info
iconfalse
titleTools
NGS: QC and manipulation Personalized medicine -> Flexbar flexible barcode and adapter removal

...